Participating organizations (WP leader in bold): P1. HCMR, P7. IMR, P17. NIFES and P22. SWH
Task 5.1 Documentation of reproductive performance in wild-captured vs cultured female Atlantic halibut (led by IMR). Even though empirical data suggest a significant difference in spawning performance between wild-captured and farmed Atlantic halibut females, there currently is a lack of systematic documentation. The Atlantic halibut is a group-synchronous, periodic spawner and in captivity wild-captured females will release 6-12 batches of eggs during a period of 2-4 weeks in the spawning season, which lasts from late February to late April. In order to obtain eggs with high viability, females have to be stripped according to their individual ovulatory rhythms, to prevent over-ripening and deterioration of the eggs. While wild-captured females generally adapt well in captivity, displaying high fecundity with egg batches spawned at regular intervals, hatchery-produced F1/F2 females appear to suffer from a reproductive dysfunction, releasing small batches of eggs at irregular intervals. Consequently, individual spawning cycles will be documented in cultured and wild-captured females (IMR, SWH). At each location (IMR and SWH), 4-6 individual females will be tagged and followed. Fertilized eggs will be photographed using a using a dissecting microscope, for measurements of egg diameter, blastomere symmetry and fertilization rate. The following parameters will be registered: Length of spawning period; number of batches; ovulatory interval; volume and number of eggs per batch; egg diameter; fertilization and hatching rate; cell symmetry, egg steroid concentration (E2, T, cortisol).
Task 5.2 GnRH implant therapy as a means to improve spawning performance (led by HCMR). Long-term release implants for gonadotropin releasing hormone agonist (GnRHa) will be produced (HCMR) using a non-degradable Ethylene-Vinyl Acetate copolymer (EVAc) using established procedures. The release kinetics of the implants will be confirmed by HCMR using an in vitro release assay, with GnRHa implants embedded in low-melting point agarose and maintained at 6°C. Mature farmed, F1/F2, female Atlantic halibut will be implanted either with GnRHa or a sham implant (IMR, SWH), and spawning performance compared between the implanted and controls. Prior to implantation, appearance and degree of swelling of the ovipore will be registered and ovarian biopsies taken, in order to estimate stage of maturation. Differences in registered spawning parameters as described above (Task 5.1) will be compared by appropriate statistical methods to determine optimum dose and timing of GnRH treatment. An optimised GnRHa therapy protocol may be efficient to improve spawning performance of F1/F2 Atlantic halibut, and to increase availability of eggs of stable and predictable quality.
Task 5.3 Fecundity regulation (led by IMR). Potential disturbances in reproductive development in F1/F2 females will be assessed by fecundity analyses, ovarian histology, gene transcript levels of fshr and lhcgr and analysis of plasma steroid and vitellogenin profiles through gametogenesis in F1/F2 females and wild-captured fish (IMR). Ovarian samples will be taken by biopsy of anaesthetized fish (maintained by SWH) at the following stages: late vitellogenesis, prespawning/hyaline, one week into spawning and two weeks into spawning. Potential and realized fecundity, and size-frequency distribution of oocytes in spawning females will be determined in wild-captured and F1 spawners (IMR). Analyses will be performed using the Auto-diametric fecundity method in NIFES. Differences between F1 and wild-captured fish will be analysed using appropriate statistical methods (IMR, NIFES).
Task 5.1 Documentation of reproductive performance in wild-captured vs cultured female Atlantic halibut (led by IMR). Even though empirical data suggest a significant difference in spawning performance between wild-captured and farmed Atlantic halibut females, there currently is a lack of systematic documentation. The Atlantic halibut is a group-synchronous, periodic spawner and in captivity wild-captured females will release 6-12 batches of eggs during a period of 2-4 weeks in the spawning season, which lasts from late February to late April. In order to obtain eggs with high viability, females have to be stripped according to their individual ovulatory rhythms, to prevent over-ripening and deterioration of the eggs. While wild-captured females generally adapt well in captivity, displaying high fecundity with egg batches spawned at regular intervals, hatchery-produced F1/F2 females appear to suffer from a reproductive dysfunction, releasing small batches of eggs at irregular intervals. Consequently, individual spawning cycles will be documented in cultured and wild-captured females (IMR, SWH). At each location (IMR and SWH), 4-6 individual females will be tagged and followed. Fertilized eggs will be photographed using a using a dissecting microscope, for measurements of egg diameter, blastomere symmetry and fertilization rate. The following parameters will be registered: Length of spawning period; number of batches; ovulatory interval; volume and number of eggs per batch; egg diameter; fertilization and hatching rate; cell symmetry, egg steroid concentration (E2, T, cortisol).
Task 5.2 GnRH implant therapy as a means to improve spawning performance (led by HCMR). Long-term release implants for gonadotropin releasing hormone agonist (GnRHa) will be produced (HCMR) using a non-degradable Ethylene-Vinyl Acetate copolymer (EVAc) using established procedures. The release kinetics of the implants will be confirmed by HCMR using an in vitro release assay, with GnRHa implants embedded in low-melting point agarose and maintained at 6°C. Mature farmed, F1/F2, female Atlantic halibut will be implanted either with GnRHa or a sham implant (IMR, SWH), and spawning performance compared between the implanted and controls. Prior to implantation, appearance and degree of swelling of the ovipore will be registered and ovarian biopsies taken, in order to estimate stage of maturation. Differences in registered spawning parameters as described above (Task 5.1) will be compared by appropriate statistical methods to determine optimum dose and timing of GnRH treatment. An optimised GnRHa therapy protocol may be efficient to improve spawning performance of F1/F2 Atlantic halibut, and to increase availability of eggs of stable and predictable quality.
Task 5.3 Fecundity regulation (led by IMR). Potential disturbances in reproductive development in F1/F2 females will be assessed by fecundity analyses, ovarian histology, gene transcript levels of fshr and lhcgr and analysis of plasma steroid and vitellogenin profiles through gametogenesis in F1/F2 females and wild-captured fish (IMR). Ovarian samples will be taken by biopsy of anaesthetized fish (maintained by SWH) at the following stages: late vitellogenesis, prespawning/hyaline, one week into spawning and two weeks into spawning. Potential and realized fecundity, and size-frequency distribution of oocytes in spawning females will be determined in wild-captured and F1 spawners (IMR). Analyses will be performed using the Auto-diametric fecundity method in NIFES. Differences between F1 and wild-captured fish will be analysed using appropriate statistical methods (IMR, NIFES).
A hatchery-produced atlantic halibut broodstock
WP5 Reproduction and Genetics - Atlantic halibut
The objective if this task is to develop an optimized GnRHa-based method to improve spawning performance in hatchery produced Atlantic halibut brood stocks.
The first experiments on the induction of ovulation in hatchery-produced Atlantic halibut were initiated on the 25 and 26 February 2014, at the Austevoll, Norway facilities of the Institute of Marine Research, as described in Task 5.2 of the project.
A group of 12 females of an average weight of 15 kg were biopsied and selected to be at the same stage of oogenesis. Fish had completed vitellogenesis and some were in early oocyte maturation, with oocytes with a diameter of >2 mm. All males were in full spermiating condition. The females were allocated randomly to three groups and were treated with one of two doses of GnRHa in a controlled-release delivery system (EVAc) or we sham implanted and were used as controls.
In the following days, females have been monitored daily for ovulation, and eggs were first collected after 5 days from treatment, and by 9 days after GnRHa treatment all females in the high GnRHa treatment entered a cycle of regular ovulations. Females in the lower GnRHa treatment did not ovulated yet, but their gonadal stage of development had advanced further. Females in the control group did not progress in development.
Below are some photos of the experiments undertaken at Austevoll.
The objective if this task is to develop an optimized GnRHa-based method to improve spawning performance in hatchery produced Atlantic halibut brood stocks.
The first experiments on the induction of ovulation in hatchery-produced Atlantic halibut were initiated on the 25 and 26 February 2014, at the Austevoll, Norway facilities of the Institute of Marine Research, as described in Task 5.2 of the project.
A group of 12 females of an average weight of 15 kg were biopsied and selected to be at the same stage of oogenesis. Fish had completed vitellogenesis and some were in early oocyte maturation, with oocytes with a diameter of >2 mm. All males were in full spermiating condition. The females were allocated randomly to three groups and were treated with one of two doses of GnRHa in a controlled-release delivery system (EVAc) or we sham implanted and were used as controls.
In the following days, females have been monitored daily for ovulation, and eggs were first collected after 5 days from treatment, and by 9 days after GnRHa treatment all females in the high GnRHa treatment entered a cycle of regular ovulations. Females in the lower GnRHa treatment did not ovulated yet, but their gonadal stage of development had advanced further. Females in the control group did not progress in development.
Below are some photos of the experiments undertaken at Austevoll.
