Indigenous technical knowledge for control of insect pest and livestock disorders

463-465The paper outlines the salient feature of traditional belief in relation to agricultural practices as well as for livestock management. The study carried out in VR Palem village of Ponnuru mandal, Andhra Pradesh revealed that the villagers use indigenous knowledge, including treatment of some of the human disease. So far as, technical knowledge in agricultural practice in the village is concerned, the insect and pest control in some of the commercially important plant like coconut tree is found to be important one. Beside, it has been also found that there exists some of the important traditional knowledge for healing the fractured bones and ligaments in livestock as well as remedies for various stomach related problems

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

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Not AvailableThe current practice of captive breeding of Clarias magur involves killing male fish to obtain sperm for fertilisation. The sperm cannot be obtained by hand stripping because of the extremely low volume of seminal fluid in the testis. This sacrificial method of seed production has been applied to most catfish species. In this study, we reported an alternative approach that allowed harvesting of C. magur spermatozoa for artificial fertilisation without sacrificing male fish. Sexually mature C. magur males were anaesthetised and placed on the surgery table. A midline incision of 2.5 cm was made on the abdomen, and the testicular lobes were carefully lifted from the coelomic cavity. Using sterilised scissors and forceps, the testicular tissue was partially harvested, weighed to nearest 0.01 g, and macerated using a mortar and pestle in physiological saline. The abdominal incision was stitched using a surgical thread and topically treated with antimycotic ointment; subsequently, the fish were resuscitated in clean oxygenated water. Using the surgical technique, we harvested a maximum of 283 mg of testicular tissue, which corresponded to a count of 9.0 × 108 spermatozoa/mL, from a male fish weighing 210 g. By using the sacrificial approach, a maximum of 500 mg of testicular tissue, corresponding to a count of 16.0 × 108 spermatozoa/mL, was recovered from a male fish weighing 220 g. The surgically obtained spermatozoa were used to inseminate C. magur eggs; the cross resulted in healthy spawn with a fertilisation rate of 80%–98%. The results were similar to those obtained using the conventional sacrificial approach, which indicated the viability of partial surgical harvest of testicular tissue in seed production in C. magur for aquaculture without sacrificing male fish.This work was supported by a Grant-in-Aid from the Indian Council of Agricultural Research, New Delhi under CRP-Agrobiodiversity program

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Not AvailableInduced 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.Financial support received from the Department of Biotechnology (BT/PR21355/AAQ/3/832/2016), New Delhi and Indian Council of Agricultural Research, New Delh

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Not AvailableSeveral 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 (40 mg/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.Not Availabl

Stem cell therapy in fishes

Not AvailableStem cell research has come into prominence because of its applications in assisted reproductive technology and the treatment of deadly diseases. In teleost fishes, spermatogonial stem cells have been effectively used to produce surrogate gametes and progeny through germ cell transplantation technique. The present study is the first report of an innovative application of stem cell therapy in fish species for revitalising the reproductive competence of senescent individuals. Senescent male goldfish, Carassius auratus, approximately 10 years of age were procured from a fish-breeding farm and were reared locally in the lab for an additional two years. The senescence of the individuals was then evaluated and confirmed using histological analysis, gonadal index assessment, and germ-cell specific vasa gene expression. Analyses revealed absence of spermatogonial cells and other germ cells in the testes of the senescent fish (n = 5). Spermatogonial cells from sexually immature C. auratus male donor were isolated using discontinuous percoll gradients, labelled with the fluorescent dye PKH-26, and transplanted into the gonads of senescent C. auratus males through urogenital papilla. Six months after the transplant, spermatozoa were collected through applying gentle manual pressure on the abdomen and were observed under a microscope. All C. auratus males with the transplant had produced spermatozoa from the transplanted cells. This was confirmed by the retention of PKH-26 in the spermatozoa and diagnostic SSR locus. Gravid C. auratus females were artificially inseminated with the spermatozoa of those senescent males and natural spawning was allowed. As a result viable progeny were produced. These observation suggests that the reproductive competence of senescent male fishes can be revitalised through spermatogonial stem cell therapy to produce functional gametes.This work was supported by a Grant-in-Aid from the National Bureau of Fish Genetic Resources (Institutional research grant # IXX10896)

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\u27 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

Surrogate Production of Eggs and Sperm by Intrapapillary Transplantation of Germ Cells in Cytoablated Adult Fish

<div><p>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 <i>Odontesthes bonariensis</i> (Atherinopsidae) were transplanted non-surgically through the genital papilla into the sexually mature gonads of Patagonian pejerrey <i>O. hatcheri</i> 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 <i>O. hatcheri</i> fathers and mothers, respectively. The crosses between surrogate fathers and <i>O. bonariensis</i> mothers yielded 12.6–39.7% pure <i>O. bonariensis</i> and that between a surrogate mother and an <i>O. bonariensis</i> father yielded 52.2% pure <i>O. bonariensis</i> 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.</p></div