Canadian Aquaculture R&D Review 2011

Finfish – Marine

Sustainable culture development for Wolf Eels

Adult Wolf Eel (Anarrhichthys ocellatus) breeding pairs (out-crossed brood years 1995 and 2001) located at the Vancouver Aquarium and the DFO – Centre for Aquaculture and Environmental Research, had histories of producing unfertilized eggs. In each of these years and at both sites, the females appeared to be ovulating and releasing normal eggs. Therefore the cause of these unsuccessful fertilizations was attributed to the males not being fully mature. This past spawning season, (2009-2010) the male Wolf Eels located at each site were implanted with Ovaplant™ (Syndel Laboratories Ltd., BC) two weeks prior to when spawnings had occurred in previous years. Within 7-10 days post–implantation, each pair had successfully produced a large, cohesive egg mass of ca. 15,000 eggs. The egg masses were incubated in the laboratory inside a high-flow venturi incubator under ambient water conditions. The fry hatched within 3 months, and were immediately feeding on artificial diets. Juveniles are currently involved in diet and density studies. In summary, the development of various incubation and husbandry techniques demonstrate that Wolf Eels can be successfully bred and reared in captivity. Implications of these results are of interest for use in culturing Wolf Eels for the live market food industry.

Apr. 2009 – Mar. 2011 • Funded by: DFO – Aquaculture Collaborative Research and Development Program (ACRDP), Island Scallops Limited
Project team: Shannon Balfry (CAER), Steve Macdonald (DFOCAER), Jeff Marliave (UBC); Rob Saunders (Island Scallops Ltd.)
Contact: Shannon Balfry ( balfry@mail.ubc.ca) • /aquaculture/acrdp-pcrda/index-eng.htm

Wolf eels

Sablefish genomics: understanding genetic variation in Sablefish

The Sablefish industry is a $300 million a year industry in North America and an important emerging fish stock for British Columbia. While commercial demand is rapidly increasing, wild stocks are remaining stable. There is a need for genetic tools to facilitate establishment of a successful Sablefish aquaculture as well as to enable better management of wild fish stocks.

Funded by Genome BC, the Sablefish Genomics project developed a preliminary suite of genomic tools that can provide important information to both aquaculture companies and management of wild fish stocks. University of Victoria's Ben Koop, in collaboration with Sablefish Canada Ltd, identified and characterized the genetic variation in Sablefish, allowing for efficient identification of individuals for monitoring wild stocks as well as for selective breeding programs for aquaculture. The preliminary characterization of genetic markers in Sablefish has led to the development of genomic tools for general gene expression analysis, thereby facilitating fisheries to specifically select for genes that are associated with growth, reproductive success and traits that will help make Sablefish aquaculture a sustainable and profitable industry for British Columbia.

Jan. 2009 - Mar. 2010 • Funded by: Genome British Columbia, Sablefish Canada Ltd., University of Victoria
Project team: Ben Koop (U of Victoria)
Contact: Ben Koop ( bkoop@uvic.ca) • http://cbr.uvic.ca/index.php?option=com_content&task=view&id=61&Itemid=83

Stress resistance selection and better growth in Atlantic Cod

One of the sub-projects of the Atlantic Cod Genomics and Broodstock Development Project (CGP) investigated whether families of Atlantic Cod vary in the magnitude of their cortisol stress response. For this study, fish (30) from each family (10) were measured, weighed and PIT- tagged, and then subjected to a standardized handling stressor (20 s out of the water) which was applied once a month for 5 consecutive months. At the end of the study fish within each family were classified as consistent high cortisol responders (HR), low cortisol responders (LR), or non-consistent responders (NC). The proportion of the LR and HR phenotypes varied greatly between families. In addition, body mass, SGR and condition factor were significantly higher in LR when compared with HR fish; average LR fish body mass was 345.1 g and HR fish was 221.8 g at the end of the experiment. These results indicate that responsiveness to stress (based on cortisol levels) may be an important trait to consider in a broodstock development program.

Aug. 2006 – Sept. 2007 • Funded by: Genome Canada, Genome Atlantic, the Atlantic Canada Opportunities Agency (ACOA) through the CGP
Project team: Kurt Gamperl (MUN-OSC), Tiago Hori (MUN-OSC), Luis O.B. Afonso (BC-CAHS), Matthew L. Rise (MUN-OSC), Stewart Johnson (DFOPBS)
Contact: Kurt Gamperl ( kgamperl@mun.ca), Luis Afonso ( luis.afonso@cahs-bc.ca) http://www.codgene.ca

Sablefish nutrition

Sablefish (Anoplopoma fimbria), also known as Black Cod, have been sustainably harvested in BC for many years, but high demand has resulted in the recent establishment of a small culture industry.

A growth trial was conducted at the DFO – Centre for Aquaculture and Environmental Research (CAER) in West Vancouver to provide information about the optimum dietary combination of protein, lipid, and carbohydrate required for growth and feeding efficiency.

A hallmark of this species is the very high fat content of the flesh, which is responsible for the soft buttery texture of the meat that is highly desirable to consumers. It is expected that best performance will be achieved on a high fat diet. As Sablefish are carnivores, it is expected that they require a high protein diet.

The fish were fed for 11 weeks and exhibited excellent growth rates, indicating that the experimental conditions and protocols were suitable.

One interesting aspect of this research was that it was run as a mixture experiment, which is ideal for studies investigating how multiple dietary ingredients work together to affect a response variable (typically growth or feed efficiency). The findings are still being collated, but it is evident that juvenile Sablefish require high protein and fish oil for optimum growth.

Sept. 2010 – Dec. 2010 • Funded by: DFO – Aquaculture Collaborative Research and Development Program (ACRDP), Sablefish Canada
Project team: Ian Forster (DFOCAER), Briony Campbell (Sablefish Canada), Jamie Bridge (Sablefish Canada)
Contact: Ian Forster ( Ian.Forster@dfo-mpo.gc.ca) /aquaculture/rp-pr/acrdp-pcrda/index-eng.html

Sablefish

Mixture methodology for studying diet formulations in fish aquafeed research

Modern feeds for fish reared under intensive conditions, (e.g., salmon), are very efficient at meeting the nutritional needs of the fish while minimizing waste production. This efficiency has been achieved by applying the results of extensive research conducted using modern methods of nutritional science. Mixture methodology is a relatively new research approach to identifying optimum combinations of ingredients. In mixture experiments, combinations of specific test ingredients are combined in a series of feeds in a defined way, such that they sum to a fixed proportion of the feeds, while the remaining portion of each feed is held constant. The response (usually growth or feed efficiency) of the animals fed these feeds is then monitored. Mixture methodology assumes that the observed response is a function of the proportions of the ingredients tested. Using this methodology, relationships between ingredients or nutrients can be ascertained and optimum combinations identified. Mixture experiments can test combinations of multiple components using fewer resources (fish, tanks and labour) than factorial methods. Mixture experiments may be used to find feed protein and energy levels that maximize growth or to identify combinations of ingredients that meet the nutritional needs of fish for the least cost.

Sept. 2010 – Dec. 2010 • Funded by: DFO – Aquaculture Collaborative Research and Development Program (ACRDP), Sablefish Canada
Project team: Ian Forster (DFOCAER), Briony Campbell (Sablefish Canada), Jamie Bridge (Sablefish Canada)
Contact: Ian Forster ( Ian.Forster@dfo-mpo.gc.ca) /aquaculture/rp-pr/acrdp-pcrda/index-eng.html

The forgotten sex: paternal effects on embryonic and larval mortality of Atlantic Cod

It is important to understand parental effects on early life history of fish as manifested, for example, in individual fitness of offspring. Immediately after fertilization, parental contributions (genetic and non-genetic) to embryos will affect larval ontogeny, physiology, morphology and survival. In marine fish, rates of natural mortality are highest during early life and are negatively correlated with rates of growth and body size. In these early life stages (eggs, larvae, young juveniles) subtle differences in mortality can cause large differences in recruitment and year-class success. Therefore, it is particularly critical to understand factors that contribute to variability in mortality during early life. This study focuses on evaluating the potential influence paternity has on rates of mortality and development in eggs and larvae of Atlantic Cod (Gadus morhua). Paternity had a strong influence on mortality during the early embryonic phases for progeny of one female, whereas early embryonic mortalities of the other female were largely maternally induced. Mortalities stabilized after day 6 (end of gastrulation), with hatching success ranging from 8 to 65% for progeny of one female and nearly zero for the other. Therefore, mate choice during spawning can play a large role on offspring survivorship (fitness).

May 2009 – Apr. 2010 • Funded by: EU COST-funded Short Term Scientific Mission (STSM) in "Fish Reproduction and Fisheries"-action (FRESH; FA0601)
Project team:Muriel-M. Kroll (Hamburg University, Germany), Myron A. Peck (Hamburg University, Germany), Ed Trippel (DFOSABS)
Contact: Ed Trippel ( Ed.Trippel@dfo-mpo.gc.ca)

Growth trial of the Spotted Wolffish in pre-commercial conditions

Numerous studies have been done on the development of wolffish aquaculture in Québec, Canada. This project puts together all the recent knowledge by Canadian, Norwegian and Icelandic research groups on a full cycle production with the objective of maximizing growth performance (density, optimal temperature-at-size, feed/feeding). Another objective will be to fully evaluate the profitability of wolffish cultivation in Québec, Canada.

2008 – Mar. 2011 • Funded by: DFO – Aquaculture Collaborative Research and
Development Program (ACRDP), Société de développement de l'industries maricole (SODIM), MAPAQ
Project team: Nathalie R. Le François (Montreal Biodôme/UQAR), Denis Chabot (DFOIML), France Dufresne (UQAR), SODIM, MAPAQ
Contact: Nathalie R. Le François ( Nle_francois@ville.montreal.qc.ca, Nathalie_le-francois@uqar.qc.ca) /aquaculture/acrdp-pcrda/index-eng.htm

Effects of extended photoperiods and light intensities on growth, sexual maturation and tissue water content of Atlantic Cod in sea cages

Mariculture of Atlantic Cod (Gadus morhua) in the Bay of Fundy was initiated in the early 2000s and early puberty has been a persistent problem since its inception. The objectives of the present study were to evaluate, over separate two-year periods, whether 20 and 24 h light exposure compared to natural photoperiod in sea cages will (i) increase body growth, (ii) halt or delay sexual maturation, and (iii) affect seasonal levels of water content in muscle and liver tissue of Atlantic Cod. A positive growth response to extended daylength (20 and 24 h light) was observed, although these light treatments did not influence the incidence of sexual maturation of either sex. Greater light intensity also did not reduce the incidence of maturity. The proportion of fish sampled that were mature was often > 90%. Immature cod of each gender were reported to grow at slower rates than mature cod. Implementation of extended daylength did, however, result in delaying attainment of sexual maturation by approximately 3 months. Of particular note is the lower reproductive investment (i.e., female GSI values) observed after two years of 20 h daily illumination. The percent water content of muscle tissue of adult females was greater than of immature females. Our results suggest the use of extended daylength to improve growth and change the timing and investment in reproduction holds some promise for Atlantic Cod mariculture.

May 2007 – Mar. 2010 • Funded by: DFO – Aquaculture Collaborative Research and Development Program (ACRDP), Cooke Aquaculture Inc.
Project team: Ed Trippel (DFOSABS), S. Neil (DFOSABS), O. Puckrin (DFOSABS), L. Dickinson (DFOSABS), F. Powell (Cooke Aquaculture)
Contact: Ed Trippel ( Ed.Trippel@dfo-mpo.gc.ca) http://www.dfo-po.gc.ca/aquaculture/rp-pr/acrdp-pcrda/index-eng.html

Cryopreservation of Atlantic Cod spermatozoa

Effects of extender composition and freezing rate on sperm motility, velocity and morphology

Broodstock selection programs are currently underway for Atlantic Cod (Gadus morhua). To complement and further these selection programs we need to develop sperm cryopreservation procedures. This will allow genomic DNA from males from selected individuals or stocks to be frozen and conserved in perpetuity. In our study we examined the effects of diluent (Mounib's sucrose-based diluent, Hanks' Balanced Salt Solution, Mounib's sucrose-based diluent + hen's egg yolk, and Hanks' Balanced Salt Solution + hen's egg yolk), cryoprotectant (propylene glycol, dimethyl sulphoxide, and glycerol), and freezing rate (-2.5, -5.0, -7.5, and -10.0°C/min) on motility of cod frozen-thawed sperm. Sperm velocity and morphometric analyses of sperm heads and flagella were also assessed. We found that sperm motility recovery index was strongly influenced by the presence of higher-order interactions of the factors we tested. The best cryoprotection used diluents that contained hen's egg yolk. Generally, extenders containing propylene glycol yielded higher post-thaw sperm motilities than those with dimethyl sulphoxide or glycerol. In comparison to sperm from other frozen-thawed extenders, sperm from extenders supplemented with propylene glycol had significantly higher curvilinear velocity. Cryopreservation showed no impact on sperm head morphology parameters; however, considerable damage to frozen/thawed sperm flagella was observed. We believe that our experimental/statistical approach and results add significantly new information to the study of semen biology/cryobiology in fishes. Our findings are also highly relevant to the development of cod mariculture and for aiding in conservation efforts of this very important marine species.

Jan. 2007 – Dec. 2010 • Funded by: Genome Canada, Genome Atlantic, Atlantic Canada Opportunities Agency, NSERC, Marguerite and Murray Vaughan Graduate Fellowship. A complete list of supporting partners can be found at http://www.codgene.ca/partners.php
Project team: Ian A.E. Butts (DFOSABS, UNB – now U of Windsor), M. Litvak (UNB),V. Kaspar (University of South Bohemia, Czech Republic), E. Trippel (DFOSABS)
Contact: Ian Butts ( iana.e.butts@gmail.com) • http://www.codgene.ca

Measuring sperm motility

Impact of dissolved oxygen on survival, growth, and metabolism of the Spotted Wolffish

The main objectives of this project were to measure hypoxia tolerance of Spotted Wolffish using two different methods, the critical oxygen threshold or PO2crit and the LC50 after 96 h of exposure, and the impact of chronic, sublethal hypoxia on growth of Spotted Wolffish and the hybrid Spotted x Atlantic Wolffish.

Spotted Wolffish are tolerant of reduced oxygen down to about 26% air saturation (LC05,96h), below which, the mortality risk becomes significant. Our two measures of lethal threshold were similar (PO2crit: 16.8%; CL50,96h = 21% air saturation). However, higher levels of dissolved oxygen (DO) still have negative effects on wolffish growth, with growth significantly reduced at 40% air saturation. Growth was also reduced at 50 and 60% air saturation, but the reduction was not significant due to small sample sizes. A follow-up study with larger sample sizes is presently underway. Final analyses will include survival times at different low dissolved oxygen levels to ensure that the impact of equipment failure can be assessed. Our final results will also include a comparison of growth and hypoxia tolerance for the Spotted Wolffish and the hybrid wolffish.

Apr. 2009 – Mar. 2011 • Funded by: DFO – Aquaculture Collaborative Research and Development Program (ACRDP), Société de Développement de l'Industrie Maricole, Université du Québec á Rimouski (UQAR), Montréal Biodôme
Project team: Denis Chabot (DFOIML), Martin Jetté (DFOIML), François Larouche (DFOIML), Nathalie le François (UQAR), Arianne Savoie (DFOIML), Pierre Blier (UQAR)
Contact: Denis Chabot ( Denis.Chabot@dfo-mpo.gc.ca) /aquaculture/acrdp-pcrda/index-eng.htm

Spotted Wolffish in respirometers

Researchers strive for single-sex stocks of cultured Atlantic Cod

Atlantic Cod (Gadus morhua) has been selected as a potential candidate for aquaculture in Canada and internationally. However, early maturation and release of viable embryos from spawning fish in cages are major impediments to commercial viability and environmental stewardship. For these reasons, protocols to produce single-sex populations of cod are being developed to take advantage of sex-related differences in growth performance, prevent the release of fertilized eggs into the wild and aid in the development of all-female triploid (sterile) cod stocks.

Gynogenetic cod juveniles have been produced to identify the genetic mechanism of sex determination in this species. Gynogenesis is a process whereby expression of the paternal genome in viable embryos is prevented. If gynogenetic offspring are all females, then female is the homogametic sex, as in salmonids and Atlantic Halibut. Knowing the genetic basis of sex determination identifies the appropriate endocrine approach for producing single-sex populations.

Direct endocrine sex-reversal experiments were done to create broodstock capable of generating single-sex progeny. For example, if female Atlantic Cod are homogametic then all-female populations can be produced by first masculinizing genotypic females and then crossing these "neomales" with normal females to yield all-female offspring.

These techniques, as well as sex-reversed broodstock capable of generating single-sex stocks for commercial production, will be transferred to Canadian aquaculture companies to increase production performance and environmental sustainability.

Dec. 2008 – Dec. 2011 • Funded by: DFO - Aquaculture Collaborative Research and Development Program (ACRDP), Natural Sciences and Engineering Research Council of Canada (NSERC), New Brunswick Innovation Foundation (NBIF)
Project team: D. Martin-Robichaud (DFOSABS), Tillmann Benfey (UNB), Song Lin (UNB/DFOSABS), Jessica Whitehead (UNB/DFOSABS), Cooke Aquaculture Inc.
Contact: D. Martin-Robichaud( Debbie.Martin-Robichaud@dfo-mpo.gc.ca) /aquaculture/rp-pr/acrdp-pcrda/index-eng.html

Atlantic Cod sex control

Hormonal induction of ovulation and spermiation in Atlantic Cod

Captive, wild-caught female Atlantic Cod (Gadus morhua) from Georges Bank were divided into three treatment groups based on body weight and diameter of the largest clutch of oocytes present. Treatments administered were an intramuscular implant of one of two gonadotropin releasing hormone analogues (D-Ala6, Pro9- NHEt)-LHRH ethylamide (LHRHa, 150 μg) and salmon gonadotropin releasing hormone analogue (sGnRHa, 150 μg, Ovaplant®), or a saline injection (control). Over a period of 20 days, 12 of the 14 implanted and one control fish ovulated. There was no significant variation between egg volumes and percent fertilization of implant-treated and naturally ovulating females; however, significant variation did exist for the estimated number of hatched larvae among treatments. Non-spermiating captive wild-caught Atlantic Cod males were divided into two treatment groups (Ovaplant® and saline control) based on body weight. Large volumes of milt with high motility and spermatocrits were obtained from each of five Ovaplant®-treated males starting 6 days post implant. Over a 27 day period, each of six control males produced milt at least once, but in small volumes with variable motility and low spermatocrits. Milt volumes of the Ovaplant®-treated males were higher than naturally spermiating males (P = 0.0002), but had similar motilities and spermatocrits. These results demonstrate the effectiveness of GnRHa implants in inducing ovulation and spermiation in Atlantic Cod broodstock without adversely affecting gamete quality.

Collection of milt from Atlantic Cod

2007-2009 • Funded by: Genome Canada, Genome Atlantic, Atlantic Canada Opportunities Agency, the Atlantic Marine Aquaculture Center, New Hampshire Agriculture Experiment Station, New Hampshire SeaGrant program.
Project team: Amber Garber (Huntsman Marine Science Centre, SABS), Susan Fordham (Huntsman Marine Science Centre), Jane Symonds (National Institute of Water and Atmospheric Research Ltd.,New Zealand), Edward Trippel (DFOSABS), David Berlinsky (University of New Hampshire, USA)
Contact: Amber Garber ( agarber@huntsmanmarine.ca) • http://www.codgene.ca

Impact of food deprivation on growth response and activity of juvenile Atlantic Cod

Compensatory growth is a period of accelerated growth experienced by organisms encountering abundant food after a period of partial or total deprivation. Rates during compensatory growth exceed those of continuously fed fish and indicate that fish normally do not grow at the maximum rates which are physiologically possible. It is suggested that hyperphagia is the main mechanism involved in the compensatory growth response and it is often accompanied by increased growth efficiency. Identifying the mechanisms and dynamics of compensatory growth in marine fishes has practical applications to aquaculture production as it might help to design optimal feeding schedules, increase feeding efficiency and decrease environmental nitrogen loading. This study examined if juvenile Atlantic Cod exhibit a compensatory growth response after different types of food deprivation (single bout or periodic) and also attempted to link this physiological response to behavioural aspects, i.e., changes in locomotory activity before and after a period of food shortage. Juvenile cod experiencing 3 weeks of food deprivation exhibited higher specific growth rates. Gross growth efficiency of previously deprived fish was higher during realimentation than of control fish, although they could not catch up completely in size with continuously fed fish after the completion of the experiment. Activity of juveniles differed between day and night time and were a function of type of food deprivation. The findings indicate that partial growth compensation is possible for juvenile cod and suggest that activity is directly related to feeding–level. Using the advantages of compensatory growth might help to reduce production costs and to minimize environmental impacts.

May 2009 – Apr. 2010 • Funded by: EU COST-funded Short Term Scientific Mission (STSM) in "Fish Reproduction and Fisheries"-action (FRESH; FA0601)
Project team: Ed Trippel (DFOSABS), Maja Walter (Institute of Hydrobiology and Fisheries Science, Germany), Myron Peck (Institute of Hydrobiology and Fisheries Science, Germany)
Contact: Ed Trippel ( Ed.Trippel@dfo-mpo.gc.ca)

The Atlantic Cod Genomics and Broodstock Development Project

The $18.1 million Atlantic Cod Genomics and Broodstock Development Project (CGP) aimed to develop a breeding program and fundamental genomics tools to supply the developing Atlantic Cod aquaculture industry in Canada with improved broodstock. Family-based breeding programs were initiated in Newfoundland and Labrador and New Brunswick, ensuring that local stocks were used for the benefit of industry partners. CGP data suggests that the breeding programs will be highly successful at improving growth rates, as well as other related traits such as fillet yield. Genetic variation was also found in stress response, thermal tolerance and disease resistance traits. Little variation was measured in sexual maturation or fillet quality at harvest.

The CGP dramatically improved genomic resources for this species, contributing 85.8% of publicly available DNA sequence information for Atlantic Cod. Over 100,000 genomic markers were identified and made available in GenBank. A genetic map was developed to help identify quantitative trait loci (QTL) related to key production traits such as growth, disease resistance and resistance to stress from handling. A microarray or "cod chip" was developed and used in several studies related to tissue response and early development. A combination of microarray and DNA sequence data aided selection of genes to include in genetic mapping and allowed for comparative genomics to identify possible candidate genes within QTL intervals. The genomics research coupled with breeding program data will help the cod industry continue to select fish with specific marker sequences rather than measuring and selecting based on individual fish phenotypes.

Jan. 2006 – Sept. 2010 • Funded by: Genome Atlantic, The Atlantic Genome Centre (TAGC), Huntsman Marine Science Centre, Atlantic Canada Opportunities Agency – Atlantic Innovation Fund, Newfoundland and Labrador Department of Fisheries and Aquaculture, DFO – Aquaculture Collaborative Research and Development Program (ACRDP), DFOSABS, NRC – IMB, The New Brunswick Innovation Foundation, New Brunswick, Newfoundland and Labrador, Nova Scotia, Memorial University of Newfoundland Ocean Sciences Centre, University of British Columbia, University of Guelph, University of New Brunswick, Cooke Aquaculture Inc., GreatBay Aquaculture, Newfoundland Cod Broodstock Company, The Newfoundland Aquaculture Industry Association, Northern Cod Ventures, RPC – The Technical Solutions Centre
Project team: Sharen Bowman (Genome Atlantic), Ed Trippel (DFOSABS), Keith Culver (UNB), Kurt Gamperl (MUN – OSC), Stewart Johnson (DFOPBS), Matthew Rise (MUN – OSC), Andy Robinson (U of Guelph)
Contact: Shelley King ( sking@genomeatlantic.ca) • http://www.genomeatlantic.ca

Semen characteristics and their ability to predict sperm cryopreservation potential of Atlantic Cod

There is a lack of biomarkers or indices that can be used to predict the quality of fish semen samples following the freezing and thawing cycle. In this study, we tested a series of semen indices to assess if they could accurately forecast the cryopreservation potential of Atlantic Cod semen. Fresh and frozen-thawed sperm activity variables were compared and relationships between frozen–thawed sperm activity and fertilization success were examined. In comparison with fresh sperm, our results clearly show that activity variables of cryopreserved spermatozoa are reduced. Of the 18 males examined, mean (± SEM) spermatocrit was 40.7 ± 4.23%, osmolality of the seminal plasma was 366.3 ± 4.95 mOsmol kg-1, pH 8.3 ± 0.04, protein concentration was 1.1 ± 0.08 mg mL-1, anti-trypsin activity was 153.8 ± 19.25 uL-1, and total antioxidant capacity was 0.2 ± 0.03 μmol Trolox equivalents mL-1. Fertilization success was highly variable among males with values ranging from 18.5 to 90.2%. Regressions demonstrated significant positive relationships between frozen-thawed motility, velocity, track crossing frequency, and subsequent fertilization success. Sequential multiple regressions explained up to 95% of the variation in frozen-thawed sperm activity. Spermatocrit and pH were negatively related, while osmolality and antioxidant capacity were positively related to frozen-thawed motility and velocity. Each of these indices can be measured within minutes of collecting a fresh sample of semen and are thus early indicators of the capacity of semen samples to withstand cryopreservation. These results have many benefits for conservation of wild stocks, aquaculture production, and for understanding semen biology and cryobiology of fishes.

Jan. 2009 – Dec. 2010 • Funded by: Research Council of Norway, Polish Academy of Sciences, the Natural Sciences and Engineering Research Council of Canada, Dr. John S. Little International Study Fellowship, Marguerite and Murray Vaughan Graduate Fellowship, Genome Canada, Genome Atlantic, Atlantic Canada Opportunities Agency
Project team: Ian A.E. Butts (DFOSABS, UNB – now U of Windsor), I. Babiak (Bodø University College, Norway), A. Ciereszko (Polish Academy of Sciences, Poland), M. Litvak (UNB), M. Slowinska (Polish Academy of Sciences, Poland), C. Soler (University of Valencia, Spain), E. Trippel (DFOSABS)
Contact: Ian Butts ( iana.e.butts@gmail.com) • http://www.codgene.ca

Milt collection from Atlantic Cod

Competitive spawning success and fertility of triploid male Atlantic Cod

Farmed Atlantic Cod (Gadus morhua) have been hypothesized to pose a risk to the natural environment through escape of fish or release of their gametes from sea cages. The use of sterile triploids has been suggested as a way to prevent spawning of farmed fish, whether escaped from their cages or still contained within them. This laboratory study examined the reproductive potential of triploid male Atlantic Cod, both in vivo and in vitro.We demonstrated that triploid males in competitive, size-matched mating with diploid males were successful at siring young. Spermatozoa produced by triploid males were larger than those of diploids, but did not differ in swimming velocity or ability to fertilize eggs. Hatch and larval survival rates, however, were reduced for progeny of triploid males. These diagnostic characteristics of aneuploidy suggest that although farmed triploid male cod may compete successfully with wild diploids for spawning access to wild females, their offspring will not survive.

Apr. 2008 – Dec. 2010 • Funded by: NSERC
Project team: Nathaniel Feindel (UNB/DFOSABS), Tillmann Benfey (UNB), Ed Trippel (DFOSABS)
Contact: Ed Trippel ( Ed.Trippel@dfo-mpo.gc.ca)

Gonadal development of triploid Atlantic Cod

Atlantic Cod (Gadus morhua) is a prime candidate for marine finfish aquaculture diversification, but there are a number of problems associated with its culture that are constraining the advancement of the industry. Generation of sterile Atlantic Cod by the induction of triploidy is one technique being studied to address issues associated with early puberty. The aim of this study was to examine gonadal development of adult-aged, triploid Atlantic Cod prior to and during the annual spawning season in comparison to sibling diploids. Cod were sacrificed three times: prior to spawning; and during two subsequent spawning seasons (at age 3 and 4 years). Female triploid Atlantic Cod exhibited diminished ovarian development which was illustrated at both macroscopic and microscopic levels. Significant differences occurred between diploid and triploid female gonadosomatic indices during all three sampling periods and carcass yields during the two spawning periods. In contrast, testicular development of diploid and triploid Atlantic Cod was indistinguishable through macroscopic and histological assessment. No significant differences were found in male gonadosomatic indices or head-on gutted carcass yields in relation to ploidy, with the exception of a slightly higher carcass yield for triploid males during the spawning season. As a result of suppressed oogenesis, female more so than male triploid cod have the potential to be utilized and advance the development of the cod aquaculture industry.

May 2009 – Dec. 2010 • Funded by: NSERC
Project team: Nathaniel Feindel (UNB/DFOSABS), Tillmann Benfey (UNB),Ed Trippel (DFOSABS)
Contact: Ed Trippel ( Ed.Trippel@dfo-mpo.gc.ca)

Atlantic Cod

Seminal plasma biochemistry and spermatozoa characteristics of wild and cultivated Atlantic Cod

Our objectives were to compare spermatozoa activity, morphology, and seminal plasma biochemistry between wild and cultivated Atlantic Cod (Gadus morhua). Swimming velocities of wild cod spermatozoa were significantly faster than those of cultivated males. Wild males had a significantly larger spermatozoa (i.e., the moving sperm cell) head area, perimeter, and length, while cultivated males had more circular heads. Total monounsaturated fatty acids and the ratio of n-3/n-6 fatty acids were significantly higher in sperm from wild males, while total n-6 from cultivated males was significantly higher than of wild males. Significantly higher concentrations of the fatty acids C14:0, C16:1n-7, C18:4n-3, C20:1n-11, C20:1n-9, C20:4n-3, C22:1n-11, and C22:6n-3 were observed in wild males, while significantly higher concentrations of C18:2n-6, C20:2n-6, and C22:5n-3 occurred in cultivated males. Osmolality, protein concentration, lactate dehydrogenase and superoxide dismutase activity of seminal plasma of wild males were significantly higher than of cultivated males. Antioxidant capacity of seminal plasma was significantly higher in cultivated males, while pH and antitrypsin did not differ between fish origin. Four bands of anti-trypsin activity and nine protein bands were detected in seminal plasma. Performing a discriminant function analysis, on morphology and fatty acid data showed significant discrimination between wild and cultivated fish. Results are relevant to breeding programs and aquaculture development.

Jan. 2009 – Dec. 2010 • Funded by: Research Council of Norway, Polish Academy of Sciences, Natural Sciences and Engineering Research Council of Canada, Dr. John S. Little International Study Fellowship, Marguerite and Murray Vaughan Graduate Fellowship, Atlantic Cod Genomics and Broodstock Development Project.
Project team: Ian A.E. Butts (DFOSABS, UNB now U of Windsor), I. Babiak (Bodø University College, Norway), A. Ciereszko (Polish Academy of Sciences, Poland),M. Litvak (UNB), M. Slowiska (Polish Academy of Sciences, Poland), C. Soler (University of Valencia, Spain), E. Trippel (DFOSABS), S.M.H. Alavi (University of South Bohemia, Czech Republic)
Contact: Ian Butts ( iana.e.butts@gmail.com) http://www.codgene.ca

Cod sperm

Cryopreservation of Atlantic Cod sperm in large volume straws: applications for commercial production and gene banking

In our study we used a full factorial ANOVA design to examine the effects of diluent (Mounib's sucrose-based diluent + hen's egg yolk, and Hanks' Balanced Salt Solution + EY), freezing rate (-2.5, -5.0, and -7.5°C min-1), and thawing rate (2.5, 5.0, and 7.5°C min-1) on motility and velocity of Atlantic Cod sperm cryopreserved in 2.5 mL cryogenic straws. We found that post–thaw sperm performance was strongly influenced by the presence of higher-order interactions of the factors we tested. For all models broken down by diluent, the 2.5°C min-1 thawing rate had the lowest sperm motility recovery index. Mounib's sucrose-based diluent + hen's egg yolk had the highest motility recovery index at all thawing rates. Mean percent motility for fresh sperm (87.7 ± 2.9%) was not significantly different than of sperm cryopreserved using Mounib's sucrose-based diluent + hen's egg yolk, frozen at - 2.5°C min-1, and thawed at 5.0°C min-1 (77.1 ± 2.9%). For Mounib's sucrose-based diluent + hen's egg yolk velocity was significantly higher with sperm thawed at 7.5°C min-1 than sperm thawed at 2.5°C min-1, while thawing rate had no effect for Hanks' Balanced Salt Solution + hen's egg yolk. Our findings have implications for cod mariculture and aiding in conservation efforts for a dominant marine fish species.

Jan. 2010 – Dec. 2010 • Funded by: Genome Canada, Genome Atlantic, Atlantic Canada Opportunities Agency, Natural Sciences and Engineering Research Council of Canada, the Marguerite and Murray Vaughan Graduate Fellowship. A complete list of supporting partners can be found at http://www.codgene.ca/partners.php
Project team: Ian A.E. Butts (DFOSABS, UNB now U of Windsor), N. Feindel (UNB, SABS), S. Neil (SABS), É. Kovàcs (Szent Istvàn University, Hungary), B. Urbànyi (Szent Istvàn University, Hungary), E.A. Trippel (SABS)
Contact: Ian Butts ( iana.e.butts@gmail.com) http://www.codgene.ca

Seasonal variations in seminal plasma and sperm characteristics of wild-caught and cultivated Atlantic Cod

The objective of this study was to investigate changes, throughout the spawning season, in body size attributes and quantitative semen characteristics of wild-caught and cultivated Atlantic Cod (Gadus morhua). Sperm velocity increased significantly throughout the spawning season of cod from both origins. At the beginning and end of the spawning season, mean (± SEM) curvilinear velocity (VCL) for wildcaught cod increased from 78.9 μm s-1 (± 6.5) to 128.2 μm s-1 (± 6.5), respectively. For cultivated fish, mean (± SEM) VCL increased from 26.6 μm s-1 (± 2.4) in January to 48.9 μm s-1 (± 3.1) in March. Spermatocrit did not undergo a significant seasonal change in wild-caught cod, but did increase for cultivated cod (24.6% ± 4.2 in January to 40.5% ± 4.4 in April; P < 0.01). Sperm head area, perimeter, length, and width declined significantly at the end of the spawning season of cod from both origins (all P values < 0.01). Seminal plasma osmolality and Na+ ion concentration followed a dome-shaped function through the spawning season for both wild-caught and cultivated cod (P < 0.05). For cultivated cod, seminal plasma pH was significantly lower at the start of the spawning season (P < 0.001), while Ca2+ increased and then decreased (P < 0.05). Body size attributes, spermatocrit, and seminal plasma constituents had significant relationships with sperm activity variables. These relationships varied as a function of time post-activation, month, and fish origin.

Jan. 2007 – Dec. 2010 • Funded by: Genome Canada, Genome Atlantic, Atlantic Cod Genomics and Broodstock Development Program, DFO – Aquaculture Collaborative Research Development Program (ACRDP), Natural Sciences and Engineering Research Council of Canada (NSERC), Dr. John S. Little International Study Fellowship, Marguerite and Murray Vaughan Graduate Fellowship
Project team: Ian A.E. Butts (DFOSABS, UNB now U of Windsor), M. Litvak (UNB), E. Trippel (DFOSABS)
Contact: Ian Butts ( iana.e.butts@gmail.com)

The effect of sperm to egg ratio and gamete contact time on fertilization success in Atlantic Cod

Currently, Atlantic Cod (Gadus morhua) is the primary finfish species being developed for aquaculture in North Atlantic waters. Despite the importance of this species, no research has been conducted to assess the effects of sperm density and gamete contact time on egg fertilization. In two separate experiments male and female gametes were crossed using nested factorial designs. For each male-female combination, we tested sperm to egg ratios ranging from 1 × 103:1 to 5 × 106:1. We also tested two-gamete contact times where sperm and eggs were held in contact with each other for 5 or 30 min. Mixed-model ANOVAs indicated that sperm density and gamete contact time had a significant effect on fertilization success. Below a sperm to egg ratio of 1 × 105 to 1, fertilization success significantly decreased. Therefore, a standard sperm to egg ratio of 1 × 105 sperm per egg is recommended for fertilization in Atlantic Cod. At the 1 × 103:1, 5 × 103:1, and 1 × 104:1 sperm to egg ratios, maximum fertilization occurred after 30 min sperm to egg contact time. Gamete contact time was not significant at sperm to egg ratios of 1 × 105:1, and 1 × 106:1. Both the maternal and paternal variance components were significant for fertilization success. This information has important implications for optimizing family production in selective breeding programs, conserving sperm from superior pedigree in genome banks, maximizing the use of available gametes in hatchery or research facilities, and understanding mating success in the wild.

Jan. 2007 – Dec. 2009 • Funded by: Genome Canada, Genome Atlantic, Atlantic Cod Genomics and Broodstock Development Program, DFO – Aquaculture CollaborativeResearch and Development Program (ACRDP), New Brunswick Innovation Fund (NBIF), Natural Sciences and Engineering Research Council of Canada (NSERC)
Project team: Ian A.E. Butts (DFOSABS, UNB now U of Windsor), M. Litvak (UNB),E. Trippel (DFOSABS)
Contact: Ian Butts ( iana.e.butts@gmail.com)

Atlantic Cod eggs at 26 hours post-fertilization

Optimized land-based tank farming of Atlantic Halibut in Canada

Over the next few years, Canaqua Seafoods Ltd. will improve production efficiencies of Atlantic Halibut aquaculture through the incorporation of innovative management tools and rearing facilities into their existing land-based grow out farm in Advocate Harbour, Nova Scotia. The initial phase of this project focused on enabling Canaqua Seafoods Ltd. to mechanically grade their halibut. This required infrastructure upgrades and installations. These were successfully completed, and the large halibut on the farm were graded. Populations were monitored for several months post-grading in order to assess the effect of size grading on performance. The set up for grading small halibut was also initiated. Grading had the clear and desired effect of reducing variation in the production tanks. Reduced population size variation increases feeding through the diminution of feeding hierarchies, and it improves fish stock management by improving accuracy of biomass and mean fish size estimations. It aids the determination of feed rations, the allocation of optimum feed size and the monitoring of fish performance and health. Soon after the fish were graded, growth rates seemed to increase with steady improvement through the winter months. There was a real reduction in time and effort required to grade the halibut when compared to the earlier manual grade techniques which likely would not even have been feasible at this stage of production.

Jul. 2009 – May 2010 • Funded by: DFO – Aquaculture Innovation and Market Access Program (AIMAP), CanAqua Seafoods Ltd., Scotian Halibut Ltd.
Project team: Paul Merlin (CanAqua Seafoods Ltd.), John Bailey (CanAqua Seafoods Ltd.), Hugh Snow (Scotian Halibut Ltd.), Brian Blanchard (Scotian Halibut Ltd.), Melissa Rommens (Scotian Halibut Ltd.), Philip Nickerson (Scotian Halibut Ltd.), Forest Merlin (Merlin Fish Farms Ltd.), Roland Cusack, Scotian Halibut Ltd., NSDFA, NRC– Institute for Marine Biosciences
Contact: Paul Merlin ( meraqua@msn.com)

Physical and biochemical properties of effluent leaving an onshore Atlantic Cod aquaculture facility and potential use in integrated multi-trophic aquaculture (IMTA)

Integrated multi-trophic aquaculture (IMTA) uses mussels to reduce waste and provide additional product. The physical and biochemical properties of Atlantic Cod (Gadus morhua) wastes were analyzed and the waste remediation potential of Blue Mussels (Mytilus edulis) was assessed. Waste generated daily by Atlantic Cod represented 24.9% of the feed added to the system. Effluent was composed of particles <70 μm (36%), 70-500 μm (31%) and particles >500 μm (33%). Particles <70 μm had significantly less organic matter, lipids and fatty acids and were expected to be ingested more by mussels than larger particles. The major lipid classes present in effluent were free fatty acids, triacylglycerols, phospholipids, acetone mobile polar lipids and sterol. Effluent contained two essential fatty acids, DHA and EPA, a diatom marker (16:1ω7), as well as two zooplankton markers (22:1ω11 and 20:1ω9) which accumulated in mussels and may serve as markers for aquaculture wastes. Although only 36% of the effluent was a size suitable for mussel ingestion this size fraction has the greatest potential to spread to surrounding areas. The fatty acid profile of effluent suggests performance of mussels may be impaired if reared on effluent alone; however, effluent may be used as an alternate food source when natural seston is low.

Feb. 2008 – Feb. 2010 • Funded by: Natural Sciences and Engineering Research Council of Canada (NSERC), Fisheries and Oceans Canada (DFO)
Project team: Chris Parrish (MUN), Adrianus Both (MUN), Randy Penney (DFO), Ray Thompson (MUN)
Contact: Chris Parrish ( cparrish@mun.ca)