The grading of follicles based on the dimensions of buffalo ovarian follicles in group I are depicted in Table 1. In group I where oocytes were subjected to fertilize with conventional non sorted semen, out of 155 ovaries 228 small follicles, 114 medium follicles and 75 large follicles were observed. The overall percentage of small, medium and large follicles in the ovaries were recorded as 16.29 ± 0.94, 8.14 ± 0.60, 5.35 ± 0.76, respectively as depicted in Table 1. A total, 417 surface follicles were measured using Vernier caliper out of 155 ovaries by categorizing them based on follicular size diameter as small, medium and large respectively.
In the present study, the number of small, medium and large follicles were far more than the findings of
Bhajoni et al., (2018) who reported 4.36 ± 0.31 small, 5.79 ± 0.75 medium and 0.82 ± 0.07 large follicles whereas
Elbaz et al., (2019) reported 0.95 small, 0.57 medium and 0.24 large follicles respectively when follicles of 155 ovaries were measured using vernier calliper. This could be because of the negative effect of CL which was surpassed by the presence of good number of follicles on ovary in present trial.
Aspiration of buffalo follicular oocytes and recovery percentage
The technique used in present study was aspiration of oocytes from the buffalo ovaries obtained from abattoir because it was more practical method than others which enabled quick collection of significant quantity of oocytes within no time
(Katska., 1984, Totey et al., 1992, Mehmood et al., 2011, Rao and Uma Mahesh 2012, Shahid et al., 2014). No significant difference was observed in oocyte maturation, fertilization and development
in vitro among the above mentioned methods of recovering oocytes
(Pawshe et al., 1994). Thus in present study, comparatively practical method was used for recovery of COCs from ovaries.
Number of COCs recovered when oocytes were fertilized using conventional non sorted semen was 409 and mean oocytes recovered per trail were 29.21±1.58. Overall recovery rate of COCs (cumulus oocytes complexes) in conventional non sorted semen
in vitro fertilization trail was 38% and mean oocytes recovery rate per trial was 2.74 ± 0.19% as depicted in Table 2.
Khan et al. (1997) recovered 635 COCs from 192 ovaries;
Nandi et al., (2000) recovered 425 COCs from 457 ovaries;
Jamil et al., (2008) recovered 363 COCs from 298 ovaries of riverine buffaloes;
Hammad et al., (2014) recovered 665 COCs from 206 ovaries of Egyptian buffaloes; and
Elbaz et al., (2019) recovered 335 COCs from 187 ovaries.
In the present study, mean oocyte recovery rate per trail was 2.74±0.19%. These values were in accordance with
Jamil et al., (2008) who reported oocytes recovery rate of 2.45±0.91% during peak breeding season and 1.93 ± 0.68% during low breeding season respectively.
Furthermore,
Nandi et al., (2000) recovered lower oocytes 0.12% whereas Rao and Uma Mahesh, 2012 recorded higher oocytes 4.60 ± 0.33% than the present study.
Usually buffaloes are slaughtered because of unproductiveness and poor fertility
(Selvaraj et al., 2008). The lower oocyte recovery rate in buffaloes may be due to anestrous condition as well as acyclicity and also because of presence of CL
(Totey et al., 1992, Das et al., 1996). This is because follicular development is restricted when luteal cells occupy major part of the ovary
(Kumar et al., 1997). Ovaries which do not possess CL have higher number of follicles being recovered and also higher grade COCs than those non CL bearing ovaries
(Khandoker et al., 2011). Furthermore, the stroma of buffalo ovaries are thick and follicles are deeply embedded in them and therefore aspiration of oocytes from follicles gets difficult
(Selvaraj et al., 2008, Hammad et al., 2014). The ovaries which had CL yielded lower number of oocytes as compared to non CL bearing ovary as it was discussed by
(Kumar et al., 1997). The lower number could be because of greater mechanical damage induced on the granulose cells during the procedure of recovery.
Evaluation of oocytes and subsequent grading of COCs
Oocytes were examined under stereomicroscopy and classified according to their compaction, number of cumulus cell layers and homogeneity of ooplasm
(Ravindranatha et al., 2003) into 5 categories as:
A) Dense and compact multilayered with (≥5) cumulus layers with homogenous ooplasm.
B) Dense and compact multilayered with (3-4) cumulus layers with homogenous ooplasm.
C) Less compact (1-2) cumulus layers with homogenous ooplasm.
D) Denuded cumulus layer with homogenous ooplasm.
E) Denuded cumulus layer with unevenly granulated ooplasm.
Accordance to above criteria the oocytes were classified as A, B, C, D and E
(De loos et al., 1992, Khan et al., 1997, Jamil et al., 2008).
In the present study, in conventional method, grade A oocytes were 5.07 ± 0.51, Grade B were 4.57 ± 0.56, Grade C were 7.21 ± 0.55, Grade D were 5.57 ± 0.66 and Grade E were 7.36 ± 0.83 respectively as depicted in Table 3. The percentage of these oocytes were 16.74% (A), 15.25% (B), 25.26 % (C), 18.33% (D) and 29.87% (E) respectively.
The oocyte quality plays major role in assessment of oocyte development therefore it is essential to grade the oocytes which in this study were correlated with
(Elbaz et al., 2019) who got similar results, grade A oocytes were 5 ± 0.68, Grade B were 6.1 ± 1.1, Grade C were 4.8 ± 0.84 and Grade D were 4.8 ± 0.55 respectively.
Percentage of graded oocytes based on the compactness of cumulus layers in both the groups were in accordance with
Khan et al., (1997) who recorded A (18.42%), B (15.43%), C (19.37%) and D (46.77%) whereas
Samad et al., (1998) recoded A (18.42%), B (15.43%), C (19.57%) and D (46.77%) respectively.
Bhajoni et al., (2018) recorded A (47.58%), B (37.42%), C (8.82%) and D (6.12%) oocytes which were higher than the present study and
Nandi et al., (2000) recorded A (0.10%), B (0.13%) and C (0.11%) oocytes which were lower than the present study.
Maturation Rate of COC’s Subjected for Maturation using Conventional Non Sorted Semen
In the present study, average number of COCs used for IVM in 14 trials of non sorted group was 29.21±1.58. Maturation rate of oocytes were81.96 ± 2.70% in conventional non sorted trail as depicted in table 4. This maturation rate was in accordance with
(Arul et al., 2017, Totey et al., 1992) who got maturation rate as 81.71 ± 1.81% and 81.7 ± 14.5% respectively as depicted in Plate 1.
Maturation rates obtained by
(Kumar and Maurya 2000, Adlak et al., 2007, Leal et al., 2007 and
Elbaz et al., 2019) was 53.35%, 71.05%, 36.68% and 28.82% respectively were lower than the present study. Furthermore maturation rates observed were higher in studies of
(De matos et al., 1995, Umadevi et al., 1998 and
Habeeb et al., 2018) was 90 ± 2 %, 83.33% and 86.5% respectively. This maturation rate was in accordance with
(Suresh et al., 2009) who got maturation rate as 78.36 ± 2.16% respectively.
In conclusion, such variations in the maturation rates could be due to variations in the number of COCs used for IVM, variation in the choice of media used must have influenced maturation rates.
The average number of oocytes used for fertilization in conventional trial was 23.78±1.33 and fertilization rate was 74.98 ± 3.87% as depicted in Table 5.
Fertilization rates were in accordance with
(Nain et al., 2006, Barkawi et al., 2007) who recorded 60.70% and 80.2% of fertilization rates. Furthermore,
Bavister et al., 1992 recoded higher fertilization rates as 85±5% and
(Jamil et al., 2008, Palta and Chauhan 1998 and
Baczkowski et al., 2004) recorded 63.75 ± 2.81%, 40-55% and 57.1% respectively which were lower than present study.
Cleavage rate of potential zygotes in conventional unsorted group
In the present study, average number of potential zygotes used in IVC are 17.92 ±1.43 and the number of average cleaved embryos obtained were 7.35 ± 0.76 in conventional trial whereas cleavage rate % was 40.84±2.51% as depicted in Table 6 which was in accordance with
Chauhan et al., (1998) and
Nandi et al., (2000) who observed cleavage rate of 40%
Cleavage rate was in accordance with
(Nain et al., 2006 and
Aquino et al., 2013) was 40-50% and 40% respectively. Cleavage rates observed
(Madan et al., 1994, Mehmood et al., 2007, Suresh et al., 2009 and
Gonclaves et al., 2013) were higher 54.4%, 75.9%, 52.14 ± 1.15% and 71.8% respectively. Furthermore, cleavage rates observed
(Ocampo et al., 2015, Sadeesh et al., 2016) were lower 39.6%, 22.5±4.2% respectively than present study as depicted in Plate 2.
In the present study, average number of blastocyst formed were 3.92±0.46 and blastocyst percentage was 21.57±1.75% as depicted in Table 7 which was in accordance with
(Neglia et al., 2003) who observed blastocyst % of 19.9 ± 4.2%. Blastocyst % observed was in accordance with
(Ocampo et al., 2016 and
Sadeesh et al., 2016) as 19.6%, 20.4±2.5% respectively. Blastocyst percentage was higher than present study
(Aquino et al., 2013, Ocampo et al., 2015 and
Shabankareh et al., 2015) as 32.50%, 22.5% and 38.7% respectively and lower than present study (Palta and
Chauhan et al., 1998, Boni et al., 1999, Prasad et al., 2013) as 10-15%, 13.5%, 10.65 ± 0.45% respectively in Plate 3.
