MVSc

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).