Generation of surrogate goldfish Carassius auratus progeny from common carp Cyprinus carpio parents.

Surrogate broodstock technology can increase the production efficiency of commercially important fishes that are difficult to breed in confinement and aid the propagation and recovery of endangered populations. In this study, we report the application of germ cell (GC) transplantation (GCT) for increasing the numbers of progeny produced by small-bodied ornamental fishes by using sexually mature adult fish as recipients. The GCs isolated from prepubertal male goldfish (Carassius auratus) donors (n = 5) were transplanted through the genital papilla into the gonads of adult common carp (Cyprinus carpio) recipients. The endogenous GCs of the recipient were depleted using busulfan (40 mg/kg body weight [BW]; in five doses at 2-week intervals) and high-temperature (38 °C) treatments. Within 4 months after GCT, the donor GCs recolonised the recipients' gonads and resumed gametogenesis. The presence of donor-derived gametes was confirmed through polymerase chain reaction-restriction fragment length polymorphism analysis in all the surrogate common carp males and females. Artificial fertilisation and induced spawning between surrogate males and females yielded pure goldfish progeny; the fertilisation and hatching rates were similar to those of the controls. These results suggest that GCT could also be potentially applied in commercial aquaculture, mainly to increase the numbers of progeny obtained from small-bodied fishes those having low gamete counts.

Non-invasive Method for Collection of Clarias magur Spermatozoa for Breeding.

Induced spawning is more inefficient in Clarias magur than in other fish species such as cyprinids and salmonids. Ovulation can be induced in the female C. magur by using pituitary extract or synthetic hormones. However, milt from the male C. magur cannot be obtained by hand stripping because the volume of seminal fluid in the testes is extremely low. Notably, similar problems are observed in other male catfishes such as C. gariepinus and C. lazera. Because milt from the males cannot be obtained for use in artificial fertilization of eggs, males are invariably sacrificed, and the testicular tissue is excised and macerated to obtain spermatozoa.•We developed an alternative approach that allowed harvesting of C. magur spermatozoa through surgery for artificial fertilization without sacrificing male fish.•The surgically obtained spermatozoa were used to inseminate C. magur eggs; the cross resulted in healthy progeny with a fertilization rate of 80%-98% and hatching of up to 85% of fertilized embryos; similar to those obtained using the conventional sacrificial approach (hatching percentage range of 45%-85%) [1].•This indicated the viability of partial surgical harvest of testicular tissue in seed production in C. magur for aquaculture without sacrificing male fish.

Depletion of endogenous germ cells in striped catfish Pangasianodon hypophthalmus (Sauvage, 1878) by heat-chemical treatments.

Germ cell (GC) transplantation (GCT) is a proven powerful reproductive technique to enhance the production efficiency of domesticated animals and aid to the recovery of endangered germ lines. In mammals, several methods have been adopted for the eradication of GCs such as treatment with cytotoxic drugs, irradiation, cold ischaemia and hyperthermic treatment. Some of these methods have also been tried in fishes, and conditions for sterilization of gonads have been established. Here, we report the production of GC-depleted male striped catfish Pangasianodon hypophthalmus in 12 weeks by the combination of heat and chemical treatments. The cytotoxic drug busulphan (40 mg/kg) was intraperitoneally injected into the animals at 2-week intervals (six doses in total) and maintained in water at 38°C between weeks 1 and 12. The effectiveness of the treatment was assessed using gonadal index and histology. At the end of 12 weeks, very severe gonadal degeneration was observed in fish treated with the heat-chemical combination, and 100% of sampled fish (n = 5) were found devoid of endogenous GCs. On contrary, high temperature alone caused minor gonadal degeneration. Results obtained in this study suggest that endogenous GCs of large-bodied fish such as P. hypophthalmus can also be sterilized by heat and chemical treatments within a considerably short period.

Generation of viable progeny from dead brooders of endangered catfish Clarias magur (Hamilton, 1822).

The obligatory air-breathing catfish Clarias magur is a prime candidate for aquaculture owing to its unique taste, high growth rate, and hardy nature. However, recently the IUCN has listed the species under the endangered category because the population has critically declined in the wild. The sexually mature C. magur brooders are often collected from their natural habitats for seed production in captivity. In many cases, the brooder dies due to handling injuries or confinement stress. In this study, we demonstrated that viable progeny could be generated from freshly dead sexually mature C. magur. Three hours after death, the gonads were excised, macroscopically examined and gamete viability was evaluated. Artificial fertilization was performed by mixing the sperm suspension with the eggs. Water was added after 1 min of mixing to activate the fertilization process. We observed 85%-93% fertilization success from gametes derived from dead donors as opposed to 90%-95% from those derived from live control donors. The embryos showed normal development and resulted in the generation of 88%-92% viable progeny, which was similar to the progeny derived from control donors (92%-93%). The results obtained in this study will have profound implications in enhancing the seed production of endangered C. magur and could potentially be applied to other key commercially or endangered fish species.

Heat and chemical treatments in adult Cyprinus carpio (Pisces cypriniformes) rapidly produce sterile gonads.

Several options have been proposed for eradication of germ cells (GCs) in mammals such as treatment with cytotoxic drugs, irradiation, cold ischemia and hyperthermic treatment. Some of these methods have been also tried in fish but conditions for complete sterilisation of gonads have not been established. Here, we report the production of sterile adult common carp Cyprinus carpio in 10 weeks by the heat and chemical treatments. The cytotoxic drug busulfan (40mg/kg) was intraperitoneally injected into the animals at 2-week intervals (5 doses in total), and they were maintained in water at 38°C between Weeks 1 and 10. The effectiveness of the treatments was assessed using gonadal index, histology, and vasa gene expression. At the end of Week 10, very severe gonadal degeneration was observed in fish treated with the heat-chemical combination, and 100% of male and female fish were devoid of endogenous GCs. The average levels of vasa transcript were 0.01±0.005 and 0.02±0.016 for males and females, respectively. By contrast, high temperature alone caused minor gonadal degeneration and the gene transcript were 0.59±0.131 for male and 0.62±0.13 for female. In Week 20, after the recovery period of 10 weeks at 25°C, the gonadal germ cell did not recover from the sterile condition in any of the sampled individuals. The change in colouration of gonads was an additional useful index of the degree of gonadal sterility.

Effects of ultrasound on permeation of cryoprotectants into Japanese whiting Sillago japonica embryos.

Cryopreservation of fish embryos requires the swift uptake of considerable amounts of cryoprotectant (CPA) but this process is hampered by the low permeability of the egg chorion. This study examined the relative efficiency of ultrasound to promote the incorporation of CPAs in two different embryonic developmental stages (somites and tail elongation) of Japanese whiting Sillago japonica and performed a preliminary cryopreservation trial using the best conditions determined during the study. Embryos tolerated ultrasound densities up to 37.5 W/cm2 well for up to 3 min but had significant mortality at 50 W/cm2. Hatching rates of somites embryos sonicated at 37.5 W/cm2 for 1-3 min in 10 and 20% Me2SO solutions were comparable (61-72%) to that of sonication in artificial seawater (65-86%) but decreased sharply at the concentration of 30% (0-55%); at similar conditions, tail elongation embryos had comparatively lower survival. Me2SO content of sonicated embryos at the somites and tail elongation stages increased significantly by 58-191% and 27-123%, respectively, compared to controls exposed to Me2SO without ultrasound. Pre-exposure to Me2SO before sonication increased the CPA uptake further by 36% without impairing survival. A preliminary cryopreservation trial after ultrasound-mediated impregnation of somites embryos with a CPA solution containing 20% PG and 10% MeOH did not yield live embryos after freeze-thawing but resulted in a significant decrease of nucleation temperature and increase of the proportion of morphologically intact embryos after freeze-thawing. These results suggest that sonication might be useful for fish embryo cryopreservation although it may require combination with other techniques to enhance CPA permeation.

Surrogate production of eggs and sperm by intrapapillary transplantation of germ cells in cytoablated adult fish.

Germ cell transplantation (GCT) is a promising assisted reproductive technology for the conservation and propagation of endangered and valuable genetic resources. In teleost fish, GCT in adult gonads has been achieved only in male recipients, limiting greatly the usefulness of this technique in situations where both sexes need equal and timely attention for conservation and/or propagation. Here we describe a simplified GCT approach that ultimately leads to production of donor-derived eggs and sperm in considerably short time. Donor germ cells isolated from young pejerrey Odontesthes bonariensis (Atherinopsidae) were transplanted non-surgically through the genital papilla into the sexually mature gonads of Patagonian pejerrey O. hatcheri recipients whose gonads have been depleted of endogenous GCs by heat (26°C) and chemical treatment (four doses of Busulfan at 30 mg/kg and 40 mg/kg for females and males, respectively). Transplanted spermatogonial and oogonial cells were able to recolonize the recipients' gonads and produce functional donor origin eggs and sperm within 7 months from the GCT. We confirmed the presence of donor-derived gametes by PCR in 17% and 5% of the surrogate O. hatcheri fathers and mothers, respectively. The crosses between surrogate fathers and O. bonariensis mothers yielded 12.6-39.7% pure O. bonariensis and that between a surrogate mother and an O. bonariensis father yielded 52.2% pure O. bonariensis offspring. Our findings confirm that transplantation of germ cells into sexually competent adult fish by non-surgical methods allows the production of functional donor-derived eggs and sperm in a considerably short time. The methods described here could play a vital role in conservation and rapid propagation of endangered fish genetic resources.

Suitability of cryoprotectants and impregnation protocols for embryos of Japanese whiting Sillago japonica.

The purpose of this study was to examine the suitability of cryoprotectant agent (CPA) impregnation protocols for the embryos of Japanese whiting (Sillago japonica), a small-sized, easy-to-rear, and prolific marine fish which may constitute a suitable experimental material for the development of cryopreservation methods for fish embryos. Our immediate goals were to assess the toxicity and permeability of various CPAs to whiting embryos of different developmental stages. Exposure of gastrula, somites, tail elongation, and pre-hatching embryos to 10%, 15%, and 20% solutions of propylene glycol (PG), methanol (MeOH), dimethyl sulfoxide (Me2SO), dimethylformamide (DFA), ethylene glycol (EG), and glycerol (Gly) in artificial sea water (ASW; 33 psu) for 20 min revealed that CPA toxicity for whiting embryos increased in the order of PG