MVSc

This study was designed to evaluate the effect of different retrieval methods like aspiration, slicing and puncture on yield of different grades of oocytes and their in vitro maturation potential. The effect of cumulus oocyte complex morphology on in vitro maturation of bovine oocyte was also studied. Slaughterhouse derived bovine ovaries from South Indian breeds like Kangayam, Khillari, Hallikar and crossbred cattle of Kerala were subjected to three retrieval methods to yield different quality grades of oocytes. In this study, a total of one hundred and fifty ovaries were processed. Fifty ovaries were processed under each method of retrieval. Each quality grade of oocyte obtained through three retrieval method were subjected to maturation for 24 h in TCM-199 medium supplemented with LH, FSH, Estrogen, Pyruvate and Foetal calf serum. Culture environment was set as 38.5°C temperature, 5% carbondioxide tension and maximum humidity in standard CO2 incubator. Maturation changes were assessed by cumulus expansion, formation of M II plates and polar body extrusion.
Total yield of COCs in slicing was 5.42 ± 0.56, which was significantly higher than aspiration and puncture which yielded 3.7 ± 0.43 and 3.66 ± 0.43 COCs respectively. This could be due to recovery of oocytes from a heterogeneous population of follicles in slicing method. Aspiration, slicing and puncture yielded A class oocytes at the rate of 44.86%, 37.2% and 43.17% respectively, wherein mean yield per ovary in same methods were 1.66 ± 0.25, 2.02 ± 0.25 and 1.58 ± 0.25 respectively. Yield of B class oocytes by aspiration, slicing and puncture were 32.97%, 29.89% and 31.69% respectively. Mean yield of B class oocytes per ovary was 1.22 ± 0.12, 1.62 ± 0.20 and 1.16 ± 0.16 respectively. Proportionate yield of D class oocytes by aspiration, slicing and puncture was 3.7%, 12.10% and 6.01% respectively. Aspiration, slicing and puncture did not differ significantly in yield of A, B and C class oocytes. But D class oocytes were proportionately more in slicing method. This finding could be attributed to the fact that the slicing recovered oocytes from a heterogeneous population of follicles, which included all kinds of growing follicles. When percentage yield of different classes of oocytes in same retrieval method was compared aspiration yielded A and B class oocytes in significantly higher proportion than C and D class oocytes. The same trend was observed in slicing and puncturing. In all the methods, the yield of D class oocytes was least.
Cumulus expansion rate of A class oocytes were 83.08 per cent, 69.70 per cent and 70.37 per cent in aspiration, slicing and puncture method respectively. B class oocytes exhibited cumulus expansion rate of 68.29 per cent, 53.0 per cent and 62.07 per cent in aspiration, slicing and puncture method respectively. Retrieval method was found to have no significant effect on cumulus expansion potential of bovine COCs. Oocytes with more than three layers of cumulus cells exhibited better cumulus expansion rate than oocytes with less cumulus cell layers.
Nuclear maturation rate of A class oocytes in aspiration, slicing and puncture was 81.08, 78.84 and 80.0% respectively, whereas in B class oocytes under same methods the percentage of nuclear maturation was 57.10, 41.67 and 52.63% respectively. D class oocytes failed to mature in all methods of retrieval except one oocytes in aspiration method. Aspiration, slicing and puncture did not differ significantly in the maturation percentage of oocytes. Polar body extrusion rate of A class oocytes in aspiration, slicing and puncture method was 45.5 per cent, 42.1 per cent and 44.0 per cent respectively. B class oocytes exhibited polar body extrusion rates of 28.6 per cent, 25.0 per cent and 26.3 per cent respectively in aspiration, slicing and puncture method respectively. Oocytes with more than three layers of cumulus cells exhibited better polar body extrusion rate irrespective of their retrieval method. Staining of 18 oocytes with fluorescent dye Hoechst 33342 as a method of cross checking, revealed 66.67 per cent Metaphase II rate. Ten oocytes tested for viability with FDA stain revealed 80 per cent viability after maturation. TCM-199 medium with 5 µg FSH/ml, 0.5 µg LH/ml, 1 µl Estradiol/ml, 0.2 mM pyruvate and 10 per cent serum was found to be ideal medium for maturation of bovine oocytes in vitro.. Oocytes with more than three layers of cumulus cells matured better than oocytes with less number of cumulus cell layers or denuded oocytes. These experiments proved that cumulus oocytes complex morphology have a very significant role in maturation of oocytes rather than retrieval methods.