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S'. SOUTHERN
: 'UACULTURE
* CENTER "
S EIGHTE-NTfHANNUAL PROGRESS REPORT
FotthePeiibdPthrocugAugust 31 2005
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DeceMtber, 2005
Southeat Regional Aquaculture Center
P.O. BdIx 19
Sto eville, Mississippi 38716'
P ote;& 662-686-3428
:Fxi 662-686-3320
* :ELrail: srac@drec.msstate.edu
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Work summarized in this report was supported in part by Grant
Nos. 2000-38500-8992, 2001-38500-10307, 2002-38500-11805,
2003-38500-12997 and 2004-38500-14387from the United States
Department ofAgriculture sponsored by the Cooperative State
Research, Education, and Extension Service.
EIGHTEENTH ANNUAL PROGRESS REPORT
SOUTHERN REGIONAL AQUACULTURE CENTER
Dr. Craig S. Tucker, Director
P.O. Box 197
Stoneville, Mississippi 38776
Phone: 662-686-3285
Fax: 662-686-3320
E-mail: srac@drec.msstate.edu
http://www.msstate.edu/dept/srac
Table of Contents
TABLE OF CONTENTS
PREFACE .............................................................. ii
ACKNOWLEDGMENTS ..................................................... ii
INTRODUCTION ........................................................ 1
ORGANIZATIONAL STRUCTURE ........................................... 3
Administrative Center .................................................. 3
Board of Directors .................................................... 4
Industry Advisory Council ............................................... 5
Technical Committee ................................................... 6
Project Criteria ........................................................ 6
Project Development Procedures .......................................... 7
ADMINISTRATIVE ACTIVITIES ................................. ........... 8
PROGRESS REPORTS ......................................... ............ 9
Publications, Videos and Computer Software .............................. 10
Identification, Characterization, and Evaluation of Mechanisms
of Control of Bolbophorus-like Trematodes and Flavobacterium
columnare-like Bacteria Causing Disease in Warm Water Fish .................. 16
Improving Reproductive Efficiency to Produce Channel x Blue
Hybrid Catfish Fry ................................................... 43
Innovative Technologies and Methodologies for Commercial-Scale
Pond Aquaculture .................................................... 85
SUPPORT OF CURRENT PROJECTS ........................................ 98
SRAC RESEARCH AND EXTENSION PROJECTS .............................. 99
SRAC Eighteenth Annual Progress Report, December 2005 i
Preface and Acknowledgments
PREFACE
In 1980, Congress recognized the opportunity for making significant progress in domestic aqua-
culture development by passing the National Aquaculture Act (P.L. 96-362). The Act established
USDA as the lead agency for aquaculture coordination and called for development of a National
Aquaculture Plan. The next year, Congress amended the National Agricultural Research, Extension,
and Teaching Policy Act of 1977 (P.L. 95-113) by granting, in Title XIV, Subtitle L, Sec. 1475(d)
of the Agriculture and Food Act of 1981 (P.L. 97-98), authority to establish aquaculture research,
development, and demonstration centers in the United States.
Congress envisioned the Centers as focal points in a national program of cooperative research,
extension, and development activities that would be developed in association with colleges and
universities, state Departments of Agriculture, federal facilities, and non-profit private research
institutions with demonstrated excellence in aquaculture research and extension. Eventually, five
such Centers were established one in each of the northeastern, north central, southern, western,
and tropical Pacific regions of the country. Funding for the Centers was reauthorized in subsequent
Farm Bills (the Food, Agriculture, Conservation, and Trade Act of 1990 [P.L. 101-624]; the
Agriculture Improvement and Reform Act of 1996 [P.L. 104-127]; and the Farm Security and Rural
Investment Act of 2002 [P.L. 107-171].
Projects that are developed and funded by the Regional Centers are based on industry needs and are
designed to directly impact commercial aquaculture development in all states and territories. The
Centers are organized to take advantage of the best aquaculture science expertise, education skills,
and facilities in the United States. Center programs insure effective coordination and a region-wide,
team approach to projects jointly conducted by research, extension, government, and industry
personnel. Inter-agency collaboration and shared funding are strongly encouraged.
ACKNOWLEDGMENTS
The Southern Regional Aquaculture Center acknowledges the contributions of the Project Leaders
and Participating Scientists involved in the projects reported in this Eighteenth Annual Progress
Report. Members of the SRAC Board of Directors, Industry Advisory Council, and Technical
Committee have provided valuable inputs to the successful operation of SRAC during the past year.
We particularly appreciate the assistance of our Board, IAC and TC, and Administrative Advisors.
We also thank the scientists and aquaculturists from across the country who contributed their ex-
pertise and valuable time to review SRAC project proposals and publications. Without their help,
it would be impossible to maintain the high quality of this program.
SRAC Eighteenth Annual Progress Report, December 2005 ii
Introduction
INTRODUCTION
The farm-gate value of United States aquaculture exceeded $1 billion dollars in 2004, and nearly
70% of the crop was produced in the southeastern states. Aquaculture is an important part of
southeastern agriculture, and its importance reaches far beyond the farm gate. Most of the support
functions for the industry such as feed manufacture and equipment fabrication also take place
in the region. The total economic impact of aquaculture is therefore many times the value of
production alone.
The success of southeastern aquaculture has come with relatively little private sector support for
research and development. The larger, more developed agricultural sector such as poultry, cotton
and soybeans are supported by a vast infrastructure of agribusinesses that conduct most of the
research needed to sustain commodity growth. Aquaculture, on the other hand, receives little
private-sector R&D support, relying instead almost entirely on public-sector funds for technology
development.
Although government agencies, particularly the United States Department of Agriculture, have
provided significant support for aquaculture research and development, much of that funding is
earmarked for specific use by specific institutions. The USDA-CSREES Regional Aquaculture
Center program is the only funding mechanism with the flexibility to stay abreast of industry
development, identify problems on a region-wide scale, and implement cooperative, interstate
projects to solve those problems.
Since its inception in 1987, the Southern Regional Aquaculture Center has become the centerpiece
of aquaculture research and extension in the southeastern United States. In its 18 years of operation,
the Center has disbursed $12 million to fund 28 multi-state research and extension projects. More
than 175 scientists from 30 institutions in the southeast have participated in Center projects.
In the past year, four research projects funded at $2.5 million were in progress. Work on those
projects has been reported in 25 publications and 24 papers presented at meetings. The Center's
"Publications" project is in its tenth year of funding and is under the editorial direction of faculty
and staff at Texas A&M University. Eleven publications were printed this year, and eight more
were in various stages of production. To date, SRAC "Publications" projects have generated more
than 171 fact sheets with contributions from 158 authors from throughout the region.
The most important measure of the impact ofprojects funded by the Southern Regional Aquaculture
Center is the extent to which the results have influenced or improved domestic aquaculture. For
example, a discovery in the "Disease" project will have a dramatic impact on catfish farming.
Research conducted as part of that project led to discovery of a safe, inexpensive method to control
the intermediate host of the trematode parasite Bolbophorus damnificus. Over the last 5 years, this
SRAC Eighteenth Annual Progress Report, December 2005 1
Introduction
disease was discussed in doomsday language. In the near future, however, it may be considered no
more than a manageable nuisance.
Beginning with the first projects funded by the Southern Regional Aquaculture Center, interest
among aquaculture research and extension scientists in Center activities has been excellent. We are
pleased with the participation by our research and extension scientists in the Southern Region in ad
hoc Work Group meetings and Steering Committees, and their willingness to serve as Project
Leaders and Principal Investigators for the projects. We believe this broad-based representation has
resulted in strong, cooperative research that will be of long-lasting benefit to aquaculture producers
and consumers, and to the growth of the aquaculture industry in the Southern United States.
This Eighteenth Annual Progress Report of the Southern Regional Aquaculture Center covers the
activities of the Administrative Center during the past year. Progress reports on the four multi-year
research and extension projects supported by Southern Regional Aquaculture Center during this
reporting period cover the life of the projects from their initiation date through August 31, 2005.
2 SRAC Eighteenth Annual Progress Report, December 2005
Organizational Structure
ORGANIZATIONAL STRUCTURE
The Agriculture Acts of 1980 and 1985 authorized establishment of aquaculture research, develop-
ment and demonstration centers in the United States. With appropriations provided by Congress for
the 1987 and 1988 FYs, efforts were undertaken to develop the five Regional Aquaculture Centers
now in existence. Organizational activities for SRAC began in 1987, with the first research and
extension projects initiated in 1988.
Research and extension problem areas for the southern region are identified each year by the
Industry Advisory Council (IAC), which consists of fish farmers and allied industry representatives
from across the region. The Technical Committee (TC), consisting of research and extension
scientists from all states within the region, works with the IAC to prioritize problem areas. The two
groups then work together to develop "Problem Statements" describing objectives of work to solve
problems with the highest priority. Using inputs from industry representatives, regional Work
Groups of the most qualified research and extension scientists are formed. The Work Groups then plan
and conduct the work in conjunction with an Administrative Advisor appointed by the Board. Regional
aquaculture funds are allocated to participants in SRAC projects approved by the Board and CSREES.
Reviews of project proposals, progress reports, and recommendations for continuation, revision, or
termination of projects are made jointly by the TC and IAC and approved by the Board.
The thirteen states and two territories represented by SRAC are Alabama, Arkansas, Florida,
Georgia, Kentucky, Louisiana, Mississippi, North Carolina, Oklahoma, Puerto Rico, South Carolina,
Tennessee, Texas, U.S. Virgin Islands, and Virginia.
ADMINISTRATIVE CENTER
The Administrative Center is located at the Delta Research and Extension Center, Stoneville,
Mississippi. Mississippi State University serves as the Host Institution. All necessary support services
for the Board, IAC, TC, Steering Committees and project Work Groups are provided by the
Administrative Center. This includes monitoring status and progress of projects, preparing and
executing Letters of Agreement, tracking administrative and project expenditures, reviewing progress
reports, and assisting Project Leaders and participating institutional Grants Offices as needed.
Operation and funding are approved by the Board for inclusion in the Grant Application submitted
annually to USDA/CSREES. The Center staff also prepares and submits to USDA/CSREES an Annual
Plan of Work covering Center activities and projects to be funded. Following final approval, Letters of
Agreement are prepared and executed with all participating institutions. The Center acts as fiscal agent
to disburse and track all funds in accordance with the provisions of the grants. Additional Administrative
Center responsibilities are detailed in the "Administrative Activities" section of this report.
SRAC Eighteenth Annual Progress Report, December 2005
Organizational Structure
BOARD OF DIRECTORS
The Board is the policy-making body for SRAC. Membership provides an appropriate balance among
representatives from State Agricultural Experiment Stations, Cooperative Extension Services, 1890
Institutions, and the Administrative Heads Section (AHS) of the Board on Agriculture Assembly (BAA)
of the National Association of State Universities and Land Grant Colleges (NASULGC).
The structure of the Board is as follows:
Three members of the 1862 Southern Extension Service Directors Association
Three members of the 1862 Southern Experiment Station Directors Association
One member of the 1890 Association of Research Administrators
One member of the 1890 Association of Extension Administrators
One AHS administrator from the host institution
Members of the Board are:
Ivory Lyles, Arkansas Cooperative Extension System
Ken Roberts, Louisiana Cooperative Extension Service
Gaines Smith, Alabama Cooperative Extension System
David Morrison, Louisiana State University
Vance Watson, Mississippi State University, Chairman
Greg Weidemann, University of Arkansas
Harold R. Benson, Kentucky State University
W. S. Clarke, Virginia State University
Ex-officio Board members are:
Chair, Industry Advisory Council
Vice-chair, Industry Advisory Council
Co-chair for Extension, Technical Committee
Co-chair for Research, Technical Committee
Director, SRAC
The Board is responsible for 1) overall administration and management of the regional center
program; 2) establishment of overall regional aquaculture research and extension goals and
allocations of fiscal resources to ensure that the center develops strong programs in both research
and extension; 3) establishment of priorities for regional aquaculture research and extension
education activities based on inputs from the TC and IAC and guidance from the National
Aquaculture Development Plan; 4) review and approval of annual plans of work and
accomplishment reports; and 5) final selection of proposals for funding by SRAC.
4 SRAC Eighteenth Annual Progress Report, December 2005
Organizational Structure
INDUSTRY ADVISORY COUNCIL
The IAC, which meets at least annually, is composed of representatives of state and regional
aquaculture associations, federal, territorial and state agencies, aquaculture producers, aquaculture
marketing and processing firms, financial institutions, and other interests or organizations as deemed
appropriate by the Board of Directors.
The IAC provides an open forum wherein maximum input from private and public sectors can be
gained and incorporated into annual and ongoing plans for SRAC. The chairman serves for two
years and is elected by IAC members.
Members of the IAC are:
Neal Anderson, AR
Bill Cheek, LA
Jane Corbin, TN
Richard Eager, SC
Theop Inslee, OK
Austin Jones, MS
Shorty Jones, MS
Joey Lowery, AR
Robert Mayo, NC
Steve Minvielle, LA
Steve Price, KY
Brent Rowley, TX
Robert Schmid, TX
Dan Solano, FL
Marty Tanner, FL
Rafe Taylor, AL
David Teichert-Coddington, AL
IAC members serve up to four-year appointments having staggered terms with options for
reappointment.
The IAC 1) identifies research and extension needs; 2) works with the TC to prioritize research and
extension needs; 3) works with the TC to develop problem statements and recommend funding
levels for projects addressing priority research and extension needs; 4) reviews project proposals,
progress reports, and termination reports; and 5) recommends to the Board, jointly with the TC,
actions regarding new and continuing proposals, proposal modifications and terminations.
SRAC Eighteenth Annual Progress Report, December 2005
Organizational Structure
TECHNICAL COMMITTEE
The TC consists of representatives from participating research institutions and state extension
services, other state or territorial public agencies as appropriate, and private institutions.
Membership of the TC includes research and extension scientists representing essentially all states
in the region. The TC meets as needed, but at least annually, and has a co-chairman for research and
a co-chairman for extension. Co-chairmen serve for two years and are elected by TC members.
Members of the TC for research are:
David Brune, SC
Frank Chapman, FL
Allen Davis, AL
Lou D'Abramo, MS
Carole Engle, AR
Delbert Gatlin, TX
Conrad Kleinholz, OK
Ray McClain, LA
Steve Mims, KY
J. L. Wilson, TN
Members of the TC for Extension are:
Jimmy Avery, MS
Jesse Chappell, AL
Dennis DeLong, NC
David Heikes, AR
George Luker, OK
Greg Lutz, LA
Michael Masser, TX
Craig Watson, FL
Jack Whetstone, SC
Forrest Wynne, KY
Technical Committee members serve up to four-year appointments having staggered terms with
options for reappointment.
The TC 1) works with the Industry Advisory Council to prioritize research and extension needs;
2) works with the Industry Advisory Council to develop problem statements and recommend
funding levels for projects addressing priority research and extension needs; 3) reviews proposals,
progress reports, and termination reports; and 4) recommends to the Board, jointly with the IAC,
actions regarding new and continuing proposals, proposal modifications and terminations.
PROJECT CRITERIA
Projects developed within SRAC should meet the following criteria:
* Addresses a problem of fundamental importance to aquaculture in the Southern
Region;
* Involves participation by two or more states in the Southern Region;
* Requires more scientific manpower, equipment, and facilities than generally
available at one location;
6 SRAC Eighteenth Annual Progress Report, December 2005
Organizational Structure
* Approach is adaptable and particularly suitable for inter-institutional cooperation,
resulting in better use of limited resources and a saving of funds;
* Will complement and enhance ongoing extension and research activities by
participants, as well as offer potential for expanding these programs;
* Is likely to attract additional support for the work which is not likely to occur
through other programs and mechanisms;
* Is sufficiently specific to promise significant accomplishments in a reasonable
period of time (usually up to 3 years);
PROJECT DEVELOPMENT PROCEDURES
The IAC initiates the project development process by identifying critical problems facing
aquaculture in the region. The TC and IAC then jointly prioritize problem areas and recommend the
most important research and extension needs to the Board. Writing teams selected from the TC-IAC
membership develop "problem statements" for each of the selected priority areas. Problem
statements briefly describe the problem area and general objectives of the work to be conducted. The
problem statement also includes a recommended funding level and project duration. Draft problem
statements are then forwarded to the Board for approval to release project development funds.
Once an area of work has been approved, the Executive Committee (the SRAC Director, the co-
chairs of the TC, and the chair and vice-chair of the IAC) appoints a Steering Committee to develop
the "Call for Statements of Interest" and oversee development of the project proposal and the
conduct of the regional project. The "Call for Statements of Interest" is distributed to state, territorial
or federal institutions and private institutions within the Southern Region with demonstrated
competence in aquaculture research and development. Interested parties respond by submitting a
"Statement of Interest" to the SRAC Adminstrative Office. After careful review of the Statements
of Interest, the Steering Committee recommends a Work Group consisting of selected project
participants and the Steering Committee. The Work Group is responsible for preparing the regional
project proposal and conducting work outlined in the proposal.
Project proposals are reviewed by the Steering Committee, IAC, TC, all project participants and
designated peer reviewers from within the region and from outside the region. The SRAC Director
submits the project proposal and peer reviews to the Board of Directors for review and approval.
Proposals not approved by the Board are returned for revision or eliminated from consideration.
The Director prepares an annual plan of work, including all project proposals approved by the
Board, and submits the plan to CSREES for approval. Pending a successful review of the project
plan and budget, CSREES notifies SRAC of final approval. Letters of Agreement (subcontracts)
between SRAC and participating institutions are then prepared and forwarded for approval and
execution by the authorized institutional official. At that point, formal work on the project begins.
SRAC Eighteenth Annual Progress Report, December 2005
Administrative Activities
ADMINISTRATIVE ACTIVITIES
The SRAC administrative staffconsists of the Center Director and Administrative Assistant. A wide
variety of support functions for the various SRAC components, including the Board, TC, IAC,
Steering Committees and project Work Groups are provided:
* Center Director serves as an ex-officio member of the Board, TC, and IAC.
* Monitor research and extension activities sponsored by SRAC.
* Solicit and receive nominations for memberships on the TC and IAC.
* Coordinate submission of written testimony to the House Agriculture, Rural Development,
and Related Agencies Subcommittee on Appropriations regarding RAC support.
* The Director of SRAC serves as a member of the National Coordinating Council for
Aquaculture which consists of the Directors of the five Regional Centers and appropriate
USDA/CSREES National Program staff.
* Prepare and submit Grant Application to USDA/CSREES entering into funding
agreement for each fiscal year, Annual Plan of Work and Amendments.
* Develop and execute appropriate Letters of Agreement with participating institutions in
each funded proposal for the purpose of transferring funds and coordinating and
implementing projects approved under each of the grants.
* Serve as fiscal agent to review and approve invoices and distribute funds to participating
institutions as approved under the grants and as set forth in the Letters of Agreement.
* Prepare budgets for the Administrative Center, track administrative expenditures, and
obtain USDA/CSREES approval for project and budget revisions.
* Prepare budget reports for the Board of Directors, tracking expenditures and status of
funded projects and the Administrative Center.
* Assist Steering Committees and Work Groups with preparation and revision of proposals
for technical and scientific merit, feasibility and applicability to priority problem areas.
* Solicit and coordinate national reviews of project proposals.
* Distribute fact sheets and videos to research and extension contacts throughout the
Southern Region, other RACs, and USDA personnel.
* Produce and distribute the "SRAC Annual Progress Report," which includes editing and
proofreading the project reports and producing camera-ready copy.
* Produce and maintain the web site for SRAC which provides downloadable copies of all
SRAC fact sheets, the Operations Manual and Annual Reports, as well as lists of other
research publications and extension contacts in the Southern Region.
* Prepare and distribute Calls for Statements of Interest to research and extension directors
and other interested parties throughout the Southern Region.
* Respond to requests from aquaculture producers, the public, and research and extension
personnel for copies of fact sheets, research publications and videos produced by SRAC
and the other Centers, as well as requests for general aquaculture-related information.
8 SRAC Eighteenth Annual Progress Report, December 2005
Progress Reports
PROGRESS REPORTS
The following cumulative reports detail the progress of research and extension work accomplished
for the duration of the respective projects through August 31 of the current year. These reports are
prepared by the Project Leaders in conjunction with the institutional Principal Investigators.
Publications, Videos and Computer Software .............. Page 10
Identification, Characterization, and Evaluation of
Mechanisms of Control of Bolbophorus-like Trematodes
and Flavobacterium columnare-like Bacteria Causing
Disease in Warm Water Fish ........................ Page 16
Improving Reproductive Efficiency to Produce
Channel x Blue Hybrid Catfish Fry ..................... Page 43
Innovative Technologies and Methodologies for
Commercial-Scale Pond Aquaculture .................... Page 85
SRAC Eighteenth Annual Progress Report, December 2005
Publications, Videos and Computer Software
PUBLICATIONS, VIDEOS AND
COMPUTER SOFTWARE
Funding Level
Participants
Administrative
Advisor
Year .
Year 2.
Year 3.
Year 4.
Year 5.
Year 6.
Year 7.
Year 8.
Year 9
Year 10
Total ..
Reporting Period
April 1, 1995 August 31, 2005
............... $ 50,000
................. 60,948
................. 45,900
................. 60,500
................. 67,000
................. 77,883
................. 83,850
................. 77,600
................. 84,500
................ 78,700
............... $686,881
Texas A&M University System serves as Lead Institution, with Dr.
Michael Masser as Project Leader. Participants in this project include
authors and co-authors from all states in the region as shown in the
listing of publications at the end of this report.
Dr. Joe McGilberry, Director
Mississippi State University Extension Service
Mississippi State, Mississippi
PROJECT OBJECTIVES
1. Review and revise, as necessary, all SRAC extension printed and video publications.
2. Establish an ongoing project location to develop and distribute new SRAC educational
publications and videos for Southern Region aquaculture industries. This project will be
responsible for preparation, peer review, editing, reproduction, and distribution of all
Extension and popular-type publications for all SRAC projects.
3. Place current, revised, and new publications in electronic format (e.g., Internet or
compact disk) for more efficient use, duplication, and distribution.
10 SRAC Eighteenth Annual Progress Report, December 2005
Publications, Videos and Computer Software
ANTICIPATED BENEFITS
The most direct benefit from this project to
the aquaculture industry is the widespread and
ready availability of detailed information on
production and marketing of aquacultural
products. SRAC fact sheets, videos, and other
publications are distributed worldwide to a
diverse clientele.
Extension Specialists. When this project was
initiated, fewer than half the states had edu-
cational materials covering the major aqua-
cultural species in their state. The concept of
using the SRAC program to produce timely,
high-quality educational materials is based
upon the benefit of utilizing a region-wide
pool of expertise to develop materials for
distribution through the nationwide network
of Extension Specialists and County Agents.
This process makes efficient use of personnel
at the State level, and results in high-quality
educational materials that are readily available
RESULTS ATA GLANCE...
S158 authors from across the United
States have contributed to SRAC's
publication projects.
to scientists, educators, producers, and the
general public.
Educators. Many colleges and universities in
the United States use SRAC technical fact
sheets as reference material in aquaculture
and fisheries courses. Educational institutions
at the elementary and secondary level use
SRAC extension materials in the classroom to
make students aware of aquaculture production
and associated trades as a possible vocation.
Consumers. Information is readily available
for consumers who are seeking background
information on aquaculture.
Producers. Information on the use of thera-
peutants, pesticides, methods of calculating
treatment rates, and possible alternative crops
and marketing strategies is in constant demand
by aquaculturists. Videos that demonstrate
such techniques are a ready source of"how-to"
information.
Potential investors. Detailed information on
production and marketing constraints and ways
to alleviate or manage those constraints are
particularly helpful to people making decisions
about entering the aquaculture business. Eco-
nomic information is used by lending agencies
and potential investors, as well as established
producers who use the information to help
make day-to-day decisions on farm manage-
ment.
Internet access. Availability of SRAC pub-
lications via the Internet and compact disk
makes access faster and easier, facilitates
searching for needed information, and reduces
storage space requirements for printed
documents.
SRAC Eighteenth Annual Progress Report, December 2005 11
Publications, Videos and Computer Software
PROGRESS AND PRINCIPAL ACCOMPLISHMENTS
During this current project year, eleven new
fact sheets were completed and the Aquaplant
web site updated. All have been distributed
throughout the Southern Region and to
interested Extension Specialists in other
regions. Five fact sheets are currently in some
stage of writing, production, or revision. Four
fact sheets have currently not had drafts
submitted.
All SRAC publications are based on research
conducted within the region or in surrounding
areas. Research funding from universities
within the region, as well as funding from
private sources, has been used to support the
work on which the fact sheets are based.
Copies of all SRAC fact sheets are available
at and
.
RESULTS AT A GLANCE...
* Eleven fact sheets and a video were
completed this year with four fact sheets
in progress.
k Twenty-seven scientists from across the
Southern Region contributed to
publications completed by SRAC this
year.
t SRAC has now published 171 fact
sheets and species profiles, 4 project
summaries, 19 research publications,
and 20 videos.
" Educators in schools and colleges use
SRAC publications in classrooms
throughout the U.S. and the world.
WORK PLANNED
During the next project year, four fact sheets
will be revised, and five new fact
sheets/species profiles, two project sum-
maries, and a DVD on crawfish aquaculture
will be produced. The new fact sheets will
address 1) hard clam hatcheries, 2) sperm
cryopreservation, 3) softshell crab shedding
techniques, 4) new seining technologies, and
5) in-pond grading techniques.
The four fact sheets to be revised are: 1) three
fact sheets on forage species, and 2) one fact
sheet on aquatic herbicides.
Final project summaries from the projects
1) Management of Environmentally Derived
Off-Flavors in Warmwater Fish Ponds, and
2) Optimizing Nutrient Utilization and Waste
Control through Diet Composition and
Feeding Strategies will be developed.
A DVD on crawfish aquaculture will also be
developed.
12 SRAC Eighteenth Annual Progress Report, December 2005
Publications, Videos and Computer Software
IMPACTS
This is a highly productive project with sig-
nificant regional, national, and international
impact. Fact sheets and videos are requested
and used by clientele in all 50 states on a
regular basis. Within the Southern Region,
more than 80 fact sheets and 6 videos are
distributed on request daily. Fact sheets
generated within the Southern Region are also
widely distributed by RACs and extension
personnel in other regions. An average of 5 to
20 SRAC fact sheets and three videos are
distributed daily from each of the other four
regions. This means that about 20,000 fact
sheets and 3,200 videos per year are used by
interested producers or consumers. In addition
to direct requests for printed material, fact
sheets and other informational materials are
accessed daily from the SRAC web site by
people searching for technical information. In
the period April through September of 2005,
more than 39,000 fact sheets were downloaded
(printed or saved) off the SRAC web site. Since
the fact sheets are also accessible through
numerous other university research and exten-
sion web sites, the total usage and impact is
undoubtedly several times greater.
RESULTS AT A GLANCE...
All fact sheets completed by this project
to date are available on the Internet at
and
.
In the months from April through
September 2005, more than 39,000 fact
sheets were downloaded from the
SRAC web site.
Publications and videos produced by SRAC
are increasingly used in educating high school
and college students about aquaculture. In
RESULTS AT A GLANCE...
Titles of some recent SRAC publications:
Controlling Bird Predation at Aquaculture
Facilities
r Constructing a Simple and Inexpensive
Recirculating Aquaculture System for
S Classroom Use
S* Anesthetics in Aquaculture
Production of Crawfish in Earthen Ponds
without Planted Forage
k Guidelines for Developing Aquaculture
Research Verification Programs
Managing Ammonia in Fish Ponds
Cobia
Biology and Culture of the Hard Clam
Channel Catfish Fingerling Production
Koi and Goldfish
\ Liming Ponds for Aquaculture
recent years there has been a rapid expansion
of aquaculture curricula in high schools.
These programs heavily utilize our publi-
cations and videos for educational purposes
but usage is impossible to measure because
many people access the information from
Internet sites. Aquaculture and fisheries
courses taught at many colleges and
universities also use SRAC technical fact
sheets as part of their course reference
material.
Another important impact is the education of
local, state, and federal regulators about the
SRAC Eighteenth Annual Progress Report, December 2005 13
Publications, Videos and Computer Software
aquaculture industry. This impact is difficult
to measure but feedback from personnel in
two states indicates that the fact sheets are
recommended reading for all new employees
dealing with aquaculture water quality, exotic
species, and other permitting duties. This
should be a positive influence toward making
aquaculturists better understood and the
development ofmore enlightened regulations.
The impact on consumers of aquaculture
products is also likely significant, although it
has not been quantified. Consumers are
primarily interested in a wholesome, safe, and
inexpensive product, and it has been reported
that the consumer-oriented fact sheets and
videos developed within SRAC have gen-
erated more interest than the producer-
directed materials. The fact sheets are in
demand in both the English and Spanish
versions and, as more information becomes
available, extension materials on food safety
will be in increased demand by health
conscious consumers.
PUBLICATIONS, MANUSCRIPTS OR PAPERS PRESENTED
Fact Sheets Completed (9/1/04 8/31/2005)
Barras, Scott C. and Kristina C. Godwin. Controlling bird predation at aquaculture facilities: Frightening
techniques. SRAC Fact Sheet 401 (Revision).
Cline, David. Constructing a simple and inexpensive recirculating aquaculture system (RAS) for
classroom use. SRAC Fact Sheet 4501.
Coyle, Shawn D., Robert M. Durborow and James H. Tidwell. Anesthetics in aquaculture. SRAC Fact
Sheet 3900.
D'Abramo, Louis R., Cortney L. Ohs, Terrill R. Hanson and Jose L. Montanez. Semi-intensive
production of red swamp crawfish in earthen ponds without planted forage. SRAC Fact Sheet
2401.
Engle, Carole, Jimmy Avery, Harry Daniels, David Heikes and Greg Lutz. Guidelines for developing
aquaculture research verification programs. SRAC Fact Sheet 5000.
Hargreaves, John and Craig S. Tucker. Managing ammonia in fish ponds. SRAC Fact Sheet 4603.
Kaiser, Jeffrey B. and G. Joan Holt. Species profile: Cobia. SRAC Fact Sheet 7202.
Whetstone, Jack M., Leslie N. Strumer and Michael J. Oesterling revised from Lorio, Wendell J. and
Sandra Malone. Biology and culture of the hard clam (Mercenaria mercenaria). SRAC Fact
Sheet 433 (Revision).
Steeby, Jim and Jimmy Avery. Channel catfish fingerling production. SRAC Fact Sheet 1803.
Watson, Craig A. Jeffrey E. Hill, and Deborah B. Pouder. Species profile: Koi and goldfish. SRAC Fact
Sheet 7201.
Wurts, William A. and Michael Masser. Liming ponds for aquaculture. SRAC Fact Sheet 4100.
Manuscripts in review
Dasgupta, Siddhartha. Economics of freshwater prawn farming in the United States.
Romaire, Robert and Ray McClain. Crawfish marketing.
14 SRAC Eighteenth Annual Progress Report, December 2005
Publications, Videos and Computer Software
Small, Brian C. Managing egg death and disease in catfish hatcheries.
Tucker, Craig S. Pond aeration (Revision).
Manuscripts in preparation
Hinshaw, Jeff and Anita Kelly. Species profile: Yellow perch.
Rakocy, Jim, Michael Masser and John Hargreaves. Aquaponics.
On-going project
Updating of the AQUAPLANT web site on aquatic weed management. Michael Masser.
,,*
SRAC Eighteenth Annual Progress Report, December 2005 15
Identification, Characterization, and Evaluation of Mechanisms
of Control of Bolbophorus-like Trematodes and Flavobacterium
Columnare-like Bacteria Causing Disease in Warm Water Fish
IDENTIFICATION, CHARACTERIZATION, AND
EVALUATION OF MECHANISMS OF
CONTROL OF BOLBOPHORUS-LIKE
TREMATODES AND FLAVOBACTERIUM
COLUMNARE-LIKE BACTERIA CAUSING
DISEASE IN WARM WATER FISH
Reporting Period
March 1, 2003 August 31, 2005
Funding Level
Participants
Administrative
Advisor
Year 1 ........................ $224,800
Year 2 ........................ $227,377
Year 3 ........................ $146,770
Total .........................$598,947
Louisiana State University
(Lead Institution) ............... John Hawke (Project Leader),
Richard Cooper
University of Tennessee .......... Andrew Mathew, Richard J. Strange
University of Arkansas at Pine Bluff Andrew Goodwin
USDA/APHIS/WS (Starkville) .... Brian Dorr, D. T. King
USDA/ARS (Stuttgart) .......... Andrew J. Mitchell
Mississippi State University College
of Veterinary Medicine (Starkville) Linda Pote, Larry Hanson, Mark
Lawrence
Auburn University .............. John Grizzle, Joe Newton
Mississippi State University,
Delta Research and Extension
Center (Stoneville) .............. David Wise
Southern Illinois University ....... Anita Kelly
North Carolina State University .... Michael Levy, James Flowers
Dr. Jerald Ainsworth, Associate Dean
College of Veterinary Medicine
Mississippi State University
Mississippi State, Mississippi
16 SRAC Eighteenth Annual Progress Report, December 2005
Identification, Characterization, and Evaluation of Mechanisms
of Control of Bolbophorus-like Trematodes and Flavobacterium
Columnare-like Bacteria Causing Disease in Warm Water Fish
PROJECT OBJECTIVES
1. Identify and characterize all of the life stages of the digenetic trematode (tentatively identified
as Bolbophorus sp.) that infects channel catfish using both classical and molecular methods.
2. Evaluate integrated methods for snail control in catfish ponds.
a. Monitor populations of catfish infected with Bolbophorus spp. to document the
effect of parasite loads on growth and survival of the fish.
b. Examine the efficacy of chemical control methods on snail populations.
c. Examine the efficacy of biological control methods (snail-eating fish) on snail
populations in ponds.
3. Develop and implement standardized methods for the isolation, culture, and antimicrobial
susceptibility testing of strains of columnaris-like bacteria isolated from diseased fish.
4. Characterize archived strains of columnaris-like bacteria based on the following
conventional and molecular techniques.
a. Morphology
b. Enzyme analysis
c. Biochemical analysis
d. Sequencing 16S ribosomal RNA and ribotyping
5. Develop challenge models for columnaris-like bacteria isolated from major warmwater
aquaculture species in the southeast.
6. Using the challenge model for each species, correlate virulence with biotype and/or
genotype of columnaris-like bacteria.
PROGRESS AND PRINCIPAL ACCOMPLISHMENTS
Objective 1. Identify and characterize all of the life stages of the
digenetic trematode(tentatively identified as Bolbophorus sp.) that
infects channel catfish using both classical and molecular methods.
Confirmation of Bolbophorus life cycle confirm the life stages of Bolbophorus
damnificus in American white pelicans and its
Mississippi State University and USDA/ snail host, Planorbellatrivolvis. Three pelicans
APHIS/WS. Two studies were conducted to were pretreated with praziquantel, challenged
SRAC Eighteenth Annual Progress Report, December 2005 17
Identification, Characterization, and Evaluation of Mechanisms
of Control of Bolbophorus-like Trematodes and Flavobacterium
Columnare-like Bacteria Causing Disease in Warm Water Fish
with B. damnificus metacercaria to establish
patent infections, and were subsequently used
to artificially infect P. trivolvis. Catfish were
exposed to these infected snails, metacercaria
from this challenge were fed to parasite free
pelicans, and patent B. damnificus infections
were established. Each life stage of this
parasite was confirmed to be B. damnificus
morphologically and molecularly. Data are
being analyzed on cercaria and ova shedding.
A second study was conducted to determine
potential snail hosts for B. damnificus and its
life cycle in the snail. Ova from pelicans
infected in Study 1 were used to artificially in-
fect several snail species housed in aquaria at
800F. Snails were checked weekly for cercaria
shedding, and checked daily when they were
positive. Time and number of cercaria shed
was recorded and data are being analyzed.
Collections to Evaluate the Avian Host
Range for Bolbophorus
USDA/ARS. A total of 106 aquatic birds have
been collected and trematodes harvested from
their alimentary canals for identification.
Some of these trematodes have potential to be
transmitted to cultured fish species. In 2003,
25 aquatic birds were collected including
5 pelicans, 10 cormorants, and 10 great egrets.
In 2004, 54 birds were collected including
17 great egrets, 12 great blue herons, 11 snowy
egrets, 6 cattle egrets, 6 green herons, 4 belted
kingfishers, and 1 little blue heron. In 2005,
27 birds were collected including 6 belted
kingfishers, 5 white pelicans, 2 great egrets,
9 black-crowned night herons, and 5 little blue
herons. It appears that Bolbophorus spp. have
been recovered only from white pelicans
collected in 2003 and 2005. The trematode
Clinostomum spp., one species of which is
responsible for the yellow grub in fish, was
found in great egrets, great blue herons,
snowy egrets, black-crowned night herons,
little blue herons, and cattle egrets. The gill
trematode Centrocestus formosanus was re-
covered from green herons and great egrets.
Identification of the trematodes is ongoing.
Confirmation of the Definitive Final
Host of Bolbophorus
North Carolina State University. Adult
Bolbophorus damnificus and immature Bol-
bophorus sp. type 2 have been recovered and
identified from the American white pelican.
Mature ovogenous Bolbophorus sp. type 2
have not been recovered from any avian
species and identification of its definitive
host remains a priority.
Mississippi State University. Birds (two
each of American white pelicans, double-
crested cormorants, great blue herons, great
egrets) were live-captured in the Mississippi
Delta. They were individually housed in
pens with recirculating water tanks and fed
catfish ad libitum daily until challenge.
Birds were acclimated for at least 1 week.
Fecal samples were collected daily starting
at 48 hours prior to anti-helmintic treatment
and continued until necropsy. At 7 days
pre-challenge, birds were administered
praziquantel at 34 mg/kg BW per os to
eliminate all trematodes. At 7 days
post-treatment birds were fed live fish
naturally infected with Bolbophorus
damnificus metacercariae (confirmed by a
B. damnificus-specific polymerase chain
reaction, PCR). Birds were necropsied 21
days post-challenge, intestinal contents of
18 SRAC Eighteenth Annual Progress Report, December 2005
Identification, Characterization, and Evaluation of Mechanisms
of Control of Bolbophorus-like Trematodes and Flavobacterium
Columnare-like Bacteria Causing Disease in Warm Water Fish
each bird were examined; all parasites were
removed, examined microscopically, identi-
fied and enumerated. A sub-sample of each
parasite type was processed for electron
microscopy and DNA analysis.
The only bird species that shed B. damnificus
ova (confirmed by PCR) during the trial was the
American white pelican. Adult B. damnificus
were found in pelican 1 (one adult trematode)
and pelican 2 (five adult trematodes). All other
bird species were negative for B. damnificus and
other trematodes.
This study confirms that the American white
pelican is a host for B. damnificus. Results
from this study demonstrate that artificial
infections of B. damnificus could not be
established in double-crested cormorants,
great blue herons, and great egrets.
Confirmation of Intermediate Hosts
of Bolbophorus spp.
North Carolina State University,
USDA/ARS, Mississippi State University.
Planorbella trivolvis snails collected from
catfish ponds in Mississippi experiencing
outbreaks of Bolbophorus-associated
morbidity/mortality were screened for the
shedding of forked-tailed cercariae in snails
shipped to North Carolina. Two morpho-
logically distinct types ofbolbophorid cercariae
were confirmed morphologically and
genetically utilizing species-specific PCR.
These were 1) Bolbophorus damnificus, a
serious pathogen of channel catfish, Ictalurus
punctatus, and 2) Bolbophorus sp. type 2, a
species not recovered from catfish but present in
several other fish hosts. Interestingly, several
snails were shown to be shedding both
bolbophorid species simultaneously or
sequentially. This indicated that both species
were present in aquaculture ponds and they
utilized the same molluscan host. A manuscript
"Morphological description of the cercariae of
Bolbophorus damnificus and Bolbophorus sp.
with notes on North American Bolbophorids"
by J. R. Flowers, M. F. Poore, L. M. Pote,
R. W. Litaker and M. G. Levy was submitted
to Comparative Parasitology in June 2004.
Information in this manuscript will allow
identification and speciation of bolbophorid
cercariae based on light microscopic details.
RESULTS AT A GLANCE...
Studies on the various life stages of
S Bolbophorus damnificus have
revealed that the adult trematode
resides in the gut of the American
white pelican. The parasite has not
been found in wild cormorants, great
egrets, great blue herons, snowy
egrets, cattle egrets, green herons,
belted kingfishers and little blue
herons. Attempts to artificially infect
cormorants, great blue herons and
great egrets failed whereas the white
North CarolinaPlanorbella duryi snails were
sent to Dr. L. Pote at Mississippi State
University who was successful in infecting
them with B. damnificus, indicating that the
North Carolina snails are a permissive
intermediate host. This indicates that in the
presence of a suitable avian host, this
infection is capable of further spread to the
southeastern United States. Dr. Pote also
provided several shipments of P. trivolvis
positive for B. damnificus and Bolbophorus-
SRAC Eighteenth Annual Progress Report, December 2005
Identification, Characterization, and Evaluation of Mechanisms
of Control of Bolbophorus-like Trematodes and Flavobacterium
Columnare-like Bacteria Causing Disease in Warm Water Fish
type 2 to Dr. Michael Levy at North Carolina
State University, and has maintained the
P. trivolvis snail colony which provided
negative snails for other cooperators in this
project.
The Bolbophorus trematode has been found in
wild fish species including channel catfish and
several centrarchids in Lake Chicot, Arkansas.
Metacercarie recovered from a variety of fish
demonstrated the following distribution: Only
B. damnificus was recovered from catfish in
aquaculture ponds. Bolbophorus species
type 2 was recovered from white crappie and
longer sunfish and largemouth bass. The
fathead minnow was found to harbor both
B. damnificus and species type 2. This is the
first finding of a B. damnificus in a fish
species other than catfish.
Both patent and pre-patent infections in
infected snails were identified using PCR.
Using PCR we also identified snails
shedding either B. damnificus or type 2
exclusively. Cercariae were then fixed in hot
10% neutral buffered formalin. Ten cercariae
of each type were examined for body length,
body width, tail-stem length and width,
furcae length and width, and oral sucker size.
An additional large number of living
cercariae were held under a cover slip and
examined for the following characteristics:
penetration glands, flame cells, organ
primordial and tegumental spine arrange-
ments. Differences between the two species
strongly suggest that cercariae have
distinguishing morphologic characteristics.
Confirmation of these observations will be
accomplished by examining additional
cercariae during the coming season in order
to rule out individual snail variation.
Fish Challenge Trials with
Bolbophorus spp.
North Carolina State University. The
potential pathogenic effect of both trematode
species was investigated in a series of
preliminary experiments. Hybrid striped bass
(Morone saxitalis x M. chrysops), and
channel catfish fingerlings were obtained
from commercial farms in North Carolina
where Bolbophorus is not known to be
present. Snails were divided into two groups
based on PCR identification of the
Bolbophorus species that they shed. Infection
rates were based on available numbers of
cercariae less than 2 hours after emergence
from the snails. Catfish were 2- to 3-inch
fingerlings and hybrid striped bass were
1.5-inch fingerlings. An aliquot of cercariae
was retained from each infection time and the
challenge species reconfirmed using PCR.
These results were not available until after
challenge was completed due to the time
involved in running the PCR assay.
Five groups of five bass were infected with
300, 500, or 550 B. damnificus, and two
groups of bass were infected with either 40
or 285 Bolbophorus sp. type 2. Eight groups
of five catfish were infected with 175, 350,
637, or 700 B. damnificus cercariae/fish.
Three groups were infected with 300, 700, or
1,000 Bolbophorus species type 2. One group
of fish was infected with 700 cercariae of a
mixture of the two species due to a "switch"
in the species shed by one or more snails in
this group. All fish were necropsied and
metacercariae removed and identified as to
type using PCR.
All catfish infected with any dose of
20 SRAC Eighteenth Annual Progress Report, December 2005
Identification, Characterization, and Evaluation of Mechanisms
of Control of Bolbophorus-like Trematodes and Flavobacterium
Columnare-like Bacteria Causing Disease in Warm Water Fish
B. damnificus developed the typical hem-
orrhagic lesions and most died beginning on
day 4 post-infection. Several fish exposed to
the lowest numbers of cercariae survived and
were euthanized 6 weeks post-infection.
Although catfish exposed to only Bol-
bophorus sp. type 2 failed to exhibit obvious
signs of infection such as hemorrhagic lesions
typical of a B. damnificus challenge, expo-
sure to B. sp type 2 cercariae did result in
these fish going 'off feed' for several weeks. A
few degenerate metacercariae, none con-
taining intact immature adult worms, were
recovered. These were identified as type 2 by
PCR.
Hybrid striped bass challenged with type 2
cercariae exhibited hemorrhagic lesions
similar to those observed with B. damnificus-
challenged catfish and mortality rates were
similarly high. No morbidity or mortality was
observed with hybrid striped bass challenged
with B. damnificus. OnlyBolbophorus species
type 2 metacercariae were recovered from
hybrid bass.
In Year 2 experimental infection of fish was
continued with the two bolbophorid species.
The potential pathogenic effect of both
trematode species was investigated in a series
of additional experiments complementing
those performed in Year 1.
Laboratory-reared hybrid striped bass
fingerlings and raceway-reared channel
catfish fingerlings were purchased from
commercial fisheries. Snails that had
identified bolbophorid infections were placed
in separate groups by species of cercariae as
determined by species-specific PCR and
cercariae were allowed to escape from their
snail host into the water column. Samples of
cercariae from each snail group were counted
and cercarial yields were calculated. Within
4 hours of escape from the snails, cercariae
were added to fish tanks containing experi-
mental fish.
Groups of ten hybrid striped bass were
separately exposed to Bolbophorus damnifi-
cus cercariae and Bolbophorus sp. cercariae
at the following exposure rates: 500, 250, or
100 cercariae/fish. Groups of ten channel
catfish were also separately exposed to
Bolbophorus damnificus cercariae and
Bolbophorus sp. cercariae at the following
exposure rates: 250, 100, or25 cercariae/fish.
At 5-days post-exposure, hemorrhagic lesions,
lethargy, and decreased appetite were noted in
the hybrid striped bass exposed to the
Bolbophorus sp. cercariae at the rates of 500,
250, and 100 cercariae/fish. Mortality of the
hybrid striped bass exposed to 500 cercariae/
fish began at 6 days post-infection and all fish
were dead by 11 days post-exposure.
Hemorrhagic lesions in hybrid striped bass
exposed to 250 and 100 cercariae per fish
disappeared by 19 days post-exposure;
however, large bumps under the skin were
noted. None of the hybrid striped bass
exposed to Bolbophorus damnificus
developed lesions.
Conversely, channel catfish reacted to the
Bolbophorus damnificus cercarial exposures.
Hemorrhagic lesions and bumps were
observed on catfish exposed to B. damnificus
cercariae at the rates of 250 and 100
cercariae/fish in the morning of day 9 post-
exposure. In the afternoon of day 9 post-
exposure, three catfish exposed to 250
SRAC Eighteenth Annual Progress Report, December 2005
Identification, Characterization, and Evaluation of Mechanisms
of Control of Bolbophorus-like Trematodes and Flavobacterium
Columnare-like Bacteria Causing Disease in Warm Water Fish
cercariae/fish and one catfish exposed to 100
cercariae/fish had died. Later, one catfish
exposed to 250 cercariae/fish and one exposed
to 100 cercariae/fish died at 14 and 23 days
post-exposure, respectively. Some of the
catfish exposed to 250 cercariae/fish
developed exophthalmia and abdominal
distension. None of the catfish exposed to the
Bolbophorus sp. cercariae developed lesions.
Also, lesions were not seen in the catfish
exposed to Bolbophorus damnificus cercariae
at the rate of 25 cercariae/fish.
The pathological consequences of
B. damnificus infection in channel catfish
were less severe compared with those seen in
past experiments. This may be due to the
larger size of fingerlings or the presence of
fewer non-trematode pathogens. In previous
challenges, fingerlings were collected from
ponds and may have harbored other path-
ogens, whereas for this experiment we
collected swim-up fry hatched in well water
and laboratory-reared the fish.
These results demonstrate a high degree of
specificity for the intermediate hosts for these
two bolbophorid trematodes. Bolbophorus
damnificus caused lesions only in catfish
where bolbophorus sp. caused lesions only in
hybrid bass.
Fish growth rates at the different parasite-
exposure rates are currently being statis-
tically analyzed. The number of encysed
metacercariae for each fish (for each
challenge parasite species and number) will
also be determined. Development of methods
for quantification and estimation of parasite
loads in molluscan populations are also in
progress.
Objective 2. Evaluate integrated methods for snail control in catfish ponds.
Objective 2a. Monitor populations ofcatfsh infected with Bolbophorus sp.
to document the effect ofparasite loads on growth and survival of the fish.
Mississippi State University. Laboratory and
field studies were conducted to evaluate the
effects of sub-lethal trematode infections on
growth, performance and disease resistance of
channel catfish fingerlings. Trematode in-
fections were established in populations of
fish stocked in four, 0.1-acre ponds. A
reservoir of trematode-infected snails was
maintained in recirculating 300 gallon tanks
located on the bank of each pond. Pond water
was recirculated through each tank at a rate of
2 gallons per minute. The effluent (containing
Bolbophorus cercariae) from the tank was
directed back into the pond and served as the
source of infection. Four additional ponds
were used as control ponds. After 40 days,
each population of fish was sampled to
evaluate health status and 120 fish from each
pond were transferred to 30-gallon aquaria to
evaluate growth rates under controlled labora-
tory conditions using well water free of
Bolbophorus cercariae. Only fish containing
visible cysts (1 to 5 cysts per fish) were
selected and used to evaluate growth
potential. Fish were acclimated to laboratory
conditions at 31 to 320C for 3 weeks before
22 SRAC Eighteenth Annual Progress Report, December 2005
Identification, Characterization, and Evaluation of Mechanisms
of Control of Bolbophorus-like Trematodes and Flavobacterium
Columnare-like Bacteria Causing Disease in Warm Water Fish
the start of the study. Following the con-
ditioning period, fish were fed once daily for
9 weeks. Total weight gain, percent weight
gain, specific growth rate, and feed efficiency
were used to evaluate growth.
Evaluation of health and growth of
channel catfish continually exposed to the
cercarial stage of Bolbophorus damnificus
throughout a production cycle
Mild trematode infections were established in
pond populations of experimental fish by
exposing fish to trematode cercaria. The percent
of infected fish in each pond ranged between
20.4% and 1.6%. Mortalities directly related to
trematode infections were not observed.
Edwardsiella ictaluri and Flavobacterium
columnare infections were diagnosed from all
populations of fish and no differences in
mortality were observed between trematode
infected and non-infected fish. At the end of
the production cycle, trematode infected fish
consumed approximately 40% less feed
compared to fish in control ponds.
Evaluation of health and growth of fish
that have been infected with Bolbophorus
damnificus cercariae by a single-pulse
exposure
At the start of the acclimation period,
trematode infected fish were significantly
smaller compared to fish collected from
control ponds. No differences in any of the
measured parameters were observed between
trematode-infected and non-infected fish at
the end of 9 weeks. Although the final weight
of trematode-infected fish was numerically
smaller than control fish, percent weight gain
and specific growth rate demonstrated a
tendency towards compensatory growth of
trematode-infected fish. Feed efficiency
(0.86) was identical between treatment
groups. Data indicates that once fish are
removed from the source of infection, chronic
trematode infections do not affect the growth
potential of channel catfish.
Evaluation of health status and growth
potential of channel catfish fingerlings
infected with Bolbophorus damnificus
under controlled laboratory conditions
Trematode infections were established under
laboratory conditions by placing fingerlings in
triplicate tanks containing Planorbella
trivolvis snails shedding cercariae. Fish were
left in the tank for 24 hours and snails were
shedding cercariae at a rate of 770 82 per
24 hours. Unexposed fish were maintained in
three tanks under similar conditions. From
each tank, trematode-infected or non-infected
fish were transferred to six aquaria (30
fish/aquaria). Three aquaria from each
replicate treatment tank received 7.5 x 105
CFU E. ictaluri/mL of water for 30 minutes
(Bolbophorus-ESC and ESC-only groups).
Fish in the remaining three aquaria were not
exposed to E. ictaluri and served as
Bolbophorus-only and negative control
groups. The later two treatment groups were
used in a second study and were challenged
with E. ictaluri 28 days after exposure to
Bolbophorus sp. cercariae.
No mortalities were observed in the
Bolbophorus-only and negative control groups.
Twenty-one days following exposure to
E. ictaluri, the percent cumulative mortalities
were 84.1 16.2% in the Bolbophorus-ESC
treatment and 45.9 3.2% in the ESC-only
SRAC Eighteenth Annual Progress Report, December 2005
Identification, Characterization, and Evaluation of Mechanisms
of Control of Bolbophorus-like Trematodes and Flavobacterium
Columnare-like Bacteria Causing Disease in Warm Water Fish
treatment. Mortalities were significantly
different between the two groups. In the
second study, when E. ictaluri exposure was
delayed 28 days following Bolbophorus sp.
infection, there was no difference in
mortalities between the ESC-only (17.8 +
4.0%) and combined Bolbophorus-ESC (21.5
1.7%) exposed groups. Apparently, once
fish are removed from the source of additional
infections, chronic trematode infections do
not increase the susceptibility of fish to ESC.
RESULTS AT A GLANCE...
Mild, sub-lethal trematode infections can
significantly reduce catfish growth by
reducing feed consumption and
increasing mortality associated with
concurrent bacterial infections.
Findings in these studies have significant
implications for management strategies to
control losses associated with trematode
infections. Data collected from laboratory and
field trials indicated that mild sub-lethal
active trematode infections, commonly
observed in channel catfish production
systems, can significantly reduce production
by reducing feed consumption and increasing
losses associated with ESC. These studies also
indicated that the presence of fully developed
metacercariae does not compromise growth
and health status of fish. These data support
the contention that the deleterious effects of
trematode infection are associated with
penetration of the parasite and initial stages of
encystment. Findings point to the need for
increased surveillance for this disease and the
benefit of initiating management protocols at
the earliest stages of infection. In addition,
breaking the trematode's life cycle by moving
fish to non-infested water or by eradicating
snails in the pond will eliminate the adverse
effects associated with this disease.
RESULTS ATA GLANCE...
The presence of fully developed
Bolbophorus metacercariae does not
affect growth or health of catfish. The
deleterious effects of this infectious
agent are therefore associated with
penetration of the parasite and initial
stages of encystment.
Evaluation of the farm-wide economic
impact of Bolbophorus damnificus
infections of channel catfish
The economic impact of trematode infections
was evaluated by conducting a disease-
monitoring and production-efficiency study
on a commercial catfish operation with ponds
containing trematode-infected fish. Fish were
sampled from each food fish production pond
and examined for the presence of cysts that
contain the metacercariae. Each pond was
placed into one of four categories based on the
percentage of infected fish in the sample.
Ponds with trematode-infected catfish were
placed into categories of light, moderate, or
severe when the percentage of trematode-
infected fish in the sample ranged from 0% to
33%, 33% to 66% or 66% to 100%, respec-
tively. Ponds that did not contain trematode-
infected fish were categorized as negative.
Production records were grouped by in-
festation level for analysis. Of the 40 pond
populations sampled, 17 were categorized as
24 SRAC Eighteenth Annual Progress Report, December 2005
Identification, Characterization, and Evaluation of Mechanisms
of Control of Bolbophorus-like Trematodes and Flavobacterium
Columnare-like Bacteria Causing Disease in Warm Water Fish
negative, 6 as light, 6 as moderate, and 11 as
severe. Fish from trematode-positive ponds
consumed significantly less feed compared to
fish from ponds that were categorized as
trematode-negative. Fish from ponds in the
trematode-negative category consumed on
average 73.4 pounds/acre per day, and fish
from ponds categorized as light, moderate and
severe consumed 62.2, 47.5, and 47.2
pounds/acre per day, respectively. Similarly,
production decreased as severity of infection
increased. Compared to trematode-negative
ponds, ponds in the light, moderate and severe
categories produced 16.8%, 36.4%, and
Obective 2b. Examine the efficacy of chemical
populations.
USDA/ARS. A total of seven trials were
conducted from 2003 through 2005 to test the
effectiveness of pond-shoreline treatments in
controlling aquatic snails. Initially, four trials
were conducted to compare a slurried-
hydrated lime treatment with an established
copper sulfate treatment. Copper sulfate and
hydrated lime were applied at 4 pound and 80
pounds, respectively, per 100 feet of shore-
line in a 6-foot swath. Trials were run under
conditions of variable wind speed (0 to 16
mph) and treatment temperature (24 to 320C).
Both treatments effectively lowered the snail
populations in the test cages. It appears that
copper sulfate was more effective than lime
in most trials, that hydrated lime treatments
appeared to increase in effectiveness at
higher temperatures (320C vs 25C), and that
strong winds negatively impacted both
treatments. Snail survival under all con-
ditions in the four trials ranged from an
average of 3.4% to 27.8% and 10.4% to
44.5% fewer pounds of fish per acre,
respectively. Net returns from ponds in the
light category were reduced by 80.8% and
production from ponds in the moderate and
severe categories were not shown to cover
variable costs of production. Ponds in the
moderate category produced a net loss of
$506 per acre and severe ponds produced a
net loss of $631 per acre. These data from a
commercial setting support results from
experimental ponds and demonstrate that
Bolbophorus infestations, regardless of sever-
ity, are a significant risk to commercial
production of channel catfish.
control methods on snail
41.5% for copper sulfate and hydrated lime,
respectively.
The goal of the second part of the study
changed from comparing lime and copper
sulfate to optimizing the hydrated lime
treatment. Trials 5, 6, and 7 were conducted
using only hydrated lime treatments at
temperatures of 24 to 260C and under low
wind conditions. In Trial 5, the rate of
hydrated lime was increased to 175 pounds
per 100 feet of shoreline in a 6-foot swath. At
that rate, effectiveness was increased and snail
survival was less than 2%. Further optimi-
zation of the hydrated lime treatment was
made by narrowing the treatment swath to
3 feet and reducing the amount of lime to 80
and 100 pounds per 100 feet of shoreline.
Average snail survivals for the 80 and 100
pound treatments were 13.3% and 2.7%,
respectively. The optimum pond shoreline
treatment with slurried hydrated lime is
SRAC Eighteenth Annual Progress Report, December 2005 25
Identification, Characterization, and Evaluation of Mechanisms
of Control of Bolbophorus-like Trematodes and Flavobacterium
Columnare-like Bacteria Causing Disease in Warm Water Fish
100 pounds of lime per 100 feet of shoreline
applied in a 3-foot swath.
RESULTS ATA GLANCE...
r A shoreline treatment with slurried
hydrated lime applied in a 3-foot swath
at 100 pounds of lime per 100 feet of
shoreline reduced snail populations by
over 95%.
Mississippi State University. The toxicity of
copper sulfate to ram's horn snails was
evaluated by establishing the 24-hour LC50
using Spearman-Karber analyses. Tests were
conducted in 300-mL glass containers
containing 200 mL of pond water (alkalinity =
235 mg/L as CaCO3, hardness = 300 mg/L as
CaCO3, pH = 7.5, temperature = 230C). Test
concentrations of copper were arranged in a
geometrically spaced dilution series. Each test
concentration consisted of 4 replications with 5
snails per replication. Copper sulfate granules
were dissolved in distilled water and delivered
as a solution. After 24 hours, snails were
removed from the test solution and placed in
fresh, untreated water. End-points for the tests
were death of the snails as determined by an
additional 96-hours post-test observation period
to confirm mortality. The effect of temperature
(15, 20,25, and 300C) and alkalinity (0, 50, 100,
and 200 mg/L CaCO3) on the toxicity of copper
to snails were also evaluated.
Laboratory tests showed copper sulfate
crystals had a 24-hour LC50 of 0.6 mg/L Cu
and, based on the alkalinity of the test water,
was below the level considered toxic to fish.
Alkalinity at the levels tested (0 to 200 mg/L
CaCO3) was not shown to effect to the
toxicity of copper to snails. The LC50
concentration at an alkalinity of 0 mg/L
CaCO3 was 0.52 mg/L Cu versus 0.67 mg/L
Cu at an alkalinity of 200 mg/L. Although
there appeared to be a trend in the LC50
values toward decreasing toxicity with in-
creasing alkalinity, these differences were not
statistically significant. Analysis of data from
this study showed a significant linear rela-
tionship between temperature and LC50
values for copper. As temperature increased
from 150C to 300C, the LC50 values
decreased from 1.1 mg/L to 0.18 mg/L Cu,
representing a ten-fold increase in toxicity.
This would be an important consideration
when treating ponds with copper sulfate with
respect to both snail and fish toxicity.
Three dose-titration trials were performed to
determine the copper concentration required to
kill snails under field conditions. Two trials
were conducted in plastic tanks containing 200
gallons of pond water. Fish and snails were
placed in three replicate tanks and dosed with a
solution of copper sulfate at 0 (control) 1.25,
2.5, 5.0, and 10.0 mg/L Cu during the first trial
and 0 (control), 0.375,0.75, 1.25, and 2.50 mg/L
Cu during the second trial. Each tank consisted
of 20 snails confined in wire mesh cages and 10
channel catfish fingerlings to evaluate the
toxicity of the treatment dose to fish.
The third trial was a non-replicated study
conducted in 0.25-acre ponds containing
approximately 1 acre-foot of water. In each
pond, three sample sites were evenly dis-
tributed along the center long axis of the
pond. At each sample site, 20 snails were
confined in cages located at the surface and
bottom of the pond. The toxicity of copper to
26 SRAC Eighteenth Annual Progress Report, December 2005
Identification, Characterization, and Evaluation of Mechanisms
of Control of Bolbophorus-like Trematodes and Flavobacterium
Columnare-like Bacteria Causing Disease in Warm Water Fish
fish was evaluated in the 0.25-acre ponds by
confining 10 channel catfish fingerlings in
floating net-pens in close proximity to each snail
sampling site. Each pond received a dose of 0
(control), 1.25, 2.5, 5.0, and 10.0 mg/L Cu by
evenly applying the copper sulfate solution (200
gallons/pond) around the margins of the pond.
Twenty-four hours after treatment, test snails
were transferred to 1-L containers (containing
the appropriate test water) and transported to the
laboratory and observed for 72 hours. Twice
daily, dead snails were removed and placed in a
separate container containing untreated pond
water. Dissolved oxygen concentrations in the
test water and fish mortality were observed for
96 hours.
Titration trials in tanks and ponds were
comparable, with laboratory toxicity trials and
indicated the minimum effect dose (snail mor-
tality >90%) of copper sulfate ranged between
0.75 and 1.25 mg/L Cu. Fish mortality was not
observed at or below 1.25 mg/L Cu in the pond
tank studies. Fish mortality (average mortality
5.3%), however, was observed at one of the
three sample locations in one of the replicate
ponds following treatment with 1.25 mg/L Cu.
Based on the results of the dose-titration trials,
triplicate 0.25-acre and duplicate 10.0-acre
experimental ponds were treated with 0.75 and
1.25 mg/L Cu to verify the minimum effective
dose. Sample site configurations and treatment
applications for tests conducted in the 0.25-acre
ponds are described above. Sample site
configurations for tests conducted in 10-acre
ponds varied to accommodate pond size. Each
10-acre experimental pond contained 12 uni-
formly distributed sample sites consisting of
20 snails confined at the surface and bottom of
the pond. An additional 20 snails were confined
along the pond bank at equal intervals. Each
10.0-acre pond was managed as a commercial
production pond and contained approximately
3,500 (average estimated size = 1 pound) fish
per acre. No mortality or signs of disease were
noted before any of the tests were conducted.
Toxicity of the copper treatment to fish was
evaluated by observing the pond stock for
behavioral indicators of toxicosis and mortality.
For each test conducted, a non-treated pond
contained similar sample site configurations and
served as a control.
Replicate single-dose pond treatments verified
that treatment doses of 0.75 and 1.25 mg/L Cu
were effective in killing snails. Average snail
mortality in trials conducted in the 0.25-acre
ponds ranged between 98.0% and 95.5% at the
low treatment doses and was 100% at the high
treatment dose. Fish mortality was observed in
1 of the 3 replicate ponds at each treatment dose.
Similar results with respect to snail toxicity were
observed in the single dose toxicity trials
conducted in 10-acre ponds. Average snail
mortality of the replicate trials at each sample
location ranged between 92 and 98% following
treatment, with 0.75 mg/L and between 98 and
100% following treatment with 1.25 g/L Cu. In
contrast to the 0.25-acre pond trials, no fish
mortality or behavioral signs of toxicosis were
observed following treatment. In all pond trials,
dissolved oxygen depletions were not observed
for up to 168 hours after treatment. Fish
mortality in the 0.25 acre ponds may be caused
by exposure of confined fish to high concen-
trations of the applied chemical before it was
completely mixed with the pond water.
Treatment efficacy was then evaluated in a
13.0-acre commercial channel catfish produc-
tion pond. Moderate numbers of snails were
SRAC Eighteenth Annual Progress Report, December 2005 27
Identification, Characterization, and Evaluation of Mechanisms
of Control of Bolbophorus-like Trematodes and Flavobacterium
Columnare-like Bacteria Causing Disease in Warm Water Fish
observed along the margins of the pond.
Seven sample sites (surface and bottom cages
containing 20 snails each) were placed a
minimum of 50 yards from the pond bank and
were distributed randomly across the pond. An
equal number of cages containing 20 snails
each were placed along the margins of the
pond. In addition, natural snail populations
along the margins of the test pond were
sampled at five locations before and after
treatment. A 20-foot section of pond bank
consisting of uniform vegetation and levee
slope was marked and divided into 2 equal
sections. Snails were collected from a 10-foot
section of the sample site before the pond was
treated, and the remaining section was
sampled 24 hours after the pond was treated.
Snails collected from each section were placed
in 4-L containers and transported to the
laboratory for observation. Live snails were
counted to estimate viable snail numbers and
used to determine the number of snails per
foot of pond bank in the sampled area.
Production fish were monitored for behavioral
signs of toxicosis and mortality. Snails
confined in a similar configuration in an
adjoining pond served as a control.
Results of the commercial field trial were
comparable to tests conducted in the experi-
mental ponds where application of copper
sulfate at 1.25 mg/L was shown to be
effective in killing snails. Average mortality
of snails confined in cages ranged between
95.4 and 97.7%. The treatment was also
shown to be effective against natural popula-
tions of snails along the margins of the pond.
The average number of snails per foot of pond
bank decreased from 21.5 snails to 0.18 snails
24 hours after treatment, representing a 99%
reduction in viable snail populations in the
habitat along the pond bank.
Treatment of the commercial pond resulted in
changes in fish behavior and mortality that
was likely related to the copper treatment.
Within 4 hours of treatment application, an
increase in the number of moribund fish were
observed. Affected fish appeared lethargic or
exhibited a spiraling swimming pattern.
However, it is not thought that these ob-
servations were solely related to the chemical
treatment. Prior to treatment, moribund and
dead fish were present in the pond that was
diagnosed with bacterial (Edwardsiella
ictaluri and Flavobacterium columnare) and
parasitic infections (Bolbophorus sp.). Mori-
bund fish also exhibited clinical symptoms
consistent with visceral toxicosis of catfish.
Mortality rates in the pond were characterized
as chronic and were estimated to be 150 to
200 fish per day. Farm management estimated
total mortality in excess of 20,000 pounds.
Following treatment, fish mortality increased
within the first 24 hours. It was estimated that
approximately 2,000 pounds of fish were lost
following treatment, however, a majority of
the fish had clinical signs ofESC. In addition
to infectious disease, analysis of water quality
2 hours after treatment revealed low chloride to
nitrite ratios and examination of fish gills and
blood indicated acute nitrite toxicosis. On the
RESULTS ATA GLANCE...
SA low-dose, full-pond treatment with
copper sulfate was developed that
safely eradicates the trematode's
intermediate host-the ram's horn snail.
28 SRAC Eighteenth Annual Progress Report, December 2005
Identification, Characterization, and Evaluation of Mechanisms
of Control of Bolbophorus-like Trematodes and Flavobacterium
Columnare-like Bacteria Causing Disease in Warm Water Fish
day of treatment, salt was added to the pond
water and, after the first 24 hours, the daily
Objective 2c. Examine the efficacy of biological
(snail-eating fish) on snail populations in ponds.
Effect of diet conditioning on prey selection
by blue catfish and redear sunfish
Mississippi State University, Southern
Illinois University. Four aquaria were stocked
with 300 juvenile blue catfish and four addi-
tional aquaria were stocked with 300 juvenile
redear sunfish. The fish were given an initial
"conditioning diet" which consisted of only one
of the following: fish food insect larvae, ram's
horn snails or red-rimmed melania snails. After
2 weeks of feeding the "conditioning diets," 100
fish of each species were stocked into eight
separate aquaria (16 aquaria total) and offered a
known amount of their conditioning diet and a
known amount of one of the other conditioning
diets used above. Prey selection was determined
by the frequency of selection of the various
diets. A Chi-square contingency test was used
to determine the influence of conditioning on
food selection. A two-tailed test of binomial
proportion was used to examine the signifi-
cance of food preference in each conditioning
experiment. Because fish that consume large
amounts of food may bias results, the percentage
of the total number of prey items the percentage
of food for each fish was determined either by
video or by examination of the stomach con-
tents. For video analysis, a video camera was
used to record a 2-hour segment of feeding. The
data obtained was analyzed using the Observer
(1997), a computer program specifically
programmed for behavior analysis. Data
obtained from this program was analyzed
mortality rate returned to pretreatment levels.
control methods
using a Chi-square contingency test.
Prey selection studies with the blue catfish
revealed that regardless of the training or
conditioning diet, blue catfish readily
RESULTS AT A GLANCE...
Ponds stocked with redear sunfish had
S significantly fewer snails than ponds
without sunfish. Redear sunfish
S preferentially consumed snails and
midge larvae when available, even
when trained on pelleted diets prior to
stocking ponds.
converted to catfish feed when it was offered.
Redear sunfish preferentially consumed snails
when available. Even redear sunfish trained to
eat commercial fish food readily consumed
snails and chironomids when available.
Determination of the ability of redear
sunfish to withstand conditions of
commercial catfish culture
Southern Illinois University. In the spring,
redear sunfish (small, medium, and large)
were stocked at a rate of 300 fish/acre into
four, 0.0-acre experimental ponds (12 ponds
total) stocked with channel catfish at a
production rate of 8,000 pounds/acre. Water
quality variables such as dissolved oxygen,
SRAC Eighteenth Annual Progress Report, December 2005
"" '
Identification, Characterization, and Evaluation of Mechanisms
of Control of Bolbophorus-like Trematodes and Flavobacterium
Columnare-like Bacteria Causing Disease in Warm Water Fish
temperature, alkalinity, carbon dioxide,
ammonia, and pH were measured on a daily
basis. The number of dead fish (catfish and
sunfish) was recorded. In September, the
ponds were seined and harvest size channel
catfish removed. The number of surviving
individuals of each species was recorded. The
ponds were under stocked with 5-inch
fingerlings to replace the fish removed for
market. Survivability of redear sunfish was
analyzed to determine if any correlation with
water quality variables exist. The number of
redear sunfish that died due to seining was
recorded. In the spring of 2004, the ponds
were seined and all redear were counted and
weighed. Harvest size catfish were removed
and 5-inch fingerlings were stocked to replace
those removed.
In the fall of 2004, redear sunfish and channel
catfish were removed from ponds. The
number and type of snails within a one meter
transect were counted and recorded. One
hundred channel catfish from each pond were
sampled and analyzed for the presence of
trematodes on gills and in the flesh. This
study was repeated in 2005.
In the 2004 study, ponds containing redear
sunfish had significantly fewer snails than
ponds without sunfish. The number and type
of snails remaining in the ponds did not differ
significantly when medium size or large
sunfish were stocked. Redear sunfish trained
to eat snails did not remove significantly
higher numbers of snails than fish not trained
or conditioned to snail diets. Survival of the
redear sunfish was 100 percent in all ponds.
The incidence of trematode infestation was
still evident on channel catfish. Approxi-
mately, 25% of the catfish had trematodes on
the gills and in some cases within the flesh.
In the 2005 study, the water temperatures in the
ponds at Southern Illinois University averaged
5C higher than in 2004. Catfish survival in all
ponds was greater than 98%. Survival of redear
sunfish was strongly correlated with the
decreased dissolved oxygen and increased
temperature in the ponds. Ponds that routinely
had dissolved oxygen concentrations of 3 mg/L
and temperatures of 320C at sunrise had
significantly higher mortality rates than ponds
with higher morning dissolved oxygen con-
centrations. No significant correlations between
alkalinity, carbon dioxide, ammonia, nitrite, or
pH on survival ofredear sunfish were observed.
The incidence of trematode infestation was still
evident on channel catfish. Approximately,
31% of the catfish had trematodes on the gills
and in some cases within the flesh. The increase
observed in the number of channel catfish with
trematodes visible on the gills or in the flesh is
likely due to the decrease in redear sunfish
numbers in the ponds due to mortalities.
Objective 3. Develop and implement standardized methods for the
isolation, culture, and antimicrobial susceptibility testing ofstrains of
columnaris-like bacteria isolated from diseased fish.
Louisiana State University. Various agar
media were evaluated for optimum primary
isolation and maintenance of Flavobacterium
columnare. Media under investigation included
30 SRAC Eighteenth Annual Progress Report, December 2005
Identification, Characterization, and Evaluation of Mechanisms
of Control of Bolbophorus-like Trematodes and Flavobacterium
Columnare-like Bacteria Causing Disease in Warm Water Fish
both selective and non-selective cytophaga agar
(CA), Hsu-Shotts (HS), Shieh (S), tryptone
yeast extract (TYE), dilute Mueller Hinton
(DMH) and Flavobacterium columnare growth
medium (FCGM). Media were made selective
by the addition of 5 gg/mL neomycin and 200
units/mL polymixin B. For primary isolation the
media were prepared as agar plates and for
maintenance the media were prepared as 20-mL
slants in 50-mL tubes with 1 mL of saline added
to preserve moisture. For the evaluation of
primary isolation media, a standardized mixture
ofF. columnare, Edwardsiella tarda, E. ictaluri,
Aeromonas hydrophila, and Streptococcus
difficilis was prepared. This mixture was
designed to mimic the mixture of aquatic
bacteria that might be present in contaminated
external sites such as the gills and skin of
diseased fish. The mixture was inoculated onto
the various test media to evaluate their ability to
produce pure colonies of F. columnare while
inhibiting contaminating bacteria.
Selective cytophaga agar (SCA) performed
the best as a primary isolation medium for
isolation of columnaris from a mixed
inoculum of aquatic bacteria. The remainder
RESULTS ATA GLANCE...
Selective cytophaga agar SCA has
performed the best as a primary
isolation medium in preliminary tests in
isolation of Flavobacterium columnare
from contaminated sites such as the gills
and skin. For maintenance following
isolation, tryptone yeast extract TYE
medium as a moist slant, held cultures
viable for as long as 84 days. For large
batch broth culture, FCGM outperforms
other formulations tested.
of the media were ranked as follows: (2) SS
(3) SHS, (4) DMH and (5) FCGM. Both
DMH and FCGM produced no isolated
F. columnare colonies.
For maintenance following isolation, TYE
slants performed the best with some cultures
maintaining viability as long as 84 days. The
remaining media were ranked as follows:
(2) CA, 52 days (3) DMH, 47days (4) HS,
34 days (5) S, 32 days and (6) FCGM, 23 days.
Some of the above-mentioned media were
evaluated as broths for batch culture of
F. columnare. A 40-mL volume of media was
inoculated with 200 giL of a McFarland #5
standard inoculum and growth performance
measured by colony forming units (cfu) per mL
and absorbance at 600 nm following 24 hours of
incubation at 280C. Flavobacterium columnare
growth medium (FCGM) out-performed other
formulations tested producing mean absorbances
of 0.2377 and colony counts of 2.2 x 109/mL.
The clumping of cells, which is a problem in
other broth media, was avoided in FCGM. The
remaining broth media were ranked as follows:
(2) Shieh (3) cytophaga and (4) DMH. For a
summary of broth culture results see Table 1.
For disk-diffusion antimicrobial susceptibility
testing, dilute Mueller Hinton (DMH) plates
prepared with different levels of agar and
nutrient were evaluated for clarity and
consistency of zone size. To insure
uniformity, one lot of each of five
anti-microbial agents, one lot of Mueller
Hinton medium, and one lot of equine serum
were used in all disk diffusion test
evaluations. The anti-microbial disks (BBL)
chosen were Sulfamethoxazole: trimethoprim
(SXT 25 ug), Sulfadimethoxine: ormetoprim
SRAC Eighteenth Annual Progress Report, December 2005
Identification, Characterization, and Evaluation of Mechanisms
of Control of Bolbophorus-like Trematodes and Flavobacterium
Columnare-like Bacteria Causing Disease in Warm Water Fish
Table 1. Growth of F. columnare in
various broth media at 280C for 24 hours.
Data is presented as absorbance at 600nm
and by colony forming units (cfu) per mL.
Test Medium
Mean
Absorbance
(600nm)
Mean
Colony
counts
(CFU/mL)
DMH* 0.0858 1.3 x 107
Cytophaga* 0.1646 6.3 x 107
Shieh* 0.2078 3.1 x 108
FCGM* 0.2377 2.2 x 109
Indicates significance at P = 0.05
(PRI-MOR 25 Fg), Oxolinic acid (OA 2 ug),
Oxytetracycline (T 30 pg), and Florfenicol
(FFC 30 pg). The basal MH broth used was
(Difco) lot # 3126187, and the degranulated
agar used was (Difco) lot #3265229. The
concentrations of MH broth evaluated were 3 g,
3.5 g, 4 g, and 5 g/L. The agar concentrations
that were evaluated were 9 g, 12 g, 15 g, and
17 g/L. Varying concentrations of MH broth
were used to determine the optimum amount of
nutrients for F. columnare growth. The varying
agar levels were evaluated for their effects on
zone appearance by limiting the gliding motility
of the bacterium. Each medium was evaluated
for thickness of growth, distinct zones, and
uniformity of zone margins. Once the optimum
concentration of broth to agar was determined,
5% equine serum lot #ANE18713 (Hyclone,
Logan, Utah) was evaluated as a replacement
for the more expensive fetal calf serum as a
growth supplement forF. columnare cultures on
MH agar plates.
The optimum media formulation for sus-
ceptibility testing was determined to contain
4 grams M-H broth and 17 grams of agar per
liter with 5% equine serum. This medium
RESULTS AT A GLANCE...
An improved medium has been
developed for antimicrobial susceptibility
testing of Flavobacterium columnare
gave the highest bacterial growth and allowed
for better definition of zones of inhibition.
Equine serum improved the growth of
F. columnare cultures on dilute M-H agar, but
not significantly different from fetal calf
serum. Because of availability and cost of
equine serum compared to fetal calf serum it
was determined to be a suitable enrichment
factor for the improved medium.
Objective 4. Characterize archived strains of columnaris-like bacteria
based on conventional and molecular techniques.
Morphologic and biochemical analysis of
columnaris-like bacteria
Louisiana State University. Forty-nine strains
of columnaris-like bacteria archived in the
LADL and UAPB collections were analyzed
using conventional biochemical testing in test
tubes, the API20E system, the API NE system
32 SRAC Eighteenth Annual Progress Report, December 2005
Identification, Characterization, and Evaluation of Mechanisms
of Control of Bolbophorus-like Trematodes and Flavobacterium
Columnare-like Bacteria Causing Disease in Warm Water Fish
and the API ZYM system. These strains were
obtained from a larger pool of yellow pigmented
columnaris-like isolates that were subjected
to screening procedures that involved a
species-specific PCR (Bader et al. 2003), the
five point characteristics of Griffin (1992) and
the physiological characteristics of Bemardet
and Grimont (1989). To conform to the
Griffin screen, the bacterium must be shown
to satisfy the following requirements:
production of flat, spreading, yellow, and
rhizoid colonies on cytophaga agar, growth in
the presence of neomycin and polymixin B,
production of gelatin degrading enzymes,
binding of congo red dye to the colony, and
production of chondroitin sulfate A de-
grading enzymes. Isolates were also tested for
the presence of flexirubin-type pigments using
the potassium hydroxide (KOH) method
outlined in Bergey's Manual of Determinative
Bacteriology 9th Edition (1994). The goal of
this part of the project is to determine if
F. columnare can be identified using con-
ventional and commercially available bio-
chemical testing schemes by labs that may not
have molecular capabilities. Prior to the start
of our study, 48 of 49 archived strains of
columnaris-like bacteria conformed to the
Griffin screen and were confirmed by PCR as
F. columnare.
Results indicate that F. columnare should be
shown morphologically to be a long, thin,
gram negative rod (3 to 10 micrometers length
and 0.3 to 0.5 micrometers in width) with
gliding and flexing motility, and form rhizoid,
yellow-pigmented colonies on agar plates.
Morphology of the cells and colony
appearance is slightly variable among strains
and is influenced by growth conditions and
age of the culture. The isolate should be a
strict aerobe, should not produce acid from
carbohydrates, and should be cytochrome
oxidase and catalase negative. Negative
reactions were obtained in the GMD and TSI
agar tests due to the lack of acid production
from carbohydrates. The API 20E, API NE
and API ZYM systems (bioM6rieux-Vitek)
were examined for usefulness in the
identification ofF. columnare. The API 20E
and API ZYM systems were determined to be
inadequate due to the lack of positive
reactions on the strips. The API NE was very
useful producing an adequate number of
positive reactions. Isolates of F. columnare
uniformly gave reactions resulting in the API
NE code number 0441455 at 24 hours. The
positive reactions in the API NE strip were as
follows: esculin, D-glucose, L-arabinose,
potassium gluconate, capric acid, malic acid,
and citrate.
Adhesion to plastic, cultured cells, and
isolated gills
Auburn University. Six types of plastic
multiwell plates (BD Biosciences, Franklin
Lakes, NJ) were compared for use in a bacterial
adhesion assay. Two hours after washed
F. columnare cells were added to the wells,
there were significant differences among the
plates. The same results were obtained with two
isolates. Adhesiveness of F. columnare was
greater for bacteria grown in Hsu-Shotts broth
rather than in Shieh broth. The addition of
calcium and magnesium to water used in the
adhesion assay increased the adhesiveness of
one isolate ofF. columnare (PL-04-02) but had
no effect on another isolate (PB-02-110). Other
waters tested, which had high concentrations of
NaC1, tended to reduce the adhesiveness of the
isolates tested.
SRAC Eighteenth Annual Progress Report, December 2005 33
Identification, Characterization, and Evaluation of Mechanisms
of Control of Bolbophorus-like Trematodes and Flavobacterium
Columnare-like Bacteria Causing Disease in Warm Water Fish
Isolates of F. columnare tested with the
multiwell plate assay had a wide range of
adhesiveness to plastic. As additional isolates
are obtained for testing, these results will be
compared to other types of results obtained by
investigators at other institutions to determine if
there is a relationship between adhesiveness and
other characteristics. Attempts to quantify the
adhesion ofF. columnare to cultured cells was
hindered by the adhesion of the bacteria to the
glass or plastic substrate used for cell culture
and by problems with accurate counting of
bacteria stained by conventional methods. To
overcome these problems, antibodies against
F. columnare were made in rabbits as a first
step in development of antibody-based methods
for bacterial quantification.
Three types of assays for adhesiveness of
F. columnare (isolated fish gills, larval fish,
and cultured cells) were developed and then
compared with a multi-well plastic plate
assay. The assay with plastic plates was
previously found to be useful for quanti-
fication ofF. columnare adhesiveness.
For the gill assay, gills were dissected from
channel catfish, bluegill, and common carp. For
each fish, one section of gill was used as a
control and two sections were exposed to
F. columnare. After a 10-minute exposure to
bacteria, gills were rinsed twice and
homogenized. Plate counts of serial dilutions of
the gill homogenate were used to quantify the
F. columnare adhering to gills. There was a
significant difference among F. columnare
isolates adhering to for gills from bluegill
(8 isolates) and common carp (3 isolates). Only
two isolates were tested with normal channel
catfish gills; however, there was a significant
increase in the number of bacteria adherent to
gills of channel catfish with proliferative gill
disease or with Aeromonas infection.
A larval zebrafish assay for adhesiveness of
F. columnare was developed. Whole fish were
exposed to F. columnare for 1 hour, rinsed for
2 minutes, and then homogenized. The adherent
F. columnare were enumerated by plate counts.
There were significant differences in adhesive-
ness of the 11 isolates ofF. columnare evaluated
with this assay. The CFU/mg offish varied over
a 150-fold range for the isolates tested.
A cultured-cell assay was used to examine
adhesiveness to EPC cells. The cell culture
medium was removed from the cells and
replaced by a suspension of F. columnare in
either phosphate-buffered saline (PBS) or well
water. After incubation at 300C for 10 minutes,
plate counts were used to determine the number
of adherent bacteria. Seven isolates of
F. columnare were tested for adhesiveness in
hypotonic conditions (well water), and there was
no significant differences among isolates. Four
isolates were tested in PBS, and one isolate had
significantly reduced adhesiveness. The assay
with EPC cells was not satisfactory because the
high concentration of sodium chloride in PBS
reduces the adhesiveness ofF. columnare. The
use of fresh water during the incubation of EPC
cells with F. columnare resulted in swelling of
the EPC cells because of the hypotonic
conditions. The variation in adhesiveness among
the F. columnare isolates tested was small for
the cultured-cell and gill assays.
Based on adhesion to plastic, the mean adhesion
of 11 isolates of F. columnare isolated from
external organs was higher than the mean for
34 SRAC Eighteenth Annual Progress Report, December 2005
Identification, Characterization, and Evaluation of Mechanisms
of Control of Bolbophorus-like Trematodes and Flavobacterium
Columnare-like Bacteria Causing Disease in Warm Water Fish
11 isolates from internal organs. The virulence
of 13 isolates (as reported by Thomas-Jinu and
Goodwin in 2004) was not correlated with
adhesion to plastic. Adhesion of 11 isolates of
F. columnare to larval zebrafish was also not
correlated with the virulence reported by
Thomas-Jinu and Goodwin (2004). The lack of
correlation between virulence and adhesion
could be the result of additional passages in
culture between the determination of virulence
and the adhesion evaluations. There could also
be differences in virulence or adhesion related to
species of fish. Additional experiments will
evaluate adhesion and virulence in simultaneous
experiments.
Molecular identification of columnaris-
like bacteria using rapid sequence analysis
of a portion of the 16S ribosomal gene and
the 16S-23S intergenic spacer region
Mississippi State University. Isolates of
columnaris-like bacteria obtained from LSU
and MSU were cultured, and DNA isolated
using Purgene DNA isolation Kit (Gentra
Systems, Inc., Minneapolis, Minnesota). A
portion of the 16S and the entire 16S-23S
intergenic spacer of one isolate was
PCR-amplified using primers to regions of the
16S and 23S ribosomal sequences that are
conserved among the gram negative bacteria.
One predominant product was obtained and
cloned into pPCR4 TOPO cloning vector
(Invitrogen) and sequenced. This was an
intergenic sequence containing the tRNA for
alanine and the tRNA for isoleucine. Several
products were expected, representing different
ribosomal operons, but as of yet only this ITS
product was found. Alignment of these
sequences with the tRNA sequences from
related organisms were used to identify con-
served sequences, and primers were developed
to allow direct PCR of the specific ITS and
direct sequencing of the products. These PCR
products have been produced and both strands
of both products sequenced for all isolates.
The fragment of DNA between the 16S and
23S ribosomal RNA encoding (ITS) of a total
of 50 Flavobacterium columnare case isolates
were amplified by polymerase chain reaction
using the common 16S-tRNA and tRNA-23S
primer sets reported in year one of the project.
The products were cloned and sequenced. The
sequences consist of a total of 748 base pairs
(bp) and include a 100 bp portion of the 16S
fragment and 200 bp overlapping region. In
sequence comparisons the 16S region was
useful in identifying isolates that were not
actually F. columnare. The ITS demonstrated
substantial variation, however, at least 3
distinct clusters of similar sequences were
identified. These clusters demonstrated 5-10%
sequence differences in the ITS region but less
than 2% divergence within a cluster. This
suggests that the isolates represent at least 3
different strains. We are evaluating an
additional 20 isolates and comparing sources
to see if sequence data correlates with host or
season. Also, the conserved sequences will be
evaluated for differentiating diagnostic PCR.
All sequencing data will be submitted to
GenBank so that other diagnostic and research
labs can use this information.
Ribotyping techniques to differentiate
isotypes of columnaris-like bacteria
University of Tennessee. A number of
Flavobacterium columnare isolates from fish
SRAC Eighteenth Annual Progress Report, December 2005 35
Identification, Characterization, and Evaluation of Mechanisms
of Control of Bolbophorus-like Trematodes and Flavobacterium
Columnare-like Bacteria Causing Disease in Warm Water Fish
disease cases were acquired, as well as several
Flavobacterium columnare-like bacteria,
which share a close taxonomic relationship to
the target organism, including Flavobacterium
hydatis, F succinicans, and F. psychrophilum.
Those isolates are currently being typed using
ribotyping methodologies, and assay com-
ponents and procedural variations that provide
the greatest fingerprint definition between the
various isolates are being determined. Once
established, the optimal methodology will be
used to generate a fingerprint database of the
above control isolates, which will then form
the basis for comparison of wild type isolates
obtained from other investigators involved
with this project.
We have replicated and validated ribotyping
methods developed to differentiate various
isolates ofFlavobacterium columnare (ATCC
strains, wild type strains isolated from
infected fish), F. hydatis, F. resiovorum,
F. aquatile, F. flevense, and suspected
Flavobacterium spp. obtained from various
sources and other diverse species of bacteria
that might inhabit aquatic environments
(Citrobacter freundii, Brochothrix
thermosphacta, Aeromona veronii,
Sphingomonas capsulate, Vibrio Cholerae,
Pseudomonas stutzeri, Micrococcus luteus,
Glaciecolia pallidula). While the Qualicon
database is quite extensive across many
species of bacteria, the database for
Flavobacterium spp. is rather limited, and
thus as a part of this process, we are building
a riboprint database, which will become
available to other users of that system. In that
analysis, we have noted good homology and
yet acceptable separation of riboprints
between various Flavobacterium species and
good separation from and between most of the
diverse isolates, including Flavobacterium-
like bacteria.
We have also progressed in studies to
determine the efficacy of pulsed-field gel
electrophoresis (PFGE) analysis for identi-
fication and differentiation of a number of the
above species, and including various strains of
pathogenic Flavobacterium columnare. The
first phase of this work has involved the
development of culture methods and PFGE
protocols, based on restriction digests, to
obtain optimal fingerprints for identifying and
separating various bacterial isolates. Because
F. colmnare colonies cannot be readily
separated from solid growth media, obtaining
an appropriate mass of cells for PFGE assays
via picks or loops, as is done with other
bacterial species, was found to be difficult.
Thus, isolates were grown in liquid culture,
followed by centrifugation to obtain a cell
pellet. Varying cell densities/concen-trations
were used for DNA isolation procedures,
following by restriction enzyme digests prior
to running digested DNA through PFGE gels
in conjunction with CHEF-mapper system
(Bio-Rad Laboratories, Hercules, California).
Test growth media have included ATCC
Nutrient Broth 3, 1839 Harpo's HTYE, 1750
Anacker and Ordal Medium. Separate trials
were conducted to test various restriction
enzymes, including SpeI, XbaI, BamHI, SmaI,
ApaI. Additionally, varying densities of
SeaKem Gold Agarose (Fisher Scientific, Far
Lawn, New Jersey) were used for PFGE
separation of bands.
For the PFGE analysis, Spel, XbaI, BamHI
restriction enzymes produced small and
numerous bands from DNA extracted from
Flavobacterium spp. and some Flavobacterium-
36 SRAC Eighteenth Annual Progress Report, December 2005
Identification, Characterization, and Evaluation of Mechanisms
of Control of Bolbophorus-like Trematodes and Flavobacterium
Columnare-like Bacteria Causing Disease in Warm Water Fish
like bacteria, and those patterns were found to be
unsuitable for PFGE type analysis; whereas
suitable fingerprints were generated for other
gram-negative species, including Salmonella
choleraesuis Braenderup control (ATCC
BAA-664). We believe that restriction sites in
Flavobacterium genome are too numerous for
the three restriction enzymes above. Smaland
Apal digests produced fewer but less distinct
bands in Flavobacterium spp. and some
Flavobacterium-like bacteria, whereas those
enzymes again produced suitable fingerprints
for other bacteria including control strains.
Varying densities of PFGE gels did not
provide better resolution of banding patterns.
Currently, we are implementing additional
protocols and alternate restriction enzymes to
develop more definitive PFGE typing
patterns. Such protocols will likely require
more lengthy DNA extraction procedures,
which will in turn require longer turn-around
times for sample analyses.
To date, our studies indicate that ribotyping
may offer a more efficient and timesaving
option for identification and differentiation of
Flavobacterium, Flavobacterium-like, and
RESULTS AT A GLANCE...
SMolecular (PCR) and conventional (API
system) methods may be used for the
identification ofF. columnare. Molecular
methods such as ribotyping, RAPD
analysis, and sequencing of the
intergenic spacer region between the
16S-23S ribosomal RNA genes can
allow for discrimination between
different genotypic strains.
non-Flavobacterium isolates.
Determine the presence of unique outer
membrane proteins of various strains of
columnaris-like bacteria
Clemson University. Several outer
membrane proteins (OMP) from Flexibacter
columnare have been isolated and are
consistently found in all F. columnare. Over
the last two years we reported that a 30 kDa
OMP isolated from F. columnare is a potent
inducer of type II nitric oxide synthase
(iNOS) and inducible prostaglandin H2
synthase (cyclooxygenase-2; COX-2) in
isolated catfish phagocytes, and that these
activities can be blocked using specific
antibodies against the OMP.
The proteomics facility within the Clemson
University Genomics Institute (CUGI) helped
us to identify F. columnare OMPs, with an
initial focus on the 30 kDa, 40 kDa, and larger
OMPs. Protein fragments between 12 and 15
amino acid residues were isolated from each
band, yet were not identified because of a lack
of homology with any known proteins in
current data banks. These fragments, how-ever,
may be useful for generating PCR primer sets to
identify larger coding regions of the OMP
genes. Very recently, we constructed a cDNA
library from a virulent Clemson University
strain of F. columnare using a commercially
available kit (Ambion) to remove 18S and 20S
RNA, thereby enriching mRNA that was
subsequently used to generate a long-PCR-
based library (Clonetech). Our in-hand
antibodies against OMPs recognized
recombinant proteins expressed in the library,
and those positive plasmids are being
sequenced at the moment. Over the course of
SRAC Eighteenth Annual Progress Report, December 2005 37
Identification, Characterization, and Evaluation of Mechanisms
of Control of Bolbophorus-like Trematodes and Flavobacterium
Columnare-like Bacteria Causing Disease in Warm Water Fish
the next 5 months recombinant OMP proteins
will be expressed and purified, then screened
for both in vitro and in vivo activity in channel
catfish and tilapia phagocytes, and finally will
be used as potential vaccines against our
virulent strain of F. columnare The most
important product of this study will be the
availability of OMP-specific antibodies and
recombinant OMPs for general use by the
cooperators in this project.
Objective 5. Develop challenge models for columnaris-like bacteria
isolated from major warmwater aquaculture species in the
southeastern United States.
Internal genetic labeling of columnaris-like
bacteria for use in the development of an
effective challenge model
Mississippi State University and Auburn
University. Our objectives for this project are
to 1) ligate a Bacteroides consensus promoter
sequence upstream of gfp mut3a to allow
expression of green fluorescent protein in
Flavobacterium columnare, 2) ligate the gfp
gene and promoter into shuttle vector pCP 11,
3) transfer the pCPll-gfp plasmid into
Flavobacterium columnare for expression of
green fluorescent protein, and 4) use the
fluorescent-labeled F. columnare to develop
an effective challenge model (in cooperation
with Joe Newton at Auburn University).
Two 65 base-pair DNA oligonucleotides con-
taining a consensus promoter sequence from
Bacteroides fragilis were synthesized and
hybridized. The double stranded DNA con-
taining the promoter was then digested with
EcoRI and SacI and ligated upstream of gfp
mut3a in plasmid pFPV25. Expression of the
gfp gene from this plasmid (designated
pFCgfp) in E. coli was confirmed using a
fluorescence plate reader.
A SpeI-EcoRV fragment from pFCgfp was
ligated into pCP 11, and the resulting plasmid
was designated pMWFCgfp. Expression of
gfp from this plasmid in E. coli was also
confirmed using a fluorescence plate reader.
Another plasmid was also constructed by
amplifying the ermF (erythromycin resis-
tance) gene from pCP 11 by PCR and cloning
it into the EcoRI site of the broad host range
plasmid pBBR1MCS4. The resulting plas-
mid, pBBRermF, will allow selection in
F. columnare based on erythromycin
resistance. We then transferred the gfp gene
with the Bacteroides promoter from
pMWFCgfp into pBBRermF on a SmallSpeI
fragment to construct pBBRFCgfp.
Objective 3 has not been successfully com-
pleted despite numerous attempts to transfer
pMWFCgfp and pBBRFCgfp into multiple
F. columnare isolates. We have been utilizing
a conjugation technique using E. coli SM10
lpir as a donor strain to attempt transfer of the
plasmids into F. columnare. We have been
using 25 columnaris strains that were
collected from John Hawke (LSU-SVM) as
well as an additional 20 isolates received from
Joe Newton. This year, we have pheno-
typically characterized all the isolates to
enable optimal growth conditions for
F. columnare during the conjugation experi-
38 SRAC Eighteenth Annual Progress Report, December 2005
Identification, Characterization, and Evaluation of Mechanisms
of Control of Bolbophorus-like Trematodes and Flavobacterium
Columnare-like Bacteria Causing Disease in Warm Water Fish
ments. We have also conducted experiments
that resulted in adjustment of the erythromycin
concentration used on the selection plates. In
addition, we have obtained two additional
plasmids, pCP23 and pCP29, from Mark
McBride at University of Wisconsin,
Milwaukee that will enable us to utilize other
antibiotic selection markers. pCP23 carries a
tetracycline resistance gene that is expressed
in flavobacteria, and pCP29 carries a cefoxitin
gene. Experiments are ongoing using our
optimized growth/antibiotic conditions to
transfer pMWFCgfp, pBBRFCgfp, pCP23,
and pCP29 into the F. columnare isolates
from our panel.
A nalidixic acid resistant F. columnare mutant
(spontaneous-not recombinant) has been iso-
lated that can be used as a tagged organism (if
it is still virulent) until the gfp gene in
F. columnare is successfully expressed.
Several F. columnare isolates (including the
nalidixic acid isolate) have been used in
challenge experiments following the pro-
cedures of Andy Goodwin and S. Thomas-Jinu
at the University of Arkansas at Pine Bluff. To
date it has not been possible to cause columnaris
disease in these experiments using their
procedures.
Challenge models for channel catfish and
golden shiners
University of Arkansas at Pine Bluff. In the
first part of our study we compared the bio-
chemistry, DNA sequence (by RAPD) and
pathogenicity of a large group of columnaris
strains. Channel catfish and golden shiners
were subjected to temperature shock and then
immersed in a bath of columnaris bacteria at
a concentration sufficient to cause 60 to 70%
mortality in 2 days using the more pathogenic
of archived columnaris strains for the
respective host. Each experiment was
performed in triplicate with 20 fish per tank.
Moribund fish were necropsied and the cause
of death verified. Columnaris bacteria were
re-isolated and identified by biochemical (tube
tests) and molecular (randomly amplified
polymorphic DNA, RAPD, Promega)
techniques to verify that the fish died from
infection by the challenge bacteria. In this
work, we found that the catfish and cyprinid
fish isolates fell in different clades, but that
there was no correlation between these genetic
and biochemical results, and any other
measure including fish species of origin or
pathogenicity to catfish and golden shiners.
Another way to look at differences between
columnaris isolates is to challenge fish and
then look for differences in response of the
infections to practical disease treatments.
Columnaris disease was produced in channel
catfish, Ictalurus punctatus (Rafinesque) by
bath exposure to 4 highly virulent isolates of
Flavo-bacterium columnare. In untreated
controls, mortality began 20 hours after
exposure and was 100% by 48 hours after
exposure. Mortality in channel catfish given
antibiotic treatments with oxytetracycline
(OTC) or a combination of sulfadimethoxine
and ormetoprim (SOR) in feed prior to
bacterial challenge was 0% with all four
strains ofF. columnare. Diquatwas the most
effective bath treatment; mortality with all
four strains was 0%. With potassium
permanganate, chloramine-T, hydrogen per-
oxide, and copper sulfate bath treatments,
efficacy varied significantly among bacterial
SRAC Eighteenth Annual Progress Report, December 2005
Identification, Characterization, and Evaluation of Mechanisms
of Control of Bolbophorus-like Trematodes and Flavobacterium
Columnare-like Bacteria Causing Disease in Warm Water Fish
strains and among treatments. Bath treat-
ments with chloramine-T and potassium
permanganate reduced mortality from 100% to
75% and 69%, respectively, but copper sulfate
and hydrogen peroxide treatments were not
effective.
Based on our results, oral antibiotics pre-
vented columnaris disease but, of the bath
treatments, only Diquat produced a dramatic
reduction in the mortality of acutely infected
fish. Diquat is labeled for aquatic use as an
herbicide in the United States but in large
ponds it is prohibitively expensive.
Challenge models for hybrid striped bass
Louisiana State University. Strains of
Flavobacterium columnare, archived in the
LSU Aquatic Diagnostic Laboratory reposi-
tory, are being used in virulence studies in
hybrid striped bass. Methods that produce
uniform mortality rates of 75% or greater
following exposure are classified as virulent
strains ofF. columnare. These criteria will be
adopted for use to compare virulence of
archived strains from various locations and
various species outlined in Objective 6.
Hybrid striped bass (20 g mean weight) were
acclimated and held in the Aquatic
Pathobiology Building at the LSU School of
Veterinary Medicine. The strains (isolates)
were evaluated for virulence in a standardized
challenge procedure where scales were re-
moved and the skin scarified in a 1 cm2 area.
Cultures of the F. columnare bacteria were
swabbed on the scarified area rather than
using the immersion method. This was done
due to difficulties with clumping of the
bacteria and difficulty enumerating bacteria in
the challenge bath. Virulent strains of
F. columnare colonized the scarified skin
readily and caused infection and disease
whereas avirulent isolates did not cause
infection. Mortality was evident 96 hours after
infection with virulent strains.
Objective 6. Use challenge models for each fish species to correlate
virulence with biotype and or genotype of columnaris-like bacteria.
Channel catfish and golden shiners
University of Arkansas at Pine Bluff. Vari-
ability in Flavobacterium columnare patho-
genicity makes disease treatment difficult
because there is currently no way to easily
recognize those strains that warrant aggressive
treatments. In order to identify suitable markers,
17 isolates of F. columnare were cultured
from six different fish species. The DNA from
all isolates was analyzed using randomly
amplified polymorphic DNA (RAPD).
Bootstrap analysis of the RAPD data produced
a tree with three major groups supported by
scores of 80% to 100% similarity.
The remaining objective was to complete
challenge assays to see if there were any stain
differences in the virulence of columnaris
isolates using the golden shiner as a host. Of
the strains tested, two of catfish origin that
were highly virulent in channel catfish (100%
mortality) produced much lower mortality
(30% to 40%) in golden shiners. For the other
40 SRAC Eighteenth Annual Progress Report, December 2005
Identification, Characterization, and Evaluation of Mechanisms
of Control of Bolbophorus-like Trematodes and Flavobacterium
Columnare-like Bacteria Causing Disease in Warm Water Fish
isolates tested, pathogenicity in channel
catfish and shiners was similar. There was no
correlation between biochemical character-
istics or RAPD genogroup and pathogenicity.
Hybrid striped bass
Strains identified by the RAPD grouping
system of Farmer 2004 were used in
challenge studies. Representative isolates
from each RAPD group and host species were
obtained from our collection of archived
strains at LSU. Strains used in the challenges
were: LADL 04-046 isolated from channel
catfish (RAPD Group I), LADL 04-066
isolated from large-mouth bass (RAPD Group
I), PB-02-12 isolated from fathead minnow
(RAPD Group II), and LADL 94-141 isolated
from channel catfish (RAPD Group II).
Strains LADL 04-066 largemouth bass
(Group I) and PB-02-12 fathead minnow
(Group II) were virulent in hybrid striped bass
causing 100% mortality in 96 hours. Strains
LADL 94-141 channel catfish (Group III) and
LADL 04-046 channel catfish (Group I) were
non-virulent in hybrid striped bass. Thus far
virulence appears to be correlated more with
host source than with RAPD group.
Additional strains are currently being tested.
PUBLICATIONS, MANUSCRIPTS, OR PAPERS PRESENTED
Publications in print
Doffitt, C., L. M. Pote, and T. King. 2005. Bolbophorus damnificus infections of piscivorous birds in the
Mississippi Delta. Southeastern Biology 52:3.
Flowers J. R., M. F. Poore, L. M. Pote, R. W. Litaker and M. G. Levy. 2005. Cercariae of Bolbophorus
damnificus and Bolbophorus sp. with notes on North American bolbophorids. Comparative
Parasitology 72(2):220-226.
Labrie, L., C. Komar, J. Terhune, A. Camus, and D. Wise. 2004. Effect of sublethal exposure to the
trematode Bolbophorus spp. on the severity of enteric septicemia of catfish in channel catfish
fingerlings. Journal of Aquatic Animal Health 16(4):231-237.
Ledford, J.J. and A.M. Kelly. In press. Comparison of black carp, redear sunfish, and blue catfish as
biological controls of snail populations. North American Journal of Aquaculture.
Mischke, C.C., D.J. Wise, and L.M. Pote. 2005. Acute toxicity of chemicals to the ram's horn snail
Planorbella trivolvis. Journal of the World Aquaculture Society 36:559-562.
Thomas-Jinu, S. and A. E. Goodwin. 2004. Morphologic and genetic characteristics of Flavobacterium
Columnare isolates: Correlations with virulence in fish. Journal of Fish Diseases 27:29-35.
Thomas-Jinu, S. and A. E. Goodwin. 2004. Acute columnaris infection in channel catfish: Efficacy of
treatments practical in warmwater aquaculture ponds. Journal of Fish Diseases 27:23-28.
Theses
Zhang, Y. 2004. Adhesiveness of Flavobacterium columnare: comparison of different methods and
isolates. M.S. thesis. Auburn University, Auburn, Alabama.
SRAC Eighteenth Annual Progress Report, December 2005
Identification, Characterization, and Evaluation of Mechanisms
of Control of Bolbophorus-like Trematodes and Flavobacterium
Columnare-like Bacteria Causing Disease in Warm Water Fish
Farmer, B. 2004. Improved methods for the isolation and characterization of Flavobacterium columnare.
M.S. thesis. Department of Pathobiological Sciences, School of Veterinary Medicine, Louisiana
State University, Baton Rouge, Louisiana.
Manuscripts in preparation
Mitchell, A. J. and S. Snyder. Optimizing slurried-hydrated lime pond-shoreline treatments for the control
of rams-horn snails
Farmer, B. and J. P. Hawke. Improved methods for culture and antibiotic susceptibility testing of
F. columnare.
Kelly, A. M. Prey preference of redear sunfish and blue catfish trained on various conditioning diets.
Aquaculture.
Presentations
Farmer, B. and J. Hawke. 2004. Improved methods for the diagnosis and characterization of
Flavobacterium columnare. Annual Meeting of the Fish Health Section, American Fisheries
Society, 25-29 July, Shepherdstown, West Virginia.
Hanson, L. A., L. M. Ford, B. Scheffler and J. P. Hawke. 2004. Sequence analysis of the 16S-23S
intergenic spacer of Flavobacterium columnare to identify potential strains. Annual Meeting of
the South Central Branch of the American Society for Microbiology, 5-6 November, Mississippi
State, Mississippi.
Zhang, Y. and J.M. Grizzle. 2004. Evaluation of an assay for adhesiveness of Flavobacterium
columnare. Annual Meeting of the Fish Health Section, American Fisheries Society, 25-29 July,
Shepherdstown, West Virginia.
Goodwin, A. E. 2004. Efficacy of treatments for columnaris disease. Arkansas Aquaculture 2004, 16-17
January, Hot Springs, Arkansas.
Goodwin, A. E. 2004. Efficacy of treatments for columnaris disease. Ninth Biennial Fish Disease
Diagnosticians Meeting. 8-10 February, Biloxi, Mississippi.
Goodwin, A. E. 2004. Farm Bureau Fish Disease Research and Extension. Arkansas Farm Bureau
Aquaculture Meeting, June, Little Rock, Arkansas.
Doffitt, C., D. T. King, and L. M. Pote. 2005. Infections of piscivorous birds in the Mississippi delta by
Bolbophorus damnificus. Association of Southeastern Biologists, 13-16 April, Florence,
Alabama.
Doffitt, C., D. T. King, and L. M. Pote. 2005. Trematode infections of piscivorous birds in the
Mississippi delta The Wildlife Society-Mississippi Chapter, 13-14 October, Choctaw,
Mississippi.
Pote, L. M., M. C. Yost, C. Doffitt, B. S. Dorr, D. T. King, A. Camus, and D. Wise. 2005. Further
elucidation of the life cycle and pathology of the digenetic trematode, Bolbophorus damnificus.
Thirtieth Eastern Fish Health Workshop. Kearneysville, West Virginia.
Wise D., C. Mischke, T. Byars, and A. Camus. 2005. Evaluation of copper sulfate to control snail
numbers in catfish ponds affected by Bolbophorus trematodes. Thirtieth Eastern Fish Health
Workshop. Kearneysville, West Virginia.
42 SRAC Eighteenth Annual Progress Report, December 2005
Improving Reproductive Efficiency to Produce Channel x Blue Hybrid Catfish Fry
IMPROVING REPRODUCTIVE EFFICIENCY TO
PRODUCE CHANNEL x BLUE HYBRID CATFISH FRY
Reporting Period
March 1, 2004 August 31, 2005
Funding Level
Participants
Administrative
Advisor
Year 1 ................ $118,390
Year2 ................ $111,610
Year 3 ................ $115,000
Year4................ $115,000
Total ................. $460,000
Auburn University (Lead
Institution) ............. Rex Dunham, Allen Davis, Ron Phelps
Louisiana State University Terrence Tiersch
Mississippi State University Lou D'Abramo
University of Memphis ... Charles Lessman, Bill Simco
USDA/ARS ............ Brian Bosworth, Brian Small
Dr. John Jensen
Special Advisor to the President
Auburn University, Alabama
PROJECT OBJECTIVES
1. Develop brood stock selection and management protocols to optimize channel x blue
hybrid embryo production.
a. Determine minimum cold period required and rates and patterns of application of
thermal changes to promote synchronous gonadal development and spawning.
b. Improve hybrid embryo production by determining the best nutritional regime to
maximize fecundity and hatch rate from induced channel catfish females and blue
catfish males.
c. Improve hybrid embryo production via genetic enhancement.
2. Develop induced spawning techniques and management strategies to optimize
gamete collection and storage.
a. Develop procedures to predict ovulation of channel catfish.
SRAC Eighteenth Annual Progress Report, December 2005 43
Improving Reproductive Efficiency to Produce Channel x Blue Hybrid Catfish Fry
b. Conduct pivotal protocol studies for determining dosage rates and timing of
application of luteinizing hormone releasing hormone (LHRHa), carp pituitary extract
and catfish pituitary extract to maximize ovulation, hatch rate and fry production.
c. Improve hybrid embryo production via pheromonal manipulation of channel
catfish males and blue catfish males for improved ovulation, spermiation, egg
quality, hatch and fry production.
d. Develop extended refrigerated storage and cryopreservation of sperm.
3. Develop techniques to identify, assess and improve gamete quality.
a. Develop criteria for standardizing and classifying egg quality prior to injection
and after manual stripping and describe the morphological and physiological
condition of channel catfish eggs including evaluation of morphological changes
of oocytes during oocyte maturation in female catfish.
b. Determine the profile of estradiol hormone from serum plasma of 2-year-old
female channel catfish over a 12-month period, determine changes in oocyte
maturation during vitellogenesis and identify the different cathepsins that are
responsible for vitellogenin degradation and oocyte maturation in female catfish.
c. Develop in vitro assays to evaluate sperm quality and evaluate their predictive
ability in relation to fertilization and hatch.
4. Develop economically viable standardized hatchery procedures and fertilization
protocols to optimize hatching rate of hybrid embryos.
a. Determine optimal sperm (fresh, frozen and refrigerated)-to-egg ratios for
fertilization and hatch.
b. Determine the effects of commonly used therapeutics on hatching success.
PROGRESS AND PRINCIPAL ACCOMPLISHMENTS
Objective 1. Develop brood stock selection and management
protocols to optimize channel x blue hybrid embryo production.
Objective la. Determine minimum cold period required and rates and
patterns of application of thermal changes to promote synchronous
gonadal development and spawning.
Louisiana State University and University spawning of channel catfish. Spawning begins
of Memphis. Water temperature is the when water temperatures consistently remain
primary environmental factor affecting the above 21C at some locations such as
44 SRAC Eighteenth Annual Progress Report, December 2005
Improving Reproductive Efficiency to Produce Channel x Blue Hybrid Catfish Fry
Louisiana and west Mississippi. The spawning
season at the Aquaculture Research Station of
the Louisiana State University Agricultural
Center was lengthened by heating ponds
through addition of geothermal water (360C).
This study attempted to use degree-days (oD)
to describe and quantify the total heat
requirement for channel catfish to initiate
spawning, which should also indicate the
same requirement to initiate artificial
spawning to produce hybrid embryos. Degree-
days were calculated for 153 spawns between
1999 and 2004. Ponds from 1999 to 2002 had
four available spawning sites (cans), and in
2003 and 2004 the ponds had six sites.
Degree-days needed to obtain the first four
(1999-2002) or six (2003-2004) spawns were
calculated to prevent spawning site limitations
effects on the degree-day values.
In 2004, three heated ponds were maintained
at three different temperatures. Degree-day
values were calculated for 18 spawns using
three threshold temperatures as the starting
point to calculate the degree-days (Table 1).
The 210C threshold yielded a constant value
of 98 40D for the heat requirement of
channel catfish to initiate spawning.
Degree-days were also calculated using the
21C threshold for 135 spawns collected
during the early spawning and regular
spawning periods between 1999 and 2003.
The average D-value above the 21C
threshold was 97 33D. Spawning proba-
bilities and frequency of spawns were plotted
against oD-values (Figure 1). The probability
that a fish will spawn after 1000D was 50%
and increased to 93% after 1500D. Fifty
percent of spawns occur between 75D and
1250D and ninety percent between 500D and
1500D. These results concur with the
literature that 21C is the minimal water
temperature needed to initiate the repro-
ductive process in channel catfish.
Additionally, D-values above 210C may be
useful as a management tool to predict
channel catfish spawning times in heated
ponds, and the correct time to initiate artificial
spawning for hybrid embryo production.
Table 1. The average degree day value for spawns above three thresholds from ponds
maintained at different temperatures. Values in the same column followed by the same
letter do not differ significantly (P < 0.05).
Threshold
Target
temperature
210C 23.1 1.50C 234a 95a 8a
240C 23.1 2.60C 203ab 98a 22b
270C 24.6 3.0C 184b 102a 41c
SRAC Eighteenth Annual Progress Report, December 2005 45
240C
Actual temperature
18C
21C
Improving Reproductive Efficiency to Produce Channel x Blue Hybrid Catfish Fry
In 2005, the temperature data results from
previous years was used to compare
reproductive performance of channel catfish
females induced to spawn before and during the
natural spawning season. The goal was to
extend the documented time female channel
catfish could be induced to spawn before the
natural spawning season without affecting
reproductive performance.
In December of 2004, channel catfish
broodstock (1.17 0.38 kg; 48.1 4 cm) were
purchased from Haring Fish Farms Inc., a
commercial fingerling producer in Northern
Louisiana. Brood fish were stocked in nine,
0.04-ha earthen ponds at the Aquaculture
Research Station of the LSU Agricultural
Center. Six of the ponds were stocked with
30 females and 10 males. The remaining ponds
were stocked with 30 females only. Geothermal
water (360C) was added to three of the ponds
(2 mixed-sex and 1 all-female) beginning on
January 13, 2005. Initial pond temperatures
were approximately 200C and were increased
2C/day until the temperature reached 280C.
Ponds were heated in sets of three until natural
spawning occurred. After six spawns (egg
masses, 20% spawning) were collected from
spawning containers in the ponds, fish were
collected by seining and brought indoors for
induced spawning.
Brood fish were acclimated in an indoor
recirculating system for 48 to 72 hours.
Females were evaluated using ultrasound
imaging of ovaries and oocytes to assess
reproductive readiness. Selected females
were weighed, measured, marked for
identification, and placed into eight, 120-L
fiberglass tanks. Pairings were made based on
length and weight to minimize size
differences within the tanks. Four tanks held
one male and one female (mixed-sex pairs);
the other four tanks contained two female fish
(female pairs). Each female was given a single
injection of leutenizing hormone-releasing
hormone analog at a dosage of 100 p.g/kg.
Temperature was maintained at 270C and
spawning behavior was monitored every 2
hours. After females began releasing eggs
they were anesthetized (Tricaine Methane
Sulphonate, MS-222) and manually stripped.
46 SRAC Eighteenth Annual Progress Report, December 2005
40 1.0
35
0.0
30
25 0. 6
20
15 0.4
10
0.2
5
0 0.0
25 50 75 10 0 125 1 50 175 200
Eegaroe dlmrn
Figure 1. Spawning probabilities and spawning frequency at different degree-day values
above the 21'C threshold.
Improving Reproductive Efficiency to Produce Channel x Blue Hybrid Catfish Fry
Objective lb. Improve hybrid embryo production by determining
the best nutritional regime to maximize fecundity and hatch rate
from induced channel catfish females and blue catfish males.
Auburn University. Since nutrition can play
a key role in maturation, as well as egg and
fry quality, proper nutrition could be a key
factor in the development of hybrid rearing
technologies. Hence, the primary goal of this
component of the project is to improve hybrid
embryo production through nutrient mani-
pulations.
Protein Level and Feed Frequency
The first subobjective was to evaluate the
influence/interaction of dietary protein level
and feeding rate on egg production of channel
catfish. As there are numerous interactions
and it is difficult to obtain brood stock, a
holistic approach was initially used to help
identify important factors to control.
A total of 495 females were stocked in 16
ponds, using four ponds per treatment. The
females were divided in three strains
considering previous spawning behavior (high
spawning, low spawning, and NWAC103),
and based on that characteristic they were
assigned proportionally in a randomized
manner to each pond. Fish were pit-tagged
and heat-branded for identification. The fish
were stocked on February 13, 2004 in 0.04-ha
ponds at a density of approximately 1,500
kg/ha, followed by an acclimation period of
approximately one month. During the
acclimation period, fish were offered a
commercial floating feed (32% protein) diet
three times a week at 1.5% of their body
weight. The two test diets were a 32% typical
practical catfish feed and a 42% high fish
meal practical catfish feed, and the feed was
offered either three or six times a week to
apparent satiation. A fifth treatment utilized
32% protein 3 days per week with supple-
mental feeding of liver 2 additional days per
week at a rate similar to the dry feed. Females
were spawned in three periods (early, middle
and late spawning periods). Dietary protein
level and feeding rate treatments were
evaluated using the following indicators: egg
mass, number of eggs, fecundity (number of
eggs per kilogram female), egg diameter, and
fertilization rate 48 hours after fertilization.
Results for this experiment are presented for
"High" and "Low" spawning strains, as strain
103 had a low number of individuals per
treatment per spawning period. High spawn-
ing and low spawning females had a survival
of 92% (414 females), of which 63.5%
spawned.
Based on logistic analyses by strain, the
following results were determined. For strain
High, the dietary treatment did not have a
significant effect on spawning percentage, but
age and spawning period had a significant
effect on spawning percentage. For this strain,
the odds of females that were 5 years old
spawning were 9.4-times higher than females
that were 3 years old and 8.4-times higher
than females that were 4 years old. The odds
of spawning in the early spawning period
were 10.6 times higher than for the late
spawning period; while the middle spawning
period had 5.1-times higher odds of spawning
than late spawning period.
SRAC Eighteenth Annual Progress Report, December 2005
Improving Reproductive Efficiency to Produce Channel x Blue Hybrid Catfish Fry
With respect to strain Low for spawning
percentage, dietary treatment did not have a
significant effect overall, however when
comparisons between treatments were
performed, the odds of spawning for
treatment 4 (32% protein feed, 3 times/week)
were 2.5-times higher than for treatment 3
(32% protein feed, 6 times/week). The other
two variables, age and spawning period had a
significant effect on spawning percentage. For
this strain, the odds of females that were 5
years old spawning were 5.5-times higher
than females 3 years old, and was not
different than for 4-year-old females. The
odds of spawning in the early spawning
period were 13.4-times higher than for the late
spawning period; and there was no difference
between the middle spawning period and the
late spawning period. In general, the two
strains exhibited different responses. These
results indicate that brood stock management
must adjust to strain variations and age.
Although using a holistic approach has
disadvantages, we were able to evaluate the
effects of strain, age and spawning period on
spawning success and egg production. In
general, each strain responded differently with
respect to dietary treatments and spawning
period. It appears that the high-protein diet
when offered six times per week resulted in
reduced fecundity. With respect to the other
treatments there were few differences duet to
dietary treatments.
Comparison of the response of fish maintained
on the 32% protein diet with 3 feedings per
week with those that also received liver, there
were no notable improvements in egg
production. As with the other treatments strain
and age influenced the response.
When fry production data for the various ages,
strains and spawning period are pooled to
represent only the dietary treatments, overall
production can be determined. Table 2
presents the combined data for the five dietary
treatments for groups of fish that could be
followed and that received LHRHa injections
(a single 20 jlg/kg priming injection and a
48 SRAC Eighteenth Annual Progress Report, December 2005
Table 2. Effect of nutrition, (% protein-number of feedings per week) 42-3, 42-6, 32-3, 32-6 and
32-3 plus 2 days of liver, on hybrid fry/kg female body weight for channel catfish, Ictalurus
punctatus, females fertilized with blue catfish, I. furcatus, sperm. Females were ovulated indi-
vidually in bags within tanks. GMP grade LHRHa injections (20 pg/kg priming dose with a 100
pg/kg resolving dose). A mixture of low and high producing lines of channel catfish was utilized.
Treatment N Fry/kg
32-6 51 1,732a
42-3 48 1,505ab
32-3 54 1,476ab
42-6 54 1,032b
32-3-2L 54 969b
ab Means followed by different letters are significantly different (P < 0.05) Duncan's multiple range test.
Improving Reproductive Efficiency to Produce Channel x Blue Hybrid Catfish Fry
100 gg/kg resolving injection). Based on
mean separation there were significant
differences between treatments with fish
maintained on the 32% protein diet offered six
times per week, producing more fry/kg than
those maintained on the 32% protein diet fed
3 times per week with a liver supplement
2 times per week as well as fish offered the
42% protein diet fed six time per week. The
treatment with the highest observed mean was
32% protein diet offered 6 times per week
although there were no significant differences
between fish offered the 32% protein diet 3 or
6 times per week or those offered the 42%
protein diet 3 times per week. For unknown
reasons, supplementation with liver was
detrimental to fry production.
Lipid Source and Ratios (n3:n6)
The second subobjective was to evaluate
different ratios of polyunsaturated fatty acids,
and their influence on fecundity and on egg
quality. A total of 190 females were stocked
in 8 ponds, using two ponds per treatment. All
females were 4-year-old Kansas strain. The
fish were stocked on January 7, 2005, giving
an acclimation period of approximately 2
months. The trial period was 73 days to 87
days depending on the spawning period.
Female brood stock were maintained in 0.04-
ha ponds at a density of approximately 600
kg/ha. They were offered a commercial
floating feed (32% protein, 5% lipid) diet
three times a week at 1.5% of their body
weight. Water temperature and dissolved
oxygen were measured daily in the early
morning and after sunset. After the
acclimation period, test diets were offered.
The four test diets were based on a commercial
catfish feed containing 5% lipid that was top-
coated with an additional 2% lipid. Three test
diets used a combination of vegetable oil
sources proportioning different 18:3n-3:18:2n-6
ratios. The lipid sources were mixed in the
following ratios: Diet 1 contained soybean oil
and linseed oil in a ratio of 0.90:1.00; Diet 2
also contained soybean oil and linseed oil, but
in a ratio of 7.00:1.00; Diet 3 contained linseed
oil only. The fourth diet was based on fish oil
and high DHA and ARA oil sources with an
n3:n6 ratio of 3:2. Diet 4 contained menhaden
fish oil, high DHA, and high ARA in a ratio of
2:1:1. Thus diets contained a range ofn3 and n6
fatty acid combinations (Diets 1-3) as well as
one diet (Diet 4) which contained HUFA
supplements. The n3:n6 ratios of the oil
supplements were approximately: 1:1, 1:4, 4:1,
3:2, respectively.
Dietary lipid treatments were evaluated using
the following indicators: total number of eggs
produced, fecundity (number of eggs per
kilogram female), fry production, fry per kg
female and overall fry survival. Biochemical
analysis will also be conducted on egg samples
as indicators of egg quality. Data for treatments
evaluating lipid source and different ratios
between essential fatty acids (Table 3) is
restricted to the first spawning period (early),
since there was a hatchery problem for the
second spawning period, with a minimum of
fertilization across all the treatments.
Additionally, one pond of fish evaluated in
treatment four during the second spawning
period were stressed (due to a broken water
line) at their harvest causing around 30%
mortality.
Based on initial analyses of the data there are
few indications that the dietary treatments had
SRAC Eighteenth Annual Progress Report, December 2005 49
Improving Reproductive Efficiency to Produce Channel x Blue Hybrid Catfish Fry
a significant effect on the percentage of fish the best results. Fish maintained on this diet
spawning or the number of eggs produced. produced almost twice the fry as fish
However, the various lipid combinations did maintained on diet supplemented with lipids
have a strong effect on fry production. Using (primarilyl8:3n-3 and 18:2n-6) producing n-
lipid supplement with a 4:1 ratio of 18:3n- 3:n-6 ratio's of 1:1 (Diet 1) or 1:4 (Diet 2).
3:18:2n-6 (linseed oil, Diet 3) resulted in very These results indicate that highly unsaturated
poor fry production. The use of menhaden fish fatty acids, HUFA, are probably a key factor in
oil with DHA and ArA supplements and a ratio proper brood stock nutrition.
of 3:2 for n-3:n-6 fatty acids (Diet 4) produced
Table 3. Egg and fry production in the early spawning period from broodfish fed four
diets containing different lipid sources. Diets are described in the text.
Diet Female No. No. Eggs/ No. fry Fry/ % Fry
weight females eggs kg female kg survival
(kg) spawned female
1 1.91 12 198,679 8,680 23,901 1,044 12
2 2.15 10 201,949 9,397 27,841 1,296 14
3 1.86 8 111,294 7,500 7,522 507 7
4 1.64 13 186,056 8,731 47,045 2,208 25
Objective lc. Improve hybrid embryo production via genetic
enhancement.
Auburn University. The channel x blue AU-1 channel catfish females produced
catfish hybrid grows faster, has more efficient greater numbers of hybrid fry than AU-7 for
feed conversion, has a higher tolerance for 4 consecutive years (Table 4). AU channel
low dissolved oxygen concentrations, and catfish lines 1, 3, 5 and 11 consistently
better survival compared to channel catfish, produced high numbers of hybrid fry
However, economic production of hybrid compared AU lines 6-10, 12 and strain 103
embryos is problematic. Some strains of over a three year period (Tables 5 and 6). AU
channel catfish females or blue catfish males lines 4 and 13 were only evaluated 2 years,
may have reproductive characteristics more and were high performers one year and low
suited for production of channel catfish another. Fry/kg for 103 was very low the first
female x blue catfish hybrid catfish embryos 2 years, but improved to average performance
than others. levels the third year. Additionally, strain of
50 SRAC Eighteenth Annual Progress Report, December 2005
Improving Reproductive Efficiency to Produce Channel x Blue Hybrid Catfish Fry
male blue catfish affected hatching rate of
hybrid embryos and sperm production.
Genotype-environment interactions were also
observed for sperm production. Utilization of
genetic variation has the potential to double
efficiency and productivity of hybrid embryo
production.
Table 4. Fry/kg for AU-1 and AU-4 channel catfish female when injected with LHRHa
and hybridized with blue catfish males over a 4 year period.
Fry/kg female body weight
2002
Genotype
2001
2003
2004
AU-1 4,598 4,300 1,857 3,105
AU-7 2,638 2,550 693 1,045
Table 5. Percentage of females ovulating, fecundity, and fry/kg for channel catfish
female strains when injected with LHRHa and hybridized with blue catfish males in
2003.
Channel catfish female % ovulation fecundity (eggs/kg) fry/kg
AU-1 100 11,047 1,857
AU-2 75 7,133 2,154
AU-3 100 11,997 1,283
AU-4 82 6,545 1,005
AU-5 100 8,790 858
AU-7 73 10,179 693
AU-8 100 9,122 625
AU-9 75 9,438 492
AU-6 90 7,814 395
103 80 7,425 257
AU-10 45 9,575 163
SRAC Eighteenth Annual Progress Report, December 2005 51
Improving Reproductive Efficiency to Produce Channel x Blue Hybrid Catfish Fry
Two strains of channel catfish were selected
for increased body weight for six generations.
The resulting females of the two select lines
were compared to their corresponding
randomly bred controls for hybrid fry
production when they were 3 years old.
Random control 1 produced more hybrid fry
than random control 2. In both cases, select
lines had reduced hybrid fry production
compared to controls (Table 7). Several
possible explanations exist. Selection for body
weight decreased reproductive performance
and the ability to generate hybrid fry via
artificial fertilization techniques and/or
reduced the age of first sexual maturity.
Alternatively, these effects could be attributed
to inbreeding depression from mass selection
in the select lines that may be accumulating in
these relatively small research populations.
52 SRAC Eighteenth Annual Progress Report, December 2005
Table 6. Fry/kg for different lines channel catfish female when injected with LHRHa and
hybridized with blue catfish males over a 3 year period.
Fry/kg female body weight
Line 2002 2003 2004
AU-11 5,570 -3,421
AU-5 8,500 858 3,136
AU-1 4,300 1,857 3,105
AU-13 3,500 3,042
AU-3 4,800 1,283 2,844
103 679 257 2,680
AU-6 -395 2,570
AU-12 3,550 -2,566
AU-8 625 2,427
AU-4 -1,005 2,332
AU-9 4,500 492 2,178
AU-10 -163 1,902
AU-7 2,550 693 1,045
Improving Reproductive Efficiency to Produce Channel x Blue Hybrid Catfish Fry
Table 7. Fry/kg for two lines of channel catfish females selected for increased body weight
for six generations and their randomly bred controls when injected with LHRHa and
hybridized with blue catfish males when 3 years old.
Line Fry/kg
Select 1 2,391
Random 1 3,105
Select 2 832
Random 2 1,265
The same two select lines were compared to one Mississippi State University. Groups of nine,
of their crossbreeds, select 2 female x select 2-year old female channel catfish brood stock
1 male. The crossbreeding resulted in no heterosis, obtained from each of four different strains/
and the fry output of the crossbred females was sources were tagged and stocked into four,
the same as the maternal strain females (Table 8). 0.04-ha earthen ponds (36 fish per pond, 9
This would be indicative ofdominance for hybrid fish per strain) in April. Blood and egg
fry/kg or a genetic maternal effect. This can only samples were collected from twelve fish in
be completely evaluated by examining the each pond (3 fish/strain) every month for 11
performance of both parental types and both and 9 months for blood and eggs, respec-
reciprocal crossbreeds. The results also suggest tively. No individual fish within a strain was
that the lower fry output by the select lines subject to sampling more than once every four
compared to their random controls is not due to months. Plasma estradiol, plasma testosterone,
inbreeding unless there is a very strong maternal cathepsins, protein content of eggs and egg
effect on reproduction as if inbreeding depression size were measured. No noteworthy differ-
was the major explanation for the decreased fry ences in the mean values of the physiological
output, the mean of the crossbreed should be at indices monitored were observed among the
least higher than the lower performing parent. four strains during each month.
Table 8. Fry/kg for 2 lines of channel catfish females selected for increased body weight for
6 generations and their crossbreed (femalexmale) when injected with LHRHa and hybridized
with blue catfish males when 3 years old. Means followed by different letters are significantly
different (P-0.05).
Genotype Fry/kg female body weight
Select 1 (S 1) 2,391a
Select 2 (S 2) 832b
S2 x Sl 835b
SRAC Eighteenth Annual Progress Report, December 2005
Improving Reproductive Efficiency to Produce Channel x Blue Hybrid Catfish Fry
Objective 2. Develop induced spawning techniques and
management strategies to optimize gamete collection and storage.
Objective 2a. Develop procedures to predict ovulation of channel
catfish.
Auburn University. Hybrid channel x blue
catfish can be obtained by induced spawning
and artificial fertilization but with variable
results. A threshold degree of maturity must
be reached before brood fish can be induced
to spawn, but selection of such fish can be
very subjective. Temperature of the sur-
rounding environment affects the rates of
physiological processes in fish. Response time
to applications of induced spawning hormones
such as LHRHa is thought to be related to
water temperature.
Female broodfish (Marion strain channel cat-
fish) were given a subjective ranking of poor,
fair or good as well as measurements of body
weight, total body length, body width and
girth were taken. Brooders were held at 24,
26, and 280C in 60-gallon aquaria and injected
with LHRHa at 20 tg/kg as a preparatory
injection followed 12 hours later with 100
jg/kg. Fish were monitored hourly as
ovulation approached, and the time of the first
RESULTS ATA GLANCE...
r Hatch rate of hybrid embryos is
improved if LHRH-injected channel
catfish females are stripped within
2 hours of first observation of egg
release. Waiting longer will increase
the number of eggs stripped, but this
is more than offset by much lower
hatch rate.
egg deposit and when approximately 100 eggs
were found were recorded. Approximately
half the females were manually stripped soon
after the first egg was observed, and the other
fish were stripped 4 to 6 hours after the first
egg was observed. Eggs were artificially
fertilized with blue catfish sperm and
incubated. For each egg mass, the percentage
of viable embryos at 24 hours after
fertilization, the percent hatch, and percent
survival at swim-up was determined.
The overall mean degree-hour response time
(temperature in C multiplied by the time in
hours to first egg release) was 1,156 275.
The mean degree hour response time was
1,416 107 at 240C, 1,228 211 at 260C and
981 278 at 280C. The percentage of
females that ovulated were 58, 62.5 and
87.5% at 24, 26, and 280C, respectively. The
majority of females which did ovulate did so
between 58 to 64 hours at 240C, 48 to 52
hours at 260C and 24 to 40 hours at 280C with
the fish classified as "good"spawning sooner
than the "poor" classification at all
temperatures. When only the good quality
females were considered, the weight of eggs
released/kg female varied by water
temperature. At 24C an average of 70 60 g
were obtained/kg, at 260C 126 41, and at
280C 154 34. The number of eggs/g of eggs
also varied by temperature, 71 11, 53 6,
and 48 10 at 24, 26 and 280C respectively.
Egg quality varied with how soon eggs were
taken after the first egg was released. For
54 SRAC Eighteenth Annual Progress Report, December 2005
Improving Reproductive Efficiency to Produce Channel x Blue Hybrid Catfish Fry
females at 280C, when eggs were taken within
2 hours of being observed the % viable
embryos averaged 76 13% and the % hatch
was 31 16%. When eggs were taken at 4 or
more hours of being observed, the % viable
embryos averaged 66 19% and the % hatch
was 9.7 6.6%. When a female was stripped
within 2 hours after the first eggs were
released, a lower weight and total number of
eggs/kg (107.3 46.6 and 5,739.8 2174)
were obtained relative to fish stripped 4 or
more hours after the first eggs were released
(147.7 36 and 7,724 2,120, respec-
tively).
Proper selection of broodfish for induced
spawning can help insure a high rate of
spawning success and good egg quality.
However, the brood selection is often
subjective based on general appearance of the
fish and the culturist's experience. Appro-
priate quantitative criteria can reduce
individual bias and assists the less experience
biologist in brood selection. Five trials were
conducted using 3- and 5-year-old channel
catfish females where the physical char-
acteristics of total length, weight, and width
were measured and ratios calculated.
Development and pulsation of the genital
papilla was also used as a point of evaluation.
Females were induced spawned using LHRHa
at 120 tg/kg and manually spawned. Eggs
were artificially fertilized with blue catfish
sperm. Spawning success, and egg production
characteristics were evaluated as to their
relationship to brood stock characteristics.
Age of broodfish had a significant effect on
spawning success. Of the 5-year-old fish, 91%
spawned while only 26% of the 3-year-old
fish spawned. The two age groups also
differed in average weight which was a factor
influencing spawning success. Fish weighing
over 3 kg had an average spawning rate of
80% while fish weighing 3 kg or less
averaged 20%. Fish that were 60 cm, or more,
in total length, had a spawning rate of 80%.
Fish with a length (cm)/weight (kg) ratio less
than 15 also averaged an 80% spawning rate.
The length (cm)/width (cm) ratio did not
exhibit a well-defined pattern, however, fish
with a ratio less than 5 had a 60% success
rate. Fish with a width (cm)/weight (kg) ratio
less than 4 have 75% chance of spawning.
Whether or not the genital papilla was
pulsating at the time of the first LHRHa
injection had no relationship to spawning
success. Brood age affected egg
characteristics. Younger fish had more eggs
per gram of eggs. The mean number of
eggs/kg for 3-year-old fish was 8274 2868,
while the mean for 5-year-old females was
4842 1130. The 3-year-old fish ovulated,
on average, 9.7 hours later than 5-year-old
fish. In general, variations in other brood
stock descriptors were not associated with
variations in eggs per gram, number of eggs
per kilogram body weight, time of ovulation,
or viability. However, egg diameter was
related to length (cm)/weight (kg) ratio with
larger fish producing larger egg diameters
than smaller fish.
In this study, brood stock age was the most
important consideration for spawning
success. Related to age were brood weight
and length and their effect on spawning
success. Ratios of body proportions also
were related to spawning success with fish
having a width (cm)/weight (kg) ratio of less
than 4 having a >70% success rate. To obtain
the best induced spawning success brood fish
SRAC Eighteenth Annual Progress Report, December 2005
Improving Reproductive Efficiency to Produce Channel x Blue Hybrid Catfish Fry
should be selected to be 5 years old, with a
weight of over 3 kg, and a length greater than
60 cm. Such fish should give a 94% spawning
rate.
Objective 2b. Conduct pivotal protocol studies for determining
dosage rates and timing of application of luteinizing hormone
releasing hormone (LHRHa), carp pituitary extract and catfish
pituitary extract to maximize ovulation, hatch rate and fry production.
USDA-ARS. The effectiveness of catfish
pituitary extract, carp pituitary extract, and
LHRHa for inducing spawning in female
channel catfish and subsequent production of
channel catfish x blue catfish hybrid fry was
compared. Mature female catfish (3 to 5 years
old) were injected with carp pituitary extract
(n = 66), catfish pituitary extract (n = 51), or
LHRHa (n = 58). Catfish pituitaries were
collected in March and April at a commercial
catfish processing plant from fish > 3 pounds,
dried in acetone, and ground to a powder.
Carp pituitary and LHRHa were purchased
from commercial vendors (Stoller Fisheries,
Spirit Lake, Iowa and Syndel International,
Inc., Vancouver, British Columbia, Canada,
respectively). Injection regimes were 2 mg/kg
female body weight (BW) initial injection and
8 mg/kg 20 hours later for carp and catfish
pituitary extract or 40 jg/kg female BW
initial injection followed by 80 .g/kg 20
hours later for LHRHa. Females were
checked for ovulation 24 hours following the
final injection. Ovulating females were
tranquilized and eggs were manually stripped
into Hank's Balanced Salt Solution (HBSS).
Eggs were weighed and then fertilized with
blue catfish sperm. Blue catfish sperm was
prepared by macerating testes from 4 to 5 blue
catfish males and pooling the sperm in HBSS.
Approximately 25 mL of sperm-solution was
used to fertilize each 400 g sample of eggs.
Egg masses were placed in hatching troughs
following fertilization and percent viable
embryos was determined at 48 hours post-
fertilization. Fry numbers at hatch were
estimated volumetrically. Data collected for
each treatment included: weight of females
injected, percent of injected females that
ovulated, fecundity (number of eggs/kg
female body weight), percent viable embryos
at 48 hours, fry/kg body weight of all females,
fry/kg body weight of ovulated females, and
total fry.
There were no differences among treatments
for any of the variables measured (Table 9).
Results demonstrate that catfish pituitary
extract was as effective as carp pituitary
extract or LHRHa for inducing ovulation in
channel catfish females. Catfish pituitary is
readily available from commercial catfish
processing facilities, although regulatory
issues associated with using it to induce
spawning in fish are not known.
Eggs from females ovulated with LHRH
flowed much easier and more completely, but
it seemed their time-frame for ovulation was
wider. The LHRH may have done better if a
longer period of time would have been
allowed for ovulation. The pituitary-treated
fish seemed to ovulate more synchronously,
but never flowed as well as a good LHRH
56 SRAC Eighteenth Annual Progress Report, December 2005
Improving Reproductive Efficiency to Produce Channel x Blue Hybrid Catfish Fry
fish. This observation that CPE-treated fish
ovulate more synchronously has been con-
firmed at Auburn University. Latency time
for LHRH-treated fish is longer, and the
observations observed at USDA are consistent
with observations at other locations.
Auburn University. The 2004 research was
conducted with Good Manufacturing Practices
(GMP) grade LHRHa for injections and
research grade LHRHa for implants at Auburn
University. The dose 30/150 (jg/kg priming/
resolving dose) was the most effective
injection treatment confirming earlier results
with research grade LHRHa. (All doses are
reported as total microgram of product in-
jected. The peptide content of the product is
82% LHRHa. Thus, a 100 jg dose of ingre-
dient is actually 82 pg of LHRHa.). This
peptide content is the same for all experiments
for all institutions conducting research in this
SRAC project. The efficacy of the 30/150
injection and the 100-ug implant were not
different. Females that were not hormone
induced and held in ponds or for short periods
in tanks did not ovulate. All LHRHa
treatments were effective yielding a minimum
of 64.7% ovulation.
The highest observed means for ovulation
percentage were the 125-pg implant and the
20/100 injection (Table 10). However, the
treatments yielding the most fry/kg female
body weight were the 100-jg implant and the
30/150 injection.
Early in the spawning season implants gave
more consistent results than injections, but the
best injection regime (30/150) was not different
from the best implant regime (100 gig)
(Table 11). Ovulation of females in individual
units was more effective than in a communal
group, and in absence ofconspecific males was
more effective than in the presence of
conspecifics males. In terms of gamete release,
all treatments were highly effective with
ovulation % ranging from 66.6 to 100.0% with
a grand mean of 86.1%. However, gamete
quality differed among treatments as indicated
by the variation in fry/kg (Table 11).
During the peak spawning season, the
ovulation rates decreased (Tables 11 and 12).
In terms of gamete release, most treatments
were effective with ovulation % ranging from
28.6 to 71.4 % with a grand mean of 54.9%.
However, gamete quality differed among
treatments as indicated by the variation in
fry/kg. Again, 30/150 injection and the 100-
jg implant were the most effective treatments
and not different from each other. Fry output,
approximately 1700 fry/kg, of these two better
treatments was similar to the output in the
early spawning period. During this peak
period of the spawning season, little
difference existed between fry output of
individually bagged fish and those spawned in
group tanks. Results were similar for the early
and peak spawning (Tables 11, 12, and 13).
During the late spawning period, the injection
regime with the highest ovulation rate was
10/50 at 100% using high line females
(Table 14). The implant with the highest
ovulation rate was 75 ig at 85.7 %, again for
the high line females. No hatch was obtained
in the last spawning period probably because
of poor quality sperm or an error in sperm
preparation. Genetics had an impact on
ovulation. High line females had higher
ovulation than low line females. However,
egg quality data was obtained (Tables 15-19).
SRAC Eighteenth Annual Progress Report, December 2005
Improving Reproductive Efficiency to Produce Channel x Blue Hybrid Catfish Fry
Table 9. Comparison of catfish pituitary extract, carp pituitary extract, and LHRHa for
inducing spawning in channel catfish females and production of channel catfish x blue
catfish fry.
Eggs/kg
female
BW
Treatment # of
females
injected
Mean
weight of
females
(kg)
%
females
ovulating
%
viable
embryos
Fry/kg
BW all
females
Fry/kg
BE
ovulated
females
Carp PE 66 2.9 71 6,482 55.5 1,348 1,788 239,100
Catfish PE 51 2.8 68 6,767 64.1 1,128 1,600 190,100
LHRHa 58 3 65 6,482 66.3 1,527 1,999 254,100
Standard Error 0.2 8.4 720 8.6 344 350
Table 10. Ovulation percentage and hybrid fry/kg female body weight for channel
catfish, Ictalurus punctatus, females fertilized with blue catfish, I. furcatus, sperm.
Females were ovulated communally in tanks, individually in bags within tanks or
individually in aquaria with or without channel catfish males. GMP grade LHRHa
injections (priming dose/resolving dose, pg/kg) or research grade LHRHa implants
(pg/kg) were used. Dose 0 is primarily from females held in ponds and some in tanks.
LHRH Dose N ovulation % Fry/kg
0 555 0
10/50 24 75 500
20/100 500 76.3 1,260
30/150 24 66.6 1,750
75 implant 32 68.9 580
100 implant 34 64.7 1,728
125 implant 10 100 955
58 SRAC Eighteenth Annual Progress Report, December 2005
Total
fry
Improving Reproductive Efficiency to Produce Channel x Blue Hybrid Catfish Fry
Table 11. Early season ovulation percentage and hybrid fry/kg female body weight for channel
catfish, Ictaluruspunctatus, females fertilized with blue catfish, I. furcatus, sperm. Females were
ovulated communally in tanks, individually in bags within tanks or individually in aquaria with
or without channel catfish males. GMP grade LHRHa injections (priming dose/resolving dose,
pg/kg) or research grade LHRHa implants (pg/kg) were used. The hatching environment was
extreme as fungal infection was extremely heavy. Low-producing line of channel catfish was
utilized.
LHRH Dose Environment N ovulation % Fry/kg
10/50 tank 10 80 0
20/100 tank 10 100 0
20/100 bag 9 78 2,293
30/150 tank 10 90 1,727
75 implant tank 10 80 492
100 implant aquaria w/male 7 71 1,650
100 implant aquaria no male 6 67 1,831
125 implant tank 10 100 955
Table 12. Peak season ovulation percentage and hybrid fry/kg female body weight for channel
catfish, Ictalurus punctatus, females fertilized with blue catfish, I. furcatus, sperm. Females were
ovulated communally in tanks, individually in bags within tanks or individually in aquaria with
or without channel catfish males. GMP grade LHRHa injections (priming dose/resolving dose,
pg/kg) or research grade LHRHa implants (pg/kg) were used. Low-producing line of channel
catfish was utilized.
LHRH Dose Environment N Ovulation % Fry/kg
10/50 bag 7 71.4 800
20/100 tank 7 28.6 502
20/100 bag 7 71.4 855
30/150 bag 7 57.1 1,736
75 implant tank 7 42.8 858
100 implant bag 7 71.4 1,704
75 implant bag 7 42.8 536
SRAC Eighteenth Annual Progress Report, December 2005 59
Improving Reproductive Efficiency to Produce Channel x Blue Hybrid Catfish Fry
Table 13. Comparison of early and peak season hybrid fry/kg female body weight for channel
catfish, Ictalurus punctatus, females fertilized with blue catfish, I. furcatus, sperm. GMP grade
LHRHa injections (priming dose/resolving dose, pg/kg) or research grade LHRHa implants
(pg/kg) were used. Low-producing line of channel catfish was utilized.
Fry/kg
LHRH Dose Early Peak
10/50 0 800
20/100 0 855
30/150 1,740 1,736
75 implant 492 858
100 implant 2,000 1,704
60 SRAC Eighteenth Annual Progress Report, December 2005
Table 14. Late season ovulation percentage for channel catfish, Ictaluruspunctatus, females
fertilized with blue catfish, I. furcatus, sperm. Females were ovulated individually in bags within
tanks without channel catfish males. GMP grade LHRHa injections (priming dose/resolving
dose, pg/kg) or research grade LHRHa implants (pg/kg) were used. Low- or high-producing
lines of channel catfish were utilized.
LHRH Dose Line N ovulation %
100 implant high 7.0 71.4
75 implant high 7.0 85.7
30/150 low 7.0 28.6
20/100 low 8.0 25.0
10/150 low 8.0 25.0
100 implant low 7.0 42.8
75 implant low 8.0 25.0
10/50 high 4.0 100.0
10/50 low 3.0 33.3
Improving Reproductive Efficiency to Produce Channel x Blue Hybrid Catfish Fry
Table 15. Egg quality and hybrid fry/kg female body weight during the early season for channel
catfish, Ictalurus punctatus, females fertilized with blue catfish, I. furcatus, sperm. Females were
ovulated communally in tanks. GMP grade LHRHa injections (priming dose/resolving dose,
pg/kg) or research grade LHRHa implants (pg/kg) were used. Low-producing line of channel
catfish was utilized. R = 0.23 (P > 0.05) between egg quality and fry/kg.
LHRH Dose N Egg Score Fry/kg
10/50 10 3.7 0
20/100 10 4.3 0
30/150 10 4.3 1,727
75 implant 10 4.2 492
100 implant 13 4.0 1,831
125 implant 10 3.6 955
Table 16. Egg quality and hybrid fry/kg female body weight during the peak season for channel
catfish, Ictalurus punctatus, females fertilized with blue catfish, I. furcatus, sperm. Females were
ovulated individually in bags. GMP grade LHRHa injections (priming dose/resolving dose,
pg/kg) or research grade LHRHa implants (pg/kg) were used. Low- producing line of channel
catfish was utilized. R=0.67 (P < 0.05) between egg quality and fry/kg.
LHRH Dose N Egg Score Fry/kg
10/50 7 2.5 800
20/100 7 3.3 855
30/150 7 3.9 1,736
100 implant 7 3.9 1,704
75 implant 7 4.1 850
SRAC Eighteenth Annual Progress Report, December 2005
Improving Reproductive Efficiency to Produce Channel x Blue Hybrid Catfish Fry
Table 17. Egg quality during the late season for channel catfish, Ictaluruspunctatus,
females fertilized with blue catfish, I. furcatus, sperm. Females were ovulated
individually in bags. GMP grade LHRHa injections (priming dose/resolving dose, pg/kg)
or research grade LHRHa implants (pg/kg) were used.
20/100 low 4 3.0
30/150 low 5 3.4
75 implant low 5 3.8
75 implant high 18 4.1
100 implant low 12 3.2
100 implant high 20 3.6
Table 18. Percentage of eggs good or bloody during the early (E), peak (P) and late
(L) season for channel catfish, Ictaluruspunctatus, females fertilized with blue
catfish, I. furcatus, sperm. GMP grade LHRHa injections (priming dose/resolving
dose, pg/kg) or research grade LHRHa implants (pg/kg) were used.
Dose Line % Good % Bloody
E P L E P L
10/50 low 30 16 63 63 -
10/50 high 0 0
20/100 low 40 13 25 40 81 75
30/150 low 37 33 20 57 58 100
75 implant low 46 44 60 58 56 80
75 implant high 40 55
100 implant low 39 29 42 47 37 75
100 implant high 40 35
125 implant low 32 50 -
62 SRAC Eighteenth Annual Progress Report, December 2005
Dose
Line
Egg Score
Improving Reproductive Efficiency to Produce Channel x Blue Hybrid Catfish Fry
Table 19. Percentage of eggs either "good" or "bloody" during the early (E), peak (P)
and late (L) season for channel catfish, Ictalurus punctatus, females fertilized with blue
catfish, I. furcatus, sperm. GMP grade LHRHa injections (priming dose/resolving dose,
pg/kg) or research grade LHRHa implants (tg/kg) were used.
Line
low
high
low
% White
Dose
10/50
10/50
20/100
30/150
75 implant
75 implant
100 implant
100 implant
125 implant
high
low
high
low
E P L
0 47
10
0 0 25
- 0
0
% Clumps
E P L
10 89 -
- 0
0 72 75
8 42 20
4 28 40
15
26 50 50
25
A second experiment was conducted during
the late spawning period comparing 75-.ig
implants with 10/50 injections. Ovulation rates
were not different, 91.4 and 91.6%, for
implants and injections, respectively. Hatch,
29%, was much higher for 75-tg implant than
for 10/50 injection,12% (Table 20). In a third
run, 28 out of 32 individual fish (87.5%)
implanted with 75-ig ovulated, with 75.6%
hatch.
Table 20. Effect of LHRH implants on the late season ovulation rate and hatch rate for
hybrid catfish embryos. Means differed significantly.
Dose N Delivery Ovulation (%) Hatch (%)
75 59 implant 91.4 29.0a
10/50 25 injection 91.6 12.0b
Egg quality data was measured subjectively quality or % good, bloodiness, whiteness and
on a 5-point scale. Traits included overall clumpiness, the latter three being negative
SRAC Eighteenth Annual Progress Report, December 2005 63
Improving Reproductive Efficiency to Produce Channel x Blue Hybrid Catfish Fry
traits. Early in the season there was little
difference in egg quality obtained from fish
with different dosages of injections or
implants. There was a relationship between
subjective egg quality and hatch. Early in the
season the correlation between egg quality
and hatch was 0.23. By the peak spawning
period differences in egg quality emerged.
Egg quality was higher for implants than
injections. The correlation between egg
quality and hatch increased to 0.67.
In the late spawning season differences in egg
quality still existed. Again implants tended to
have higher mean values than injections.
Genetics impacted the results. High line females
had higher observed means than low lines.
Implanted fish had a more variable time of ovu-
lation, but females that ovulated up to 48 hours
later than the average female gave high quality
eggs, whereas late ovulating injected females
give over ripened eggs. The advantage of the
implants is greatest late in the spawning season.
Latency period after initial injection was
longer in the early season at lower
temperatures. Higher doses tended to give
shorter and more uniform latency periods.
Injections produced shorter latency periods than
implants. As the season progressed and the
water warmed, these differences diminished and
the overall latency period shortened (Table 21).
By the late season, virtually no difference in
latency existed among treatments, including no
differences between injections and implants.
University of Memphis. Channel catfish
ovarian follicles were treated in vitro with 17a,
20p-dihydroxyprogesterone and human
chorionic gonadotropin in vitro. Initial efforts
have focused on screening for potentially
effective hormones to influence oocyte
maturation and ovulation. Evaluations have
included various culture media, hormonal
concentrations, and the timing of the application
of hormones. Methods are being investigated to
adequately evaluate the oocyte response to
various treatments. Such findings will hopefully
be applicable to the evaluation ofgonadotropins
used to induce spawning of eggs of high quality
from channel catfish broodstock.
Objective 2c. Improve hybrid embryo production via pheromonal
manipulation of channel catfsh males and blue catfish males for
improved ovulation, spermiation, egg quality, hatch and fry production.
Auburn University. Reducing handling and
stress of channel catfish females may be key
factors for effective production of channel
catfish female x blue catfish hybrid catfish
embryos. Females were either left free in
tanks or confined in bags or aquaria.
Confinement increased hybrid fry production
and reduced labor involved in the production
protocol. Exposure to the scent of conspecific
males increased, decreased or did not affect
hybrid fry production (Table 22, 23, and 24).
Method of exposure appears to have an effect.
Positive effects on hybrid fry production were
obtained when water from tanks containing
males is introduced, whereas visual or actual
contact appears to have negative effects
(Tables 23 and 24).
64 SRAC Eighteenth Annual Progress Report, December 2005
Improving Reproductive Efficiency to Produce Channel x Blue Hybrid Catfish Fry
Table 21. Mean latency period (hours standard deviation, SD) for female body weight for
channel catfish, Ictaluruspunctatus, females fertilized with blue catfish, I. furcatus, sperm.
Females were ovulated communally in tanks, individually in bags within tanks or individually in
aquaria with or without channel catfish males. GMP grade LHRHa injections (priming
dose/resolving dose, jtg/kg) or research grade LHRHa implants (pg/kg) were used. No resolving
dose treatments are implants. Batch = nutrition or injection (dose) experiments.
Date Batch Priming Resolving N latency SD
5/20 Injection 20 100 3 45.72 5.56
5/20 Injection 30 150 1 43.80
5/21 Injection 10 50 8 55.81 3.35
5/21 Injection 20 100 14 51.61 1.01
5/21 Injection 30 150 8 51.17 0.31
5/21 Injection 75 0 8 57.45 0.5
5/21 Injection 100 0 9 56.42 3.77
5/21 Injection 125 0 10 56.51 0.98
5/26 Nutrition 20 100 75 43.65 1.58
5/27 Nutrition 20 100 69 40.80 1.74
6/3 Nutrition 20 100 55 50.27 1.98
6/11 Injection 10 50 5 44.06 2.14
6/11 Injection 20 100 7 40.89 4.36
6/11 Injection 30 150 3 40.43 3.52
6/11 Injection 75 0 5 43.50 3.86
6/11 Injection 100 0 4 41.75 2.2
6/16 Nutrition 20 100 64 42.35 3.17
6/17 Nutrition 20 100 57 39.85 2.1
6/18 Nutrition 20 100 41 38.88 2.72
6/23 Injection 10 150 2 40.35 4.17
6/23 Injection 20 100 2 39.35 0.07
6/23 Injection 30 150 2 43.55 2.76
6/23 Injection 75 0 8 39.8 2.81
6/23 Injection 100 0 8 38.09 1.36
6/24 Injection 10 50 5 50.28 2.42
SRAC Eighteenth Annual Progress Report, December 2005 65
Improving Reproductive Efficiency to Produce Channel x Blue Hybrid Catfish Fry
Table 22. Mean eggs/kg female body weight (BW), hatching percentage, fry/kg female body weight
and egg quality of channel catfish females (Ictalurus punctatus) exposed or not exposed to channel
catfish male after injection with luteinizing hormone releasing hormone, LHRHa, when hybridized
with blue catfish (Ictalurusfurcatus) male (mean SD) in 2001.
Spawning Egg/kg Fry/kg Latency
Percentage Female Hatching Female Time Egg
Treatment (N=24) BW Percentage BW (hour) Quality
Unexposed 90a 30 6,822a 2,268 31. la 6.7 2,246a + 652 3la 5 3.3a 0.2
Exposed 100a 0 7,358a 1,756 40.5b 1.6 3,031b 1,028 30a 5 3.7b 0.1
Means followed by the same letter are not different (P>0.05) within each column.
Table 23. Mean spawning percentage, egg/kg female body weight (BW), hatching percentage,
fry/kg female body weight and latency time at 290C for channel catfish (Ictalurus punctatus)
females injected with luteinizing hormone releasing hormone, LHRHa, with different exposures
to channel catfish males (mean SD) in 2002.
Spawning Egg/kg Hatching Fry/kg Latency
Treatment Percentage Female BW Percentage Female BW Time (hour)
(N =10) (N =10) (N= 10) (N = 10) (N = 10)
30+ 150 low male 80a 42 9,368a 1,519 14.4a 0.64 1,351a 219 31a 0.10
30 + 150 no male 80a 42 8,288a 2,671 52.9b 0.45 4,384b 1413 31a 0.10
30 + 150 high male 90a 31 8,211a 3,882 23.2c 0.11 1,901a 899 32b 0.52
Means followed by the same letter are not different (P>0.05) within each column.
Table 24. Ovulation % and fry/kg female body weight for channel catfish receiving 100-jg
LHRHa implants either in direct contact with or not exposed to conspecific males and fertilized
with blue catfish sperm.
LHRH Dose (tg/kg) Environment N ovulation % Fry/kg
100 aquaria w/male 7 71.4 1,650
100 aquaria no male 6 66.6 1,831
100 bag no male 7 71.4 1,704
66 SRAC Eighteenth Annual Progress Report, December 2005
Improving Reproductive Efficiency to Produce Channel x Blue Hybrid Catfish Fry
Objective 2d. Develop extended refrigerated storage and
cryopreservation of sperm.
Louisiana State University. Knowledge of
sperm concentration is essential for
standardization of protocol for gamete
cryopreservation and for optimizing
fertilization in artificial spawning. Currently
there is a lack of information regarding sperm
concentration and how it relates to
cryopreservation and fertilization in
essentially all species including channel
catfish. Practical methods for evaluation of
sperm concentration in channel catfish are
needed. The specific objectives of this study
were to evaluate: 1) the use of a
spectrophotometry in determining sperm
concentrations; 2) sperm concentrations
relative to gonad composition, and 3) optimal
sperm concentration for fertilization during
artificial spawning.
Channel catfish were seined from ponds
during the months of April and May 2004.
The males were killed and total lengths and
weights were recorded. Testes were surgically
removed and were suspended in calcium-free
Hanks' balanced salt solution (C-F HBSS) at
290 mOsmol/kg. The testes were cleaned by
removing adherent blood and tissue, separated
into visually estimated anterior and posterior
sections, weighed, and crushed in C-F HBSS
(lg/20mL) to release sperm.
Sperm concentrations and motility estimates
relative to gonad composition are summarized
in Table 25. Sperm concentrations varied in
relation to gonad composition.
Table 25. Summary of sperm concentrations and motility from whole testis and posterior
and anterior sections.
Concentration (/mL)
Total Concentration
Sperm/g Testis
Intact 1.73 x108'9.4 x 107a 1.78 x 10'02.0 x 101'a 3.52 x1091.89 x 109a 354.5a
Posterior 1.06 x 1072.7 x 107b 1.41 x 108+2.37 x 108b 2.09 x 108+5.4 x 108b 234.6a,b
Anterior 3.13 x 1081.18 x 108c 1.42 x 10101.5 x 1010c 5.74 x 10'92.24 x 109c 414.6b
Means in a column with different letters were significantly different (P<0.05, n=21)
SRAC Eighteenth Annual Progress Report, December 2005 67
Motility
(%)
Improving Reproductive Efficiency to Produce Channel x Blue Hybrid Catfish Fry
Objective 3. Develop techniques to identify, assess and improve
gamete quality.
Objective 3a. Develop criteria for standardizing and classifying egg
quality prior to injection and after manual stripping and describe the
morphological and physiological condition of channel catfish eggs
including evaluation of morphological changes ofoocytes during
oocyte maturation in female catfish.
University of Memphis. Initial images of
catfish oocytes and embryos were made by
automated transparency scanners. Automated
transparency scanners imaged catfish oocytes
and embryos during oocyte maturation and
embryogenesis, respectively. This technology
was developed for analysis of motility mutants
in zebrafish (Computer-Aided-Screening, CAS)
and is being adapted for analysis of catfish
oocytes and embryos. Initial trials indicate that
CAS may be used to follow catfish embryos
throughout their 6 to 7 day period of
development to hatching. The CAS system
worked quite well in spite of the prolonged
development time for catfish embryos (i.e., 6 to
7 days versus 2 days for zebrafish).
Animations of time-lapse image stacks in
ImageJ revealed a surprising amount of cell
movement in cleavage stage embryos. Other
details of embryonic development included
gastrulation/epiboly, neurulation, initiation of
motility and hatching. Arrested development
and subsequent cytolysis of abnormal embryos
could also be clearly documented, including the
developmental events prior to arrest and death.
In collaboration with Dr. Terry Tiersch at LSU,
brood fish in ponds were subjected to elevated
water temperatures early in the year in an
attempt to induce early gonadal maturation and
spawning in 2005. Eggs were stripped from
female channel catfish and fertilized from sperm
obtained from blue catfish testes. Observations
were made on development and survival of the
progeny produced. Several hybrid and channel
spawns were obtained and imaged by Computer-
Aided Screening (CAS).
The initial analysis of two parallel runs (trials 1
and 2) showed a time window of mortality that
corresponded to approximately 2000 to 2900
minutes post-fertilization (33 to 48 hours post-
fertilization). Initial analysis of the CAS images
revealed embryos undergoing cytolysis as
expected (Figures 2 and 3). However, upon
closer examination, develop-ment was arrested
in some embryos and they failed to gastrulate,
yet they continued to survive. Cleavage-arrested
embryos continued to show these movements in
spite of failed development. Developmental
arrest is not necessarily followed immediately
by cytolysis and death. We are currently
examining this surprising finding in more detail.
Lambert, Small and Chatakondi have also
observed this window of critical development,
and treat-ment of embryos at this
developmental stage is discussed in Objective 4.
During this same time period, mortality occurs
in channel catfish embryos exposed to antisense
contracts designed to disrupt dorsal-lateral
orientation. The cause of this developmental
68 SRAC Eighteenth Annual Progress Report, December 2005
Improving Reproductive Efficiency to Produce Channel x Blue Hybrid Catfish Fry
arrest needs to be ascertained and corrected.
Embryo densities of 30 or more per well were
found to be deleterious to embryos imaged by
the CAS system even with flows greater than
10mL/hour. While embryo densities of 16 or
less embryos per well allowed complete
development to hatch in the CAS system.
In addition to cytolysis, developmentally-
arrested embryos may continue to survive and
superficially appear normal and viable.
However, careful imaging of groups of hybrid
embryos at various times after fertilization
revealed cleavage stage embryos persisting
until at least 72 hours post-fertilization
(Figure 4). This surprising result may explain
some of the variability observed in hatch rates
for hybrid catfish embryos.
Large ovarian follicles were obtained from a
single channel female and were tested for
germinal vesicle (GV) position and response
to progestogen. Application of 5% acetic acid
elicited clearing of the ooplasm and
visualization of the GV. The dynamics of the
clearing process was determined and imaged
using CAS (Figure 5). GV identity was
verified by presence of numerous nucleoli
upon microscopic examination (Figure 6).
Additional images of catfish oocytes and
embryos were made by automated transparency
scanners. Daily use of 20-gL 4% formalin per
well provided protection from parasitic in-
festations that prevented complete development
of catfish embryos within the CAS system
previously. We have begun high resolution
imaging of fixed embryos to develop an embryo
staging table for channel and hybrid catfish.
A test of 0.5% bovine serum albumen (BSA)
as an egg extender in Hank's salts was found
to be less effective than Hank's salts alone for
hybrid catfish from the USDA-ARS
Stoneville, MS, facility. The CAS system
worked quite well in spite of the prolonged
development time (i.e., 6-7 days).
We are currently extracting data from stacks of
scanned images from 1) catfish oocytes treated
with various media and hormones, and 2) analy-
sis of embryonic development. We also plan to
screen catfish oocyte and ovary extracts for
reaction with cell-cycle control protein anti-
bodies (e.g., anti-cyclin B 1) that may prove use-
ful in our studies ofoocyte maturation in catfish.
Louisiana State University. Ultrasound is a
user-friendly technology capable of creating
ultra-clear images that can be captured as
movies or still images. Ultrasound has been
used extensively in human medicine and
livestock species, but has had limited
application in finfish. This non-invasive
technique has been used with female livestock
to monitor follicular growth through ovu-
lation. It has also been used as a tool for sex
identification and carcass evaluation in several
species offish (e.g., Atlantic salmon, Atlantic
halibut, striped bass, shovelnose sturgeon, and
barfin flounder). The objectives of this study
were to evaluate visibility of gonads at
different life stages, ovarian development in
strip spawned and non-spawning females,
oocyte diameter, compare ultrasound measure-
ments to physical measurements, classify
females prior to hormone injection, identify the
time to strip eggs after injection, and determine
the efficacy of stripping by use of ultrasound in
channel catfish.
SRAC Eighteenth Annual Progress Report, December 2005 69
Improving Reproductive Efficiency to Produce Channel x Blue Hybrid Catfish Fry
trial 1 data
35
S- hybrid CF channel CF
30
25 -........ .. .
& 20
i"
10
5
0
minutes poet fertlllzation
Figure 2. Viability of hybrid catfish and channel catfish embryos (trial 1) using CAS.
trial 2 data
70 SRAC Eighteenth Annual Progress Report, December 2005
10
6
0
0 16 36 66 766 96 11U6 1366 1566 1766 1966 2166 2366 2666 2766 2966 3166 3366 366
minute poet fertilitlon
Figure 3. Viability of hybrid catfish and channel catfish embryos (trial 2) using CAS.
Improving Reproductive Efficiency to Produce Channel x Blue Hybrid Catfish Fry
Figure 5. Channel catfish oocytes imaged by CAMMA and treated with 5% acetic acid.
This treatment clears the yolky ooplasm and reveals the presence and location of the
oocyte nucleus or germinal vesicle (GV).
SRAC Eighteenth Annual Progress Report, December 2005 71
Figure 4. Digital photomicrographs ofdechorionated, formalin-fixed hybrid catfish embryos
Embryos may arrest development in the cleavage stage but not immediately cytolyze.
72hr normal
Improving Reproductive Efficiency to Produce Channel x Blue Hybrid Catfish Fry
During February through June, 2004, channel
catfish gonads were evaluated at three different
life stages: fingerlings (under 0.4 kg), market-
sized food fish (0.4 to 0.8 kg), and brood stock
(more than 1.5 kg). Fish were scanned using a
linear ultrasound probe (3 to 10 mHz), and
gonadal sex was verified by dissection. To
evaluate ovarian development, 12 females
were given injections of artificial luteinizing
hormone-releasing hormone. Of these, five
were strip-spawned. Fish were scanned daily to
monitor gonadal development.
Gonads were correctly identified as testis or
ovary for fingerlings (57%), food fish (90%),
and brood stock (86%). Immature gonads were
difficult to distinguish from surrounding
tissues. Mature testes were partially visible,
but we could not quantify their development
due to lack of contrast with surrounding
tissues. Unlike testes, mature ovaries were
easily distinguished and their development
quantified by measuring ovarian diameter,
calculating the ratio of ovarian diameter to
body wall diameter (OD:BD), and measuring
oocyte diameter. There were no significant
differences in ovarian diameter or in OD:BD
between strip-spawned and non-spawning
females (Table 26). Strip-spawned females
had significantly larger oocyte diameters than
non-spawning females on days 3 and 4 after
injection. The results indicate that
ultrasonography could be a useful tool for
monitoring ovarian development in channel
catfish. This could be used in artificial
spawning of large groups of females, such as
in production of hybrids of channel catfish
females and males of blue catfish.
72 SRAC Eighteenth Annual Progress Report, December 2005
Figure 6. Isolated
channel catfish oocyte
germinal vesicle (GV)
after 5% acetic acid
fixation. Note the large
number of characteristic
nucleoli (n), a definitive
set of landmarks for the
GV.
Improving Reproductive Efficiency to Produce Channel x Blue Hybrid Catfish Fry
Table 26. Ovarian and oocyte development of strip-spawned (N = 5) and non-spawning (N = 7)
females after hormone injection. Daily means for strip-spawned and non-spawning fish within
each variable that share letters were not significantly different (P<0.01).
Ovarian Diameter (mm)
Day Strip- Non-
spawned spawning
OD:BD*
Strip-
spawned
Non-
spawning
Oocyte Diameter (mm)
Strip- Non-
spawned spawning
1 54.7 5.8a 54.2 7.4a 0.87 + 0.03a 0.84 0.05a 1.8 0.4a 1.8 0.5a
2 65.8 + 5.1a 50.4 + 9.1a 0.89 0.03a 0.85 0.04a 1.9 + 0.4a 1.9 0.5a
3 70.6 + 6.6a 63.9 + 7.4a 0.89 0.03a 0.88 0.05a 2.2 0.5a 1.8 0.4b
4 60.6 + 0.0a 64.3 + 11.3a 0.90 0.OOa 0.87 0.06a 2.0 + 0.4a 1.8 0.5b
*Ovarian diameter : Body wall diameter
During January to June of 2005, 234 channel Females were scanned at 2-8 hour intervals to
catfish females were scanned in situ using a 3 identify the proper time to strip eggs. After
to 10 mHz linear probe on a laptop ultrasound the fish were stripped, scanning was repeated
(TELAVET 1000, Classic Medical, Tequesta, to determine the efficacy of stripping.
Florida). Ultrasound measurements were com-
pared with physical caliper measurements of Ultrasound measurements were not
the body cavity and oocytes diameters for significantly different from the physical
15 female catfish. Conditioned females measurements (Table 27). With ultrasound,
(N=210) were seined from ponds, scanned by a trained technician could classify fish prior
ultrasound, and classified as "already spawned," to injection within 10 seconds. Identifying
"poor ovarian development," or "good ovarian the time to strip females involved several
development." Seventy-two females (6 sets of factors including a decrease in the space
12) classified as having "good ovarian devel- between the body cavity and the ovaries and
opment" were given injections of luteinizing an increase in ovarian edema (black areas)
hormone-releasing hormone analog (Peninsula that were coincidental to ovulation of eggs
Laboratories Inc., San Carlos, California). into the lumen of the ovary.
Table 27. Ultrasound and physical diameter measurements (mean SE) for channel catfish
females (N=15). Means sharing a letter within each variable were not significantly different
(P< 0.05).
Diameter Ultrasound measurement (mm) Physical measurement (mm) % Difference *
Body cavity 70.4 1.0a 68.4 2.0a -3
Oocyte 1.7 0.1a 1.9 0.1a 9
*[(Physical measurement Ultrasound measurement)/ Physical measurement] x 100
SRAC Eighteenth Annual Progress Report, December 2005
Improving Reproductive Efficiency to Produce Channel x Blue Hybrid Catfish Fry
Using these criteria, a trained technician could
identify when to strip eggs during induced
spawning by classification of eggs as "under-
ripe," "ripe" and "overripe." After fish were
stripped, ultrasound determined if a second
stripping was necessary. The ability to identify
proper timing of collection of ripe eggs from
females would increase efficiency of hybrid
catfish production by improving egg quality and
possibly increasing numbers of fry produced.
Auburn University. The variation in the
timing of egg collection and the act of
manually collecting eggs may impact the
quantity and quality of eggs obtained. The
ovulation process and factors that might affect
egg quality was examined.
Female channel catfish were held individually
in aquaria and induced to spawn using LHRHa
at 120 jg/kg body weight. Fish were monitored
hourly as ovulation approached. The time of the
first egg release into the aquaria was noted. Fish
releasing eggs were randomly assigned to one
of four treatments: stripped at 1 hour post-egg
release, sacrificed at 1 hour and the ovary
collected, stripped at 4 hours post-egg release,
and sacrificed at 4 hours and the ovary
collected. An additional set of injected females
were paired with male channel catfish and
allowed to spawn naturally. Data on egg
quantity and quality was collected for each
female including number of eggs/g of eggs, egg
diameter, fecundity and viability 48 hours post-
fertilization. Samples of un-ovulated eggs and
ovulated eggs from fish manually stripped at
different times were collected and frozen for
biochemical analyses.
The gonadosomatic index (GSI) index of
females at the time of first egg release averaged
21.31%. The time of stripping after the first
egg release had little effect on the quantity or
quality of eggs obtained. Fish stripped at
approximately 2 hours after the first egg release
had a mean fecundity of 6,374 2,111 eggs/kg
compared to fish stripped at 4 hours of 7,086
3,330 eggs/kg. The number of eggs/g egg mass
(42.5 9.4 and 41.1 + 12.9) were the same for
the two times of stripping groups. Egg diameter
averaged 3.51 mm for both groups. Likewise
egg viability at 48 hours post-fertilization did
not differ between strip times, 85.1 12.6 and
87.0 9.4 respectively. No differences were
seen in the characteristics of the first set of eggs
stripped from a female and the last set of eggs
stripped. Fecundity of same-age females
induced to spawn and either manually stripped
or which spawned naturally were similar, 7,804
2,954 and 6,166 4,570, respectively.
Objective 3b. Determine the profile ofestradiol hormone from serum
plasma of 2-year-old females of channel catfish over a 12-month
period, determine changes in oocyte maturation during vitellogenesis
and identify the different cathepsins that are responsible for
vitellogenin degradation and oocyte maturation in female catfish.
Mississippi State University. The catfish
industry is hampered by a chronic inefficiency
resulting from the low spawning success of
female brood stock for the annual production
74 SRAC Eighteenth Annual Progress Report, December 2005
Improving Reproductive Efficiency to Produce Channel x Blue Hybrid Catfish Fry
of fingerlings. Current estimates of spawning
success of females range from 20 to 30%. An
understanding of the relationship of annual
changes in physiological indices during a
reproductive cycle to oocyte maturation and
successful spawning in channel catfish may
contribute to an accurate prediction of
successful spawns. The objective of this
study was to evaluate the effects of plasma
steriod concentrations (estradiol and testos-
terone), egg size and protein degredation by
cathepsins D, L and B on in vivo egg
maturation in four strains of channel catfish.
The first study of profiles of plasma
estradiol and testosterone concentrations,
size and protein content of eggs in 2- to 3-
year-old channel catfish has been completed
for four commercial strains of channel
catfish, Gold Kist (2 strains), Thompson and
NWAC103 for one year (age 2 to age 3).
This study also included the first
measurements of the activities of the
proteolytic activities of cathepsins D, L, and
B and their relationships to other processes
involved in oocyte maturation. All of the
parameters collectively evaluated may serve
to assist in the selection of the best 2-year-
old channel catfish female brood stock, and
to determine the optimal timing of
treatments of hormone injection to increase
reproductive performance.
Groups of nine, 2-year-old female channel
catfish brood stock obtained from each of
four different strains/sources were tagged
and stocked into four 0.1-acre earthen ponds
(36 fish per pond, 9 fish per strain) in April.
Blood and egg samples were collected from
twelve fish in each pond (3 fish/strain) every
month for 11 and 9 months, respectively,
for blood and eggs. No individual fish
within a strain was subject to sampling
more than once every four months.
Great variation among individuals of the
same strain precluded the identification of
any significant, strain-specific differences
for the variables under investigation. For all
strains, mean plasma estradiol con-
centrations ranged from 0.02 to 0.29 ng/mL
from June through December, and in-
creased dramatically in January, peaking in
February (3.4 to 3.7 ng/mL), and remained
above 1.00 ng/mL through May.
RESUL TS ATA GLANCE...
SThe effects and interactions of
plasma steriod concentrations
(estradiol and testosterone), egg
size, protein content of eggs and
protein degradation by cathepsins D,
L and B on in vivo egg maturation
channel catfish was determined for
the first time.
Mean plasma testosterone concentrations
increased from May through September (0.03
to 1.23 ng/mL), decreased in October, and
then increased and remained at approximately
1 ng/mL through April. When variables from
fish of all strains were collectively evaluated
over time, concentrations of both plasma
estradiol and testosterone significantly in-
creased in July and again later from February
to May. The increase in hormone concen-
tration was accompanied by oocyte growth
SRAC Eighteenth Annual Progress Report, December 2005 75
Improving Reproductive Efficiency to Produce Channel x Blue Hybrid Catfish Fry
and increases in proteolytic activity of
specific cathepsins, supporting the role of
estradiol in regulating vitellogenesis.
During oocyte development, there were
sequential relationships among hormone
concentration, cathepsin activity, protein
content, and predominant oocyte proteins.
Plasma levels of vitellogenin gradually
increased from February and peaked in May.
Vitellogenin was enzymatically broken down
into smaller protein units by cathepsins L, D,
and B that individually predominated during
different months when different stages of
oocyte development occurred.
Mean activities of cathepsins D and L
steadily increased beginning in October and
were highest in March, whereas the activity
of cathepsin B was variable from month to
month. High levels of activity of cathepsin L
occurred during February and March,
suggesting its important role in protein
degradation during that time, while peak
activity of cathepsin B occurred during
November to January. Activities of
cathepsin D were the highest recorded,
peaking in March, April, and May.
Cathepsin B is more important in oogenesis
or early vitellogenesis, cathepsin L assumes
a principal role during middle vitellogenesis,
and activity of cathepsin D peaks during late
vitellogenesis.
Mean protein content of eggs was highest in
October (3.08 to 3.795) when eggs appeared
and decreased to levels of 0.54% to 2.14%
for the remainder of the year (November
through April) when eggs were present.
From October to November the mean egg
size increased by approximately 40%, to
1.0 to 1.4 mm, and remained at this size
until May and June when size increased by
approximately 75% to 100%.
Twenty hours subsequent to the injection of
fish with either carp pituitary hormone or
luteinizing hormone releasing hormone,
plasma estradiol and testosterone concen-
tration increased, activities of cathepsins L,
D, and B increased, and egg size and
protein content increased. These changes
stimulated oocyte maturation. The
percentages of spawning obtained were
18.8% of LHRH injected fish, 12.4% of
CPE injected fish, 9.4% of fish not injected,
and 0% of saline injected fish.
Injection of females with LHRH can
potentially serve as a tool to increase
spawning success in appropriate commer-
cial settings, particularly for improving 3-
year-old catfish spawning success early in
the spawning season. Low levels of plasma
estradiol in all 3-year-old fish suggest that
insufficient stimulation of vitellogenin
production by estradiol may underlie the
lack of vitellogenin incorporation into
developing oocytes. Sufficiently high
peaks in estradiol concentration in July
likely indicate a reproductively mature
female. This information should serve as a
foundation to apply in the evaluation of the
relative effectiveness of exogenous
hormone treatments in increasing the
spawning success of channel catfish for
producing both intraspecific and
interspecific embryos.
76 SRAC Eighteenth Annual Progress Report, December 2005
Improving Reproductive Efficiency to Produce Channel x Blue Hybrid Catfish Fry
Objective 3c. Develop in vitro assays to evaluate sperm quality and
evaluate their predictive ability in relation to fertilization and hatch.
Louisiana State University. Channel catfish
were seined from ponds during the months of
April and May 2004. The males were killed
and total lengths and weights were recorded.
Testes were surgically removed and were
suspended in calcium-free Hanks' balanced
salt solution (C-F HBSS) at 290 mOsmol/kg.
The testes were cleaned by removing adherent
blood and tissue, separated into visually
estimated anterior and posterior sections,
weighed, and crushed in C-F HBSS (1 g/20
mL) to release sperm. The sperm solutions
were poured through a 100-jm filter into a
50-mL conical tube. Sperm motility was
estimated after activation with deionized
water and concentrations were calculated
using duplicate hemacytometer counts.
Optical density of the sperm solutions was
measured using absorbance readings obtained
by spectrophotometry (Spectronic 20
Genesys) at wavelengths of 400, 450, 500,
550 and 600 nm.
The most accurate absorbance readings for
determining sperm concentrations from whole
testis occurred at 500 nm (y=29+1.99,
R2=0.531). These results indicate that
spectrophotometric assays can be used to
determine sperm concentrations from crushed
testis of channel catfish.
Objective 4. Develop economically viable standardized hatchery
procedures and fertilization protocols to optimize hatching rate of
hybrid embryos.
Objective 4a. Determine optimal sperm (fresh, frozen and
refrigerated)-to-egg ratios for fertilization and hatch.
Louisiana State University. The
production of hybrid catfish fry is limited
by factors including the inefficient use of
sperm from the male blue catfish. The
time, effort and expense involved in rearing
a blue catfish male to maturity requires
efficient use of the sperm obtained when
the male is killed. The objectives of this
study were to evaluate the effects of
concentration on refrigerated and
cryopreserved blue catfish sperm for: 1)
sperm motility, 2) fertilization of channel
catfish eggs, and 3) hatch of hybrid fry.
In 2004, only channel catfish males were
available, and they were used for experi-
mentation. Channel catfish were seined from
ponds during the months of April and May
2004. The males were killed and total
lengths and weights were recorded. Testes
were surgically removed and were
suspended in calcium-free Hanks' balanced
salt solution (C-F HBSS) at 290
mOsmol/kg. The testes were cleaned by
removing adherent blood and tissue,
separated into visually estimated anterior
and posterior sections, weighed, and crushed
SRAC Eighteenth Annual Progress Report, December 2005
Improving Reproductive Efficiency to Produce Channel x Blue Hybrid Catfish Fry
in C-F HBSS (Ig: 20 mL) to release sperm.
The sperm solutions were poured through a
100-jm filter into a 50-mL conical tube.
Sperm motility was estimated after
activation with deionized water and
concentrations were calculated using dup-
licate hemacytometer counts. The solutions
were diluted to contain 1x108, 1xO17 and
lxl06 sperm cells/mL and were used for
fertilization during artificial spawning with
eggs from two females and sperm from
3 males (0.5 mL/400 eggs). The sperm
concentration of 1xl06 yielded 71 16%
fertilization for fresh sperm (3 5% for
thawed sperm); 1xl07 yielded 88 9%
fertilization for fresh sperm (45 37% for
thawed), and 1 x 10 yielded 91 10% fertili-
zation for fresh sperm (48 55% for
thawed). The varied concentration of sperm
used for artificial spawning yielded
significant differences in fertilization
(P<0.05) and there is a correlation between
sperm concentration and fertilization.
Six sexually mature male blue catfish were
killed and their testes were surgically
removed during the 2005 spawning season
(May and June). The testes were cleaned of
excess blood and tissue, weighed and placed
in Ziploc bags containing 1:10 testiss
weight:volume of extender) Hanks' bal-
anced salt solution prepared without calcium
and magnesium. The testes were crushed
and poured through a 100-[pm filter. Sperm
motility was estimated and concentrations of
the samples were determined using
hemacytometer counts. The initial sperm
dilutions were divided into three groups.
The first group was used as a control at the
original concentration (3.6x108 4.6x107
cells/mL). The remainder was diluted to final
concentrations of 1x108, lxlo7 and 1xl06
cells/mL. The control and diluted samples
were divided into two aliquots; one was
cryopreserved at Genex Custom Collections,
Inc. and the other was stored in a
refrigerator (4C) until used for fertilization.
Eggs were stripped from gravid female
channel catfish that had been injected with
100 jtg/kg of synthetic luteinizing hormone-
releasing hormone (Peninsula Laboratories
Inc., San Carlos) for artificial spawning. A
monolayer of eggs was poured into 100-mL
cups and fertilized using either one 0.5-mL
straw of cryopreserved sperm or 0.5 mL of
refrigerated sperm at the three
concentrations. The fertilized eggs were
placed into cups in a hatching trough for
incubation. Neurulation was used as a
conservative measurement of fertilization
and was estimated in all cups at 24 hours
after fertilization. The number of hatched
fry was recorded at 120 hours after
fertilization.
There was a significant difference in motility
across the various concentrations and
between refrigerated and cryopreserved
sperm. In addition, there was a significant
difference in neurulation, although there was
no significant difference in hatch across the
various concentrations (Table 28). Given
that current hatchery practice is to use sperm
RESUL TS ATA GLANCE...
SSperm concentrations can be reduced
in currently used fertilization protocols
by 100-fold with little reduction in
subsequent hatch rate.
78 SRAC Eighteenth Annual Progress Report, December 2005
II
jiL.._
II
Improving Reproductive Efficiency to Produce Channel x Blue Hybrid Catfish Fry
dilutions prepared around 1:10
(weight:volume), this study suggests that
refrigerated and cryopreserved blue catfish
sperm can be diluted considerably (100
times greater) without reducing fertilization
of channel catfish eggs.
Objective 4b. Determine the effects of commonly used therapeutics on
hatching success.
USDA-ARS. The chemotherapeutic and
respective concentration yielding the greatest
hybrid hatching success was identified. Four
hybrid catfish egg masses were each divided
into thirteen equal sub-masses. Each sub-mass
was subjected to once daily chemotherapeutic
treatment as a 15-minute static bath until
eyed. The treatments were as follows:
(1) Control (no treatment), (2) 125 ppm
hydrogen peroxide, (3) 250 ppm hydrogen
peroxide, (4) 500 ppm hydrogen peroxide, (5)
50 ppm formalin, (6) 100 ppm formalin, (7)
200 ppm formalin, (8) 50 ppm povidone
iodine, (9) 100 ppm povidone iodine, (10) 200
ppm povidone iodine, (11) 2.5 ppm copper
sulfate, (12) 5 ppm copper sulfate, and (13) 10
ppm copper sulfate. Egg masses were allowed
to hatch to completion within individual
containers. When hatching was complete, the
fry were siphoned into a graduated cylinder
and the volume of fry recorded. The total
number of fry was calculated after
determining the number of fry in 1 mL then
multiplying times the total volume of fry
collected. Hatching success was calculated as
the percentage of eggs hatched.
Hatching success was high in the untreated
controls (82.8%) and highly variable within
treatments. Overall, hatching success was
not significantly improved with chemo-
therapeutic treatments; however, a tendency
SRAC Eighteenth Annual Progress Report, December 2005 79
Table 28. Mean neurulation and hatch rate for channel catfish x blue catfish hybrid
embryos fertilized with either refrigerated or cryopreserved sperm at undiluted
(approximately 4 x 10, 1 x 108, 1 x 10', or 1 x 106 sperm/400 eggs. Data for refrigerated and
cryopreserved sperm treatments were pooled as no significant differences were found
between these two sperm preparations. Means within a column sharing a letter were not
significantly different, Duncan's Multiple Range Test (P > 0.05).
Sperm/ Mean SD
400 eggs Neurulation Hatch
Control 70 14a 55 17a
1 x 108 67 18ab 58 16a
Sx 107 64 16ab 51 21a
1 x 106 61 lib 47 15a
Improving Reproductive Efficiency to Produce Channel x Blue Hybrid Catfish Fry
toward increased hatching success was
observed among eggs treated with 100 ppm
formalin (87.7%), 100 ppm iodine (88.1%),
and 2.5 ppm copper sulfate (87.0%). A
The optimal treatment frequency for maxi-
mizing hybrid hatching success was
determined. Formalin is the most common
therapeutant used to treat catfish egg
diseases, and formalin yielded one of the
highest hatching success rates in the first
experiment. For these reasons, formalin was
chosen as the therapeutant for this
experiment. Four trials were conducted with
four egg masses per trial to determine the
optimal frequency of formalin application
for maximizing hatching success. Formalin
treatments were administered 0, 2, 3, or 4
times daily as a 100 ppm static bath. Egg
masses were allowed to hatch to completion
within individual containers. When hatching
was complete, the fry were siphoned into a
significant decrease in percent hatch was
observed in eggs treated with 500 ppm
hydrogen peroxide (Figure 7).
RESULTS ATA GLANCE...
i The frequency of formalin treatments
should be three per day to maximize
hatch rate of hybrid embryos. Four treat-
ments per day is excessive. At 28"C,
hybrid embryos are chemically sensitive
to formalin between 42 to 46 hours post-
fertilization, and formalin treatments
should be avoided during this period.
graduated cylinder and the volume of fry
recorded. The total number of fry was
calculated after determining the number of
fry in 1 mL then multiplying times the total
80 SRAC Eighteenth Annual Progress Report, December 2005
120.0 b
b b b b b
100.0 b b
bab
8 0.0
40.0 -
20.0
0.0
Treatment
Figure 7. Mean hatching success (%) of hybrid catfish eggs treated daily with increasing doses of
hydrogen peroxide (HP), formalin (F), povidone iodine (I), or copper sulfate (CS). Numerals refer
to dosage rate (ppm). Bars with common letters are not statistically different.
Improving Reproductive Efficiency to Produce Channel x Blue Hybrid Catfish Fry
volume of fry collected. Hatching success
was calculated as the percentage of eggs
hatched. The optimal frequency of formalin
treatments was determined to be three times
daily (Table 29).
Table 29. Effect of daily formalin treatment frequency on hybrid hatching success.
Frequency of daily formalin treatments
Ox 2x 3x 4x
Percent hatch 12.7 4.5a 31.4 4.6b 51.6 3.6c 33.7 4.6b
Means followed by different letters are statistically different (P<0.05).
The effect of withholding formalin treatment potentially sensitive developmental period,
during a putative sensitive developmental formalin treatments (100 ppm) were ad-
stage on hybrid hatching success was deter- ministered three times daily such that treat-
mined. A preliminary study was conducted to ments occurred at 42 hours post-fertilization
ascertain the developmental stage at which (control) or were withheld from 42 to 44, 42 to
mortality most often occurs in hybrids. Briefly, 46, or 42 to 48 hours post-fertilization.
hybrid eggs were collected throughout Hatching success was calculated as previously
development, cleared in Stockard's solution described. Formalin treatments administered at
and microscopically elevated for develop- 42 hours post-fertilization significantly re-
mental differences indicative of egg mortality, duced hatching success. Withholding treat-
At 280C, mortality was observed between 42 ments until 46 hours post-fertilization at 28C
and 46 hours post-fertilization. To determine yielded the greatest percent hatch (Table 30).
the effect of withholding treatments during this
Table 30. Effect of formalin treatments administered at 42 hours post-fertilization
(control) or withheld from 42 to 44, 42 to 46, or 42 to 48 hours post-fertilization on
hybrid hatching success at 280C.
Time of formalin treatment (hours post- fertilization)
42 h 44 h 46 h 48 h
Percent hatch 19.6 + 5.3a 30.7 11.0b 58.3 3.9c 34.1 8.5b
Means followed by different letters are statistically different (P<0.05).
SRAC Eighteenth Annual Progress Report, December 2005 81
Improving Reproductive Efficiency to Produce Channel x Blue Hybrid Catfish Fry
WORK PLANNED
Louisiana State University initiated, reported
and plans to continue to evaluate ultrasound
as a means to evaluate female gonadal
development. The University of Memphis
initiated, reported and plans to continue to
evaluate various hormones in vitro for
stimulating oocyte maturation. These experi-
ments were not part of the original work
plan, and are being conducted in addition to
the original work planned.
IMPACTS
Early spawning can be accomplished by
heating water prior to the natural spawning
season without any difference in success
compared to the natural spawning season.
When 100 degree hours are reached ovulation
and fertilization should be successful. Feeding
standard 32% protein floating catfish feed 6
times per week for 2 months prior to spawning
gives equal or better fry production compared
to high protein diets. Supplementation of
brood fish diets with menhaden fish oil, DHA
and ARA two months prior to spawning can
double hybrid fry output. Utilization of the
appropriate genetic line of channel catfish
female can double and triple hybrid fry output.
Injections of 30ug/kg female body weight of
LHRHa followed by 150 jtg or the utilization
of 100 p.g/kg implants generates the greatest
number of fry/kg during the early and peak
spawning periods. Hatch rate of hybrid
embryos is improved if channel catfish
females are stripped within 2 hours of first
observation of egg release. Ultrasound may be
used to ascertain ovulation in channel catfish
females and the appropriate time for stripping
of eggs. Spectrophotometric assays can be
used to determine sperm concentrations from
crushed testis of catfish. Utilization of this tool
should result in more efficient use of sperm,
and more consistent fertilization rates. Sperm
concentrations can be reduced 100-fold, from
2 x 108 to 2 x 106 sperm/800 eggs, and still
obtain good hatch rates. The cost for the sperm
savings is a reduction of 16% for relative
hatch rate. The frequency of formalin
treatments should be three times per day to
maximize hatch rate of hybrid embryos and
four treatments per day is excessive. At 280C,
hybrid embryos are chemically sensitive to
formalin between 42 to 46 hours post-
fertilization, and formalin treatments should be
avoided during this period to maximize hatch
rate.
82 SRAC Eighteenth Annual Progress Report, December 2005
Improving Reproductive Efficiency to Produce Channel x Blue Hybrid Catfish Fry
PUBLICATIONS, MANUSCRIPTS, OR PAPERS PRESENTED
Publications
Small, B.C. and N. Chatakondi. 2006. Efficacy of formalin as an egg disinfectant for improving hybrid
catfish (Ictalurus punctatus x I. furcatus) hatching success. North American Journal of
Aquaculture. In press.
Doctoral Dissertations
Barrero-Monzon, M. 2005. Plasma steroid and vitellogenin concentrations and activity of cathepsins
during oocyte maturation, and the influence of hormone injection in four commercial strains of
channel catfish (Ictalurus punctatus) Ph.D. Dissertation. Mississippi State University, Starkville,
MS.
Kristanto, A. H.2004. Evaluation of various factors to increase the efficiency of channel-blue hybrid
catfish embryo production. Ph.D. Dissertation. Auburn University, AL.
Presentations
Ballenger, J., A. Hutson, D. Beam, G. Umali, A. Kristanto, M. Trask, M. Templeton, A. Davis, H.
Quintero, F. Wang and R. A. Dunham.2005. Effect of genetics on channel-blue hybrid catfish
embryo production. Special Session-Hybrid Catfish Reproduction. World Aquaculture Society
Meeting. New Orleans, LA. January 2005.
Barrero, M. L., R. D'Abramo, A. M. Kelly, L. A. Hanson, B. C. Small. 2005. Plasma steroid, cathpsin
activity and egg size and protein content during in vivo oocyte maturation in four strains of
channel catfish broodstock. Special Session-Hybrid Catfish Reproduction. World Aquaculture
Society Meeting. New Orleans, LA. January 2005.
Beam, D. A. Hutson, A. Kristanto, J. Ballenger, M. Templeton, G. Umali, F. Wang and R. A. Dunham.
2005. Effects of confinement in bags or aquaria and exposure to the scent of conspecific males
on the production of channel-blue hybrid catfish embryos by channel catfish females. Special
Session-Hybrid Catfish Reproduction. World Aquaculture Society Meeting. New Orleans, LA.
January 2005.
Bosworth, B. G. 2005. Comparison of carp pituitary extract, catfish pituitary extract, or LHRHa for
induced spawning of channel catfish females to produce channel catfish x blue catfish hybrid
fry. Special Session-Hybrid Catfish Reproduction. World Aquaculture Society Meeting. New
Orleans, LA. January 2005.
Campbell, W. T., P. Pawiroredjo, A. M. Guitreau, and T. R. Tiersch. 2005. Evaluating sperm
concentration in channel catfish. World Aquaculture Society Meeting. New Orleans, LA. January
2005.
Chatakondi, N., R. Yant and R. Dunham. 2005. Effects of age, strain and post-spawning protein levels in
diets of female channel catfish and male blue catfish in hybrid catfish fry production. Special
Session-Hybrid Catfish Reproduction. World Aquaculture Society Meeting. New Orleans, LA.
January 2005.
SRAC Eighteenth Annual Progress Report, December 2005 83
Improving Reproductive Efficiency to Produce Channel x Blue Hybrid Catfish Fry
Chatakondi, N., R. Yant, G. Umali, A. Kristanto and R. Dunham. 2005. Effective LHRHa dose based on
varying maturity of channel catfish (Ictalurus punctatus) to optimize channel catfish (female) x
blue catfish (male) hybrid fry production. Special Session-Hybrid Catfish Reproduction. World
Aquaculture Society Meeting. New Orleans, LA. January 2005.
Guitreau, A. M., B. E. Eilts, W. T. Campbell, P. Pawiroredjo, and T. R. Tiersch. 2005. Ultrasonography
for evaluation of gonads, gonadal development, and oocytes in channel catfish. Special Session-
Hybrid Catfish Reproduction. World Aquaculture Soc. Meeting. New Orleans, LA. January 2005.
Hutson A., Y. Zohar, E. Abraham, A. Kristanto, J. Ballenger, M. Templeton, G. Umali, D. Beam, F.
Wang, C. Ligeon, P. Waters,Jr., Z. Liu and R. A. Dunham. 2005. Evaluation of LHRHa delivered
via liquid injections or implants for the production of channel-blue hybrid catfish embryos.
Special Session-Hybrid Catfish Reproduction. World Aquaculture Society Meeting. New
Orleans, LA. January 2005.
Pawiroredjo, P. A., N. Mandhani, S. G. Hall, and T. R. Tiersch. 2005. Quantifying the thermal
requirements of catfish spawning. Special Session-Hybrid Catfish Reproduction. World
Aquaculture Society Meeting. New Orleans, LA. January 2005.
Phelps, R. P., R. Hasty, A. Pendetar, L. Linley and N. Papanikos. 2005. Effects of temperature and body
characteristics on the induced spawning of channel catfish and the production of channel x blue
catfish hybrid fry. Special Session-Hybrid Catfish Reproduction. World Aquaculture Society
Meeting. New Orleans, LA. January 2005.
Quintero, H. E., D. A. Davis, R. Dunham, R. P. Phelps, and A. Abebe. 2005. Evaluation of feeding
regime and protein content on egg quality and fecundity from induced channel catfish females.
Special Session-Hybrid Catfish Reproduction. World Aquaculture Society Meeting. New
Orleans, LA. January 2005.
Simco, B. and C. A. Lessman. 2005. Initial imaging of catfish oocytes and embryos by automated
transparency scanners. Special Session-Hybrid Catfish Reproduction. World Aquaculture Society
Meeting. New Orleans, LA. January 2005.
Small, B. C. and N. Chatakondi. 2005.Optimizing chemotherapeutic treatment of hybrid catfish eggs for
maximal hatching success. Special session-Hybrid Catfish Reproduction. World Aquaculture
Society Meeting. New Orleans, LA, January 2005.
Umali, G. M. and Rex A. Dunham. 2005. The economic significance of aquatic biotechnology in the
production of channel x blue catfish embryo. Special session-Hybrid Catfish Reproduction.
World Aquaculture Society Meeting. New Orleans, LA. January 2005.
84 SRAC Eighteenth Annual Progress Report, December 2005
Innovative Technologies and Methodologies for Commercial-Scale Pond Aquaculture
INNOVATIVE TECHNOLOGIES AND
METHODOLOGIES FOR COMMERCIAL-
SCALE POND AQUACULTURE
Reporting Period
August 1, 2004 August 31, 2005
Funding Level
Participants
Administrative
Advisor
Year 1 ............ .. $314,695
Year2 ................ $287,451
Year 3 ................ $213,168
Year4 ................ $170,096
Total ................. $985,410
Auburn University (Lead
Institution ............ Claude Boyd
Clemson University ....... David Brune, T. E. Schwedler
Louisiana State University .. John Hargreaves
Mississippi State University Doug Minchew, Charles Mischke, Filip To
University of Arkansas at
Pine Bluff ............ Carole Engle, David Heikes
USDA/ARS (Stuttgart) .... Bart Green
USDA/ARS (Stoneville) ... Les Torrans
Dr. Kenneth J. Roberts, Associate Vice Chancellor
LSU Ag Center
Baton Rouge, Louisiana
PROJECT OBJECTIVES
1. Evaluate new or improved production systems for channel catfish.
a. Continuous production and inventory control with the partitioned aquaculture system.
b. Installation of low-cost, semi-confinement systems in commercial-scale, earthen
ponds.
c. Fry and food fish production using in-pond raceways with the option for culturing
supplemental species in open-pond areas.
d. High-intensity production in heterotrophic-based culture units.
SRAC Eighteenth Annual Progress Report, December 2005
Innovative Technologies and Methodologies for Commercial-Scale Pond Aquaculture
2. Improve equipment to enhance culture.
a. Motor-powered U-tube aerator for commercial-scale channel catfish ponds.
b. Low-head, low-speed paddlewheel aerator for crawfish ponds.
c. Low-power, electrically-enhanced seine to harvest market-sized channel catfish
from commercial-scale ponds.
3. Assess energy, material, and economic efficiency of production systems.
a. Quantify energy, protein, and water use in traditional systems for channel catfish
culture.
b. Develop and evaluate economic and financial models of existing and improved
production practices and technologies.
ANTICIPATED BENEFITS
Aquaculture operations in the southeastern
United States find it increasingly difficult to
maintain profitability as production costs
increase but farm-gate prices remain low.
Solutions to the problem are complex and
multifaceted, but improved production
efficiency can decrease production costs and
improve the prospects for profitability. This
project will provide new technology for
production systems, aeration and harvesting
techniques, and use of energy, materials, and
capital. These technologies will be valuable
in improving the profitability of aquaculture
in the southeast.
PROGRESS AND PRINCIPAL ACCOMPLISHMENTS
Objective 1. Evaluate new or improved production systems for
channel catfish.
Objective la. Continous production and inventory control with the
partitioned aquaculture system.
Clemson University. Studies are being con-
ducted at Clemson University on continuous
production with inventory control in partitioned
aquaculture systems (PAS). On June 10, 2005
channel catfish fry were stocked in six cells,
6 feet x 10 feet x 4 feet deep, located within the
2-acre PAS system (Figures 1, 2, and 3). The
specific experiments for the 2005 research
focused on 1) physical holding and handling of
fry and fingerlings, 2) stocking density and
required water flow rates, 3) feed presentation
and food consumption, 4) growth response
86 SRAC Eighteenth Annual Progress Report, December 2005
Innovative Technologies and Methodologies for Commercial-Scale Pond Aquaculture
under raceway culture conditions as opposed
to an "accelerated" fingerling culture pond.
Six, 1,800-gallon PAS cells were stocked
with 5,000 fry (in three cells) and 10,000 fry
(in three cells) on June 10. The fry were
held in bins (2.5 feet x 1.5 feet x 1 foot
deep) with 1/16-inch mesh screens for one
week and then transferred to bins with 1/8-
inch mesh screens for an additional week.
After having reached an average size of 1.2
to 1.4 g, fingerlings were released into the
1/4-inch mesh net-pens held within the 6
feet x 10 feet PAS cells. Each cell was
supplied with water delivered by a 0.75-hp
submerged aerator providing between 75 to
190 gpm to individual cells (Figures 2 and
3). After initial stocking, fish were fed
starter feed of 52% to 56% protein supplied
using automated feeders (Figure 4).
SRAC Eighteenth Annual Progress Report, December 2005
Figure 1. Overview of 2-acre
Clemson PAS unit with
Fingerling Production Cells.
Figure 2. Six 60 ft2
Fingerling Production Cells
Containing Net-Pens and
Aerator-Driven Water Flow
Innovative Technologies and Methodologies for Commercial-Scale Pond Aquaculture
After 6 weeks, fingerlings had reached 11 to
14 g and hand feeding was initiated. At 7
weeks, fish in cells containing 10,000
fingerlings had reached 14 to 15 g, and were
moved to grow-out raceways in the 0.33-
acre PAS units (30 feet x 7 feet x 4 feet
deep). At the end of 8.5 weeks, fingerlings
had reached 27 to 32 g in units stocked at
5,000 per cell, and 20 to 22 g in cells
stocked at 10,000 per cell (Figure 5).
In addition to the fingerling culture trials
conducted within cells and raceways, experi-
ments were initiated to study the possibility of
using PAS cells and raceways to provide an
initial growth acceleration prior to stocking and
grow-out in conventional fingerlings ponds. A
conventional, 0.5-acre fingerling culture pond
as stocked with 34,000 fry (0.03 g/fish), while
17,000 fry of the same cohort were held in bins
for 2 weeks until reaching 1.4 g. They then
88 SRAC Eighteenth Annual Progress Report, December 2005
Figure 3. Individual
Fingerling Production Bins
Contained Within PAS Cells
with Aerator-Driven Water
Flow
Figure 4. Automatic Feeders
Used to Feed Fingerlings
During Initials Stages of
Culture
Innovative Technologies and Methodologies for Commercial-Scale Pond Aquaculture
were stocked into a conventional, 0.3-acre
fingerling culture pond. To date, the finger-
lings in both ponds are of similar size. This
suggests the importance of converting fry or
finger-lings to floating feed as quickly as
possible. Growth response in the "accelerated"
pond was delayed as a result of slow response
of the fish to hand feeding after being stocked
in the pond. In 2006, fingerlings will be moved
to floating feed on a faster schedule before
pond stocking.
Mississippi State University. Work in Year 1
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was devoted to retrofitting four, 1-acre ponds
as modified PAS ponds. The four ponds have
been surveyed and construction initiated.
Preliminary excavation has been completed
and gravel has been spread for the foundation.
The next phases consist of pouring concrete
foundations, laying concrete block walls,
building and installing paddlewheels, com-
pleting backfill and finishing dirt work, and
installing the hydraulic systems and electrical
hook-ups. The remaining construction should
be completed by spring of 2006.
Figure 5. Growth of Catfish
Fingerlings in PAS Cell 2
for First Eight Weeks of
2005 Season
Objective lb. Installation of low-cost, semi-confinement systems in
commercial-scale, earthen ponds.
University of Arkansas at Pine Bluff.
Five confinement systems were installed in
research ponds at the UAPB Aquaculture
Research facility to determine whether
physically separating fish by size group with
a pond confinement system will result in
improved yield, survival, feed conversion
ratio (FCR) and growth compared to
conventional multiple-batch pond culture.
This will be accomplished by installing a
SRAC Eighteenth Annual Progress Report, December 2005 89
Innovative Technologies and Methodologies for Commercial-Scale Pond Aquaculture
wire mesh barrier in existing earthen ponds.
This study consists of ten, 0.25-acre ponds;
five of these are control ponds and do not
have barriers. The five treatment ponds have
a 0.5 x 1.0-inch, PVC coated, wire mesh
barrier that partitions off one-third of the
pond. In the treatment ponds, fingerling
catfish are being raised in 1/3 of the pond
and larger carryover fish were stocked in the
remaining 2/3 of the pond. The fish in the
control ponds are allowed to co-mingle as in
traditional multiple-batch culture. The ponds
will be seined every 2 months during the
growing season and average weights will be
calculated. After harvest, survival and FCR
will be calculated. Yield, survival, FCR, and
growth will be compared among treatments
and controls with t-tests. If warranted a
partial budget analysis will be conducted.
Preliminary results from the first sampling
date are presented in the Table 1. Based on
the results obtained at the completion of the
current trials, a commercial-scale confine-
ment system will be installed and evaluated
on a commercial farm over the next year.
Table 1. Mean weights (g) of fingerlings and carryover fish in control and confinement
ponds. Values with the same letter in the row are not significantly different. All values
are mean SD.
Control Confinement
Fingerlings
Stocking (5/1/05) 25 Oa 25 Oa
Sampling (6/27/05) 53 4.1a 56 11.4a
Carryover
Stocking (5/1/05) 408 Oa 408 Oa
Sampling (6/27/05) 696 9.9a 708 27.9a
Objective Ic. Fry and food fish production using in-pond raceways
with the option for culturing supplemental species in open-pond areas.
Louisiana State University. A study will be ducted on a commercial farm, and a farm
conducted to evaluate the technical and had been selected for the effort. Unfor-
economic performance an in-pond raceway tunately, the producer was no longer able to
system for producing both fry and foodfish- continue in catfish farming. The LSU
sized channel catfish in raceways and principal investigator currently is seeking
various combinations of other species in the the cooperation of other producers to con-
open-pond areas. This study should be con- duct the research.
90 SRAC Eighteenth Annual Progress Report, December 2005
Innovative Technologies and Methodologies for Commercial-Scale Pond Aquaculture
Objective Id. High intensity production in heterotrophic-based
culture units.
United States Department of Agriculture- was 0.085 kg at stocking. Salt was added to
Pine Bluff. Funding for work at this location each raceway to ensure chloride concen-
does not begin until Year 2 of the main tration exceeded 100 mg/L. Fish were fed
project (2005-2006). However, work was daily to apparent satiation with a 32%
initiated in April 2005, despite the funding protein, floating feed. Dissolved oxygen,
year not being synchronized with the catfish- temperature and pH are measured daily, and
growing season. Hybrid (channel x blue) total ammonia nitrogen, nitrite, nitrate,
catfish were stocked into nine, 38-m3 race- soluble reactive phosphorus, total nitrogen,
ways on 13 April 2005. Fish were stocked at total phosphorus, and chlorophyll a concen-
25, 50, 75, 100, 125, 150, 175, 200, or 225 trations are measured weekly. Harvest is
fish/raceway. Mean individual fish weight planned for October-November 2005.
Objective 2. Improve equipment to enhance culture.
Objective 2a. Motor-powered U-tube aeratorfor commercial-
scale channel catfish ponds.
United States Department of Agriculture-
Stoneville. A prototype U-tube was
constructed and installed in a 1-acre pond at
the National Warmwater Aquaculture
Center, Stoneville, MS. The U-tube was
fabricated from a 36-inch-diameter
corrugated galvanized culvert (Figure 6) and
buried to a depth of 20 feet below the
bottom of a 1-acre pond. The unit was
powered by a 240-volt, 3-phase, 5-hp,
helical-geared Flender motor. The motor
was vertically mounted on a 36-inch-
diameter culvert elbow that was attached to
the tube with a 12-inch band clamp. The
motor turned a three-vane impeller attached
to a 24-inch long x 2-inch diameter
unsupported, steel shaft (Figure 7). Water
level was maintained at the top of the elbow.
The impeller speed was controlled by an in-
line, general purpose, open-loop vector, AC-
drive (Safetronics Model GP10). With an
impeller speed of 150 rpm at 60 Hz, the
motor drew 12.7 amps and produced 5.36
hp. The pump had an output of 8,300 gpm
(Table 2).
Pump efficiency increased as impeller speed
decreased, but both total output and water
velocity decreased. It was determined that
the higher velocity was necessary to entrain
the volume of air needed to optimize
performance. Air was provided by a 5-hp,
3-phase blower to diffusers located at or
below the mouth of the "down-leg" of the
U-tube, which was level with the pond
bottom and approximately 5 feet below the
water surface.
Oxygen transfer efficiency tests were
conducted using a variety of diffuser types
SRAC Eighteenth Annual Progress Report, December 2005
Innovative Technologies and Methodologies for Commercial-Scale Pond Aquaculture
and configurations. The optimum conditions
produced an increase in dissolved oxygen of
2.3 mg/L (outflow DO minus inflow DO)
and a standard aeration efficiency of 1.66
pounds 02/hp-hr. These results were
encouraging but less than desired for
commercial application.
Two problems were noted during testing of
the initial prototype. First, it was desired to
eliminate obstructions in the tube to enhance
water flow. Thus, the impeller shaft was
kept relatively short because it had no
supports near the end. This resulted in the
impeller being located slightly above the
bottom of the horizontal (discharge) end of
the elbow (Figure 7). As the air-to-water
ratio increased, back-flow from the pond
was observed. This decreased water flow
through the tube, and at higher ratios, flow
through the tube ceased. Second, using this
design, the water level is critical. If the
water level dropped below the top of the
discharge elbow, flow rate was affected
(Figure 8). If the water level rose above the
top of the elbow, the motor could be
damaged. For commercial application, the
unit should have at least a 2-foot
freeboardd" to allow for normal variations in
pond water level.
Figure 6. U-tube prior to installation. The tube was 35" I.D. x 20' tall and was made
with 14 gauge galvanized steel.
92 SRAC Eighteenth Annual Progress Report, December 2005
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