Studies on in vitro maturation of porcine follicular oocytes

Abstract

A study was designed and carried out to evaluate the effect of different retrieval methods like aspiration, slicing, puncturing and post aspiration slicing on yield of different grades of oocytes and their in vitro maturation potential. The effect of cumulus oocyte complex morphology and culture duration on in vitro maturation of porcine oocyte was also studied. a total of 40 follicular and 36 luteal stage ovaries obtained from Large White Yorkshire, Landrace and Duroc pigs were used for the study. Each quality grade of oocyte obtained through four retrieval methods was subjected to maturation for 42 h in TCM-199 medium supplemented with LH, FSH, estradiol, pyruvate and foetal calf serum. Culture environment was set as 38.5°C temperature, 5 per cent CO2 and maximum humidity in standard water-jacketed CO2 incubator. Maturation changes were assessed by observing the degree of cumulus expansion at 24h, 36h and 42h of incubation. There was no significant difference in oocyte yield from follicular and luteal stage ovaries even though the surface follicle distribution was different significantly. Slicing, puncturing and post aspiration slicing did not differ significantly in the yield of total number of oocytes per ovary. But the yield from aspiration was significantly lower compared to other methods (7.64 vs.33.83, 33.43, 25.42). The percentage yield of good quality (A and B) oocytes was more from aspiration than slicing, puncturing or post aspiration slicing. The percentage yield of D grade oocyte was more from post aspiration slicing method. The percentage of maturation for aspiration, puncturing, slicing and post aspiration slicing were 68.07, 64.63, 64.17 and 45.56 per cent respectively. It was found that the maturation rate for post aspiration slicing was significantly lower than other methods. The maturation rate for A grade, B grade and C grade oocytes were found to be 66.67, 65.29 and 54.84 per cent respectively. A and B grade oocytes were not significantly different in the maturation rate. At zero hour culture all the oocytes were devoid of any detectable response of maturation. At 24h, 81.33 per cent oocytes showed minimum observable response. At 36 h, 72 per cent oocytes showed expansion limited to the outer layer of cumulus cells. At 42h, about 62.28 per cent COCs showed maximum degree of cumulus expansion. The over all cumulus expansion rates was found to be 64 per cent and nuclear maturation rate was 62 per cent. Slicing and puncturing were found to be good methods for the collection of oocytes from porcine ovaries. These two methods yielded higher number good quality oocytes with satisfactory level of maturation rate. Among A, B and C grade oocytes A and B grade oocytes showed a higher maturation rate indicating that the oocytes with more than three layer of cumulus cells are better for in vitro maturation. For the completion maturation process in porcine oocytes a minimum period of 42h was found essential.