PG Thesis

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Invitro fertilization of bovine oocytes using fresh, frozen and epididymal spermatozoa
(Department of Animal Reproduction, Gynaecology and Obstetrics, College of Veterinary and Animal Sciences, Mannuthy, 2009) Binoy, V S; Vijayakumaran, V
The study was designed to assess the fertilizability of bovine oocytes matured in vitro on co-culture with fresh, frozen or epididymal spermatozoa. The yield of oocytes and effect of cumulus oocyte complex (COC) morphology on in vitro maturation (IVM) and in vitro fertilization (IVF) was also studied. A total of eighty one ovaries of abattoir origin from South Indian breeds like Hallikar, Kangayam, Khillari and crossbred cattle of Kerala were subjected to oocyte retrieval by aspiration method. Grade I (462) and grade II (138) COCs so obtained were subjected to maturation in separate groups for 24 h in Hepes modified TCM-199 enriched with sodium pyruvate, sodium bicarbonate, antibiotics, estradiol-17β, FSH, hCG and 20 per cent heat inactivated day zero estrus cow serum. Culture environment was set as 39o C temperature, five per cent CO2 tension and maximum humidity in a standard CO2 incubator. Maturation status of COCs was assessed by observing cumulus expansion and mucification. Oocytes with maximum degree of cumulus expansion from each group (407 and 95) were subjected to IVF using fresh (n=169), frozen (n=162) and epididymal (n=171) spermatozoa in Fert-TALP medium supplemented with epinephrine, hypotaurine, pencillamine and heparin (5-10 oocytes in 100 μl droplet). Good quality spermatozoa isolated by percoll density gradient separation technique were used for IVF. Culture conditions set for IVF were 39o C temperature, five per cent CO2 tension with maximum humidity in a standard water jacketed CO2 incubator. After 24 h co-culture in fertilization medium, the oocytes were evaluated for evidence of sperm penetration like presence of swollen decondensed sperm head, male pronuclei, two pronuclei, a clear second polar body and cleavage The total yield of oocytes by aspiration method per ovary was 11.59±0.10 (939 /81) and percentage yield of grade I, grade II and total culture grade oocytes were 49.20±0.31 (462), 14.70±0.41 (138) and 63.90±0.22 (600) respectively. Mean number of grade I, grade II and culture grade oocyte per ovary were 5.70±0.06, 1.70±0.05 and 7.41±0.07 respectively. The percentage and yield of grade I oocytes were significantly higher than grade II oocytes.Cumulus expansion rates of grade I, grade II and total culture grade oocytes were 88.20±0.75 (407), 69.21±1.97 (95) and 83.67±0.35 (502) per cent respectively. The mean number of oocytes showing cumulus expansion per ovary from grade I, grade II and culture grade COCs were respectively 5.04±0.06, 1.18±0.03 and 6.22±0.06. Grade I oocytes showed significantly higher maturation rate and mean yield of matured oocyte per ovary than grade II oocytes. The fertilization rates obtained with fresh, frozen and epididymal spermatozoa were respectively 36.52±1.68, 28.65±0.76, 46.53±1.32 for grade I; 45.00±5.63, 23.89±3.03, 35.20±4.62 for grade II and 37.86±0.47, 27.72±0.89, 44.51±0.57 per cent for culture grade oocytes. The mean number of oocytes fertilized per ovary by fresh, frozen and epididymal spermatozoa were respectively 1.87±0.06, 1.43±0.04, 2.33± 0.05 for grade I; 0.51±0.07, 0.31±0.05, 0.39±0.06 for grade II and 2.38±0.05, 1.73±0.07, 2.72±0.03 for culture grade oocytes. Significant difference was observed between three sources of spermatozoa for grade I and culture grade oocytes on fertilization rate and mean yield of fertilized oocytes per ovary. No significant difference could be observed between three sources of spermatozoa with respect to fertilizability when grade II oocytes were used.There was no significant difference between grade I and grade II oocytes on fertilization rate of fresh, frozen and cauda epididymal sources of spermatozoa. But the mean number of fertilized oocytes per ovary obtained from grade I oocytes was significantly higher than that from grade II oocytes for fresh, frozen and cauda epididymal sources of spermatozoa. The overall fertilization rate obtained was 36.70±1.71 per cent and in vitro fertilized oocyte per ovary was 2.28±0.10 in the present study. The mean motility (per cent), concentration (x 106 /ml), percentage of live sperms, dead sperms, normal spermatozoa, abnormal heads, abnormal tails, proximal protoplasmic droplet and distal protoplasmic droplet of epididymal semen were 49.17±9.26, 37175±7612 , 84.5±8.02, 15.5±8.02, 35.67±2.30, 3.17±1.58, 2.33±0.61, 11.67±4.01 and 47.17±3.17 respectively. The present study revealed that more number of grade I oocytes could be obtained by aspiration method from cow’s ovaries than grade II oocytes. Even though COC morphology has a significant role in maturation rate of oocytes, the fertilizing ability of grade I and grade II oocytes did not differ significantly after proper maturation. Epididymal spermatozoa retrieved from bulls after slaughter could be efficiently used for IVF of in vitro matured bovine oocytes equally or even better than the freshly ejaculated or frozen semen. Epididymal spermatozoa showed significantly more fertilization rate than fresh semen and this was closely followed by frozen semen (44.51±0.57, 37.86±0.47 and 27.72±0.89 per cent respectively).
Studies on in vitro maturation of porcine follicular oocytes
(Department of Animal Reproduction, Gynaecology and Obstetrics, College of Veterinary and Animal Sciences, Mannuthy, 2007) Deepa, S; Vijayakumaran, V
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.
Effect of ovum retrieval methods and cumulus-oocyte complex morphology on in vitro maturation of bovine oocytes
(Department of Animal Reproduction, College of Veterinary and Animal Sciences, Mannuthy, 2005) Magnus Paul, K; Sreekumaran, T

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