A comparative study on parthenogenetic activation and embryo production from in vitro matured caprine oocytes

Abstract

The objective of this study was to compare the effectiveness of different activation treatments of in vitro matured oocytes and their developmental potency in potassium simplex optimization medium. Ovaries were collected from the local abattoir and transported within 4h to the laboratory in warm saline (37°C) containing 100IU penicillin-G and 100μg streptomycin sulphate per ml. A total of 1004 cumulus oocyte complexes (COC's) were collected from 454 ovaries. Oocytes were matured in TCM-199 medium containing FSH (5μg/ml), LH (10μg/ml), oestradiol-17β (1μg/ml) supplemented with 10% fetal bovine serum and 3mg/ml BSA at 38.5°C and 5% CO2 in an incubator under humidified air for 27h. After 27h of IVM, oocytes were denuded, washed and selected 933 in vitro matured oocytes were randomly divided into four groups. Group 1 in vitro matured oocytes (n=579), were exposed to 7% ethanol for 5min followed by treatment with 2.0mM DMAP for 4h in KSOM. Group 2 in vitro matured oocytes (n=145) were exposed to 7% ethanol for 5min followed by treatment with 10μg/ml CHX for 4h in KSOM. Group 3 in vitro matured oocytes (n=100) were exposed to 7% ethanol for 5min followed by treatment with 2.0mM DMAP and 10μg/ml CHX for 4h in KSOM. Group 4 in vitro matured oocytes (n=109) were cultured for 4h without any chemical activation treatment in KSOM medium (control). After 4h of culture in different chemicals, the oocytes were washed five to ten times in the culture medium (KSOM) and cultured in 50μl drops of KSOM. Development of activated oocytes was observed at every 48h till day 10 post activation under an inverted phase contrast microscope (200x, Nikon, Japan). The cleavage rate in groups 1, 2, 3 and 4 were 42.83%, 58.62%, 74.0% and 0.00%, respectively and morula production in groups 1, 2 and 3 were 24.59%, 30.58% and 31.08%, respectively. These results indicated that the activation of in vitro matured oocytes by 7% ethanol for 5min followed by treatment with 2.0mM DMAP and 10μg/ml CHX for 4h in KSOM is most favorable for parthenogenetic caprine embryos production.