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Kharche S.D.,ICAR CIRG | Pathak J.,ICAR CIRG | Agarwal S.,ICAR CIRG | Kushwah B.,ICAR CIRG | Sikarwar A.K.S.,ICAR CIRG
Reproduction in Domestic Animals | Year: 2016

The aim of the present investigation was to study the effect of calcium ionophore activation on blastocyst production following intracytoplasmic sperm injection (ICSI) in in vitro-matured Caprine oocytes. A total of 470 in vitro-matured oocytes were selected and randomly divided in to three groups. Cumulus oocyte complexes (COCs) recovered by slicing the Caprine ovaries were matured in TCM199 supplemented with 10% foetal bovine serum (FBS) + 10% follicular fluid + FSH (5 μg/ml) + LH (10 μg/ml) + estradiol (1 μg/ml) + EGF (10 ng/ml) + BSA (3 mg/ml) for 27 h in humidified atmosphere at 38.5°C with 5% CO2 in CO2 incubator. After 27 h of culture, selected COCs (n = 470) were separated from cumulus cells by treating with 0.1% hyaluronidase enzyme and passing repeatedly through a fine pipette and randomly divided into three groups. In group 1, (n = 168) matured oocytes were injected with injection micropipette without sperm as control. In group 2, (n = 152) capacitated spermatozoa were injected into cytoplasm of in vitro-matured oocytes through injection micropipette. In group 3, (n = 150) capacitated spermatozoa were injected into cytoplasm of in vitro-matured oocytes through injection micropipette and then activated with 5 μm Ca ionophore for 5 min. The oocytes of all groups were then culture in RVCL media for embryo development. The cleavage rate was observed after 48–72 h of injection. The cleavage rate and blastocyst production in group 1, 2 and 3 were 0.00 and 0.00, 18.42 and 3.57 and 61.33% and 16.30%, respectively. The result indicated that mechanical activation failed to induce cleavage in in vitro-matured Caprine oocytes, whereas chemical activation of intracytoplasmic sperm-injected in vitro-matured Caprine oocytes showed significantly higher cleavage rate and blastocyst production as compare to non-activated oocytes. © 2016 Blackwell Verlag GmbH Source

Gangwar C.,ICAR CIRG | Kharche S.D.,ICAR CIRG | Ranjan R.,ICAR CIRG | Kumar S.,ICAR CIRG | And 3 more authors.
Small Ruminant Research | Year: 2015

Thirty six ejaculates from 6 adult Barbari bucks (2-4 years old) maintained at C.I.R.G under semi intensive management system were used to find out the freezability of buck semen at different levels of vitamin C (0.0. μM, 45.42. μM, 56.78. μM, 68.13. μM) by conventional method of freezing. The ejaculates were collected twice at weekly intervals by artificial vagina. The semen samples were diluted with tris-citric acid fructose diluents having 10% (v/v) egg yolk and 6% (v/v) glycerol as cry protectant agent. The semen samples were extended to maintain sperm concentration approximately 100-120 million per dose. Filling and sealing of straws were done at 5. °C in cold handling cabinet after 4. h of equilibration period, then straws were vapour frozen for 10. min above 2. cm of liquid nitrogen and finally stored into liquid nitrogen container. Post thaw motility, live sperm count, abnormalities, acrosomal integrity and hypo osmotic swelling test were conducted to check the freezability. Post thaw motility, live sperm count, acrosomal intergrity and hypo osmotic swelling positive spermatozoa differed significantly among groups and they were the highest in 1% group. The result indicated that vitamin C at the level of 56.78. μM can be used as an antioxidant in semen diluter in routine freezing process for better post thaw recovery of buck semen. © 2015 Elsevier B.V. Source

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