Indian Journal of Animal Research

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Full Research Article

Effect of Vitrification on Mature Buffalo Oocytes and Their Fertilization Rates using Different Concentrations of Cryoprotectants

B. Srilatha1,*, K. Ramchandra Reddy2, Sathya Velmurugan3, N. Nalini Kumari4, T. Raghunandhan5
1Department of Veterinary Gynaecology and Obstetrics, College of Veterinary Science, Garividi-535 101, Andhra Pradesh, India.
2Department of Veterinary Gynaecology and Obstetrics, College of Veterinary Science, Korutla-505 326, Telangana, India.
3National Institute of Animal Biotechnology, Hyderabad-500 032, Telangana, India.
4Department of Animal Nutrition, College of Veterinary Science, Hyderabad-500 032, Telangana, India.
5PV. Narasimha Rao Telangana Veterinary University, Hyderabad- 500 032, Telangana, India.

ABSTRACT

Background: Buffalo is an integral part of livestock agriculture in Asia since many centuries, because they provide draught power, milk, meat and hide to millions of people, particularly small-scale farmers. In addition, female buffaloes have few primordial follicles and a higher rate of follicular atresia. Therefore, the emphasis has now shifted to in vitro embryo production (IVEP). Oocyte cryopreservation expands the potential of IVF (in vitro fertilization). Cryopreservation of oocytes by vitrification, especially with the use of very high cooling rates for oocytes suspended in extremely small volumes of various additives, seems the most appropriate method. The present investigation was aimed to test the efficacy of different concentrations of cryoprotectants like EG (Ethylene glycol), DMSO (Dimethyl sulphoxide), their combinations and their effect on mature oocytes and IVF of vitrified mature buffalo oocytes and their comparison with the control/non-vitrified oocytes.

Methods: A total of 2,127 matured oocytes were used in the present study which was undertaken during the year 2015-17. For the process of vitrification, different concentrations of EG (10, 20 and 30%), DMSO (10, 20 and 30%) and EG+DMSO (15% and 20%) in eight treatments were used.

Result: Highest morphological survival rate was noticed in 30% EG (90.55±0.79) while lowest was seen in 10% DMSO (77.56±1.59 per cent). The proportion of damage was significantly higher in 10% DMSO (22.44±1.59 per cent) than 20% (14.40±2.93 per cent), 30% EG (9.44±0.79 per cent) and 20% EG+20% DMSO (10.60±2.19 per cent). The cleavage rate was significantly (P≤0.05) higher in 30% EG (41.65±0.53 per cent), 20% EG+20% DMSO (43.29±0.65 per cent) and 30% DMSO (39.75±0.69 per cent) than 20% EG (30.95±0.81 per cent), 10% EG (27.71±0.56 per cent), 20% DMSO (33.22±0.52 per cent), 10% DMSO (25.38±.60 per cent) and 15% EG+15% DMSO (38.01±1.24 per cent).

INTRODUCTION

Cryopreservation of oocytes is difficult in most animal species because of their large volume, high sensitivity to cooling, low surface area to volume ratio, high water content and low hydraulic conductivity (Leibo, 1980). Cryobiology in reproduction has revolutionized applications to preserve the genetic material of rare breeds, endangered species or transgenic animals (Dua et al., 2021). Various protocols for cryopreservation involved slow cooling, rapid cooling and ultra rapid cooling. In the past decade, various new methods for embryo cryopreservation have been published (Niemann, 1991; Rall, 1992). Among these methods, vitrification have been widely used and is now regarded as a potential alternative to traditional slow-rate freezing. Vitrification which is defined as a physical process by which a highly concentrated solution of cryoprotectants solidifies during cooling, without formation of ice crystals (Niemann, 1991). The success of vitrification was determined by subjecting vitrified-warmed oocytes to IVF and comparing fertilization and subsequent embryo development to those of fresh oocytes. Laboratory production of embryos allows mass scale production of embryos for practical and scientific purposes as well as for the exploitation of oocytes from donors (Pitroda et al., 2024).

MATERIALS AND METHODS

A series of experiments were carried out to standardize the best concentration and combination of cryoprotectants. Various concentrations and combinations of cryoprotectants using EG and DMSO for cryopreservation of mature oocytes and their effect on IVF were studied.

Group I (n=2,127): Cryopreservation of mature oocytes following in vitro maturation.

Treatment I: 10% EG (n=266/5) Treatment II: 20% EG (n=265/5) Treatment III: 30% EG (n=266/5) Treatment IV: 10% DMSO (n=265/5) Treatment V: 20% DMSO (n=266/5) Treatment VI: 30% DMSO (n=267/5) Treatment VII: 15% EG+15% DMSO (n=266/5) Treatment VIII: 20% EG+20% DMSO (n=266/5.

Group II (n=261/5): Control (fresh oocytes) without cryopreservation.
*n=Number of oocytes/no. of replicates.  All the experimental groups were subjected to IVF.

Vitrification of mature buffalo oocytes

Vitrification was done as per the standard protocol (Dutta et al., 2013). The mature oocytes were exposed to vitrification solution 1 (half of the original concentration i.e 5, 10, 15 per cent EG; 5, 10, 15 per cent DMSO and 7.5, 10 per cent EG+DMSO) for equilibration upto 3 minutes followed by 25-30 seconds in vitrification solution II (original concentration i.e 10, 20, 30 percent EG; 10, 20, 30 per cent DMSO and 15, 20 per cent EG+DMSO)  at room temperature (22-25°C). The oocytes in vitrification solution II were immediately loaded into hemi-straw and plunged into liquid nitrogen (LN2). Straws were stored for a period of 7 days.

Hemi-straw (HS) method

It was carried out according to the method used by Vandervorst et al. (2001) with slight modifications. The open end of a 0.25 ml straw (6.5 cm) was cut with a surgical blade, so that the end of the straw was open (about 1 cm in length) and it was easy to pipette a small droplet (<1.0 µ diameter) onto the open inner surface of the straw. The 0.25 ml straw with the cut end was inserted into a 5 cm length of 0.5 ml straw. The cut end of the straw (0.25 ml) was pressed with an artery forceps to become flat at 1.5 cm below the cotton plug to avoid the slipping from the covering (0.5 ml straw). Prior to loading, the COC’s were exposed to vitrification solutions. After placing a drop (0.75 µl) of VS II with COC’s into the inner surface of the cut end of the straw, the HS was directly dipped into the LN2. Then the 0.25 ml straw was pulled back (1.5 cm) to cover the open end of the straw with 0.5 ml straw. Similarly, for warming the 0.25 ml straw was pushed 1.5 cm into the 0.5 ml straw in sucrose solution, so that the cut end of the straw comes out of the covering.

Thawing/warming of vitrified oocytes

After 7 d, the straws were warmed and the oocytes were rehydrated in serial dilutions of sucrose (0.3 M and 0.15 M in holding medium) each for 1 minute and washed in holding medium (filtered Dulbecco’s phosphate buffered saline with 20% estrus buffalo serum, 0.4% bovine serum albumin and 50 µg/ml of gentamicin) at the end.

Assessment of oocyte survival

The survival of oocytes post-thaw and embryos after IVF was evaluated by morphological examination. Oocytes with homogenous ooplasm, intact membranes and zona pellucida post-thaw were recorded as living oocytes (Men et al., 2002) which were observed under stereo zoom microscope.

In vitro fertilization (IVF)

Two frozen thawed murrah buffalo semen straws were used for each experiment.

Preparation of motile sperm

By swim-up method described by Parrish et al. (1995) was used to separate the motile fraction of sperm. 0.5 ml semen was diluted with 4.5 ml of SOF (Synthetic oviductal fluid) and centrifuged at 200g for 5 min. Then the supernatant was removed and the sperm pellet was resuspended with SOF and recentrifuged. Lymphocyte tubes containing 1 ml of swim up medium was layered with 0.2 ml of sperm pellet at the bottom. After 1 h incubation at 39°C at 5% CO2 under humidified air, the top most 0.5 to 0.8 ml of medium containing the motile fraction of sperm was removed from each tube and pooled in a sterile centrifuge tube. This sample was centrifuged at 200 g for 5 min. After washing, sperm concentration was assessed by using Neubaeur counting chamber and diluted to a final sperm concentration of 2×106 sperm/ml progressive motile spermatozoa.

In vitro fertilization of oocytes

After maturation, oocytes were denuded off cumulus cells by repeated pipetting in SOF medium and were washed thrice with IVF medium. Subsequently, mature oocytes were selected for IVF and placed in 75 µl droplets (10 oocytes/droplet) of IVF medium (SOF supplemented with 20% (v/v) estrus buffalo serum, 2 mM glutamine, 2% BME- essential amino acids and 1% MEM-non essential amino acids) in 35 mm tissue culture dishes. The droplets were overlaid with pre equilibrated light weight mineral oil. 5µl aliquot of sperm suspension was transferred into each 75 µl fertilization droplet and incubated for 24 hours at 38.5°C in 5% CO2 under humidified atmosphere in air.

In vitro culture following fertilization (Day 0)

Oocytes were washed with SOF and cultured in SOF for another 6 days at 38.5°C in 5% CO2 under humidified atmosphere in air. During culture, the embryos were transferred to fresh SOF medium every 48 hours.

Evaluation of embryos

The cleavage was assessed at 24 and 48 hours post insemination and the number of embryos at each stage was recorded under stereo zoom microscope.

Statistical analysis

Fertilization rates of vitrified oocytes were analyzed statistically and compared with the non-vitrified oocytes (control). Comparisons of fertilization rates of cryopreserved mature and control groups were analyzed by one-way analysis of variance (ANOVA) using software (SPSS, 2005, Version 16).

RESULTS AND DISCUSSION

A total of 2400 immature oocytes were taken for maturation. Out of which, 2,127 matured oocytes were vitrified using different concentrations and combinations of EG and DMSO. Various post-thaw/warming oocyte quality parameters (Fig 1 Table 1) for different concentrations of cryoprotectants were observed.

Fig 1: Post warming quality parameters.


Table 1: Effect of vitrification on maturation status of in vitro matured oocytes using different concentrations of cryoprotectants.



Post-thaw recovery rate

The post-thaw recovery rate in the present study ranged between 92.19±0.73 to 95.31±1.51 per cent. These were in agreement to the findings of Sharma and Loganthasamy (2007) (89-92%), Dolakasaria et al. (2013) (91.06%), Loganathasamy and Sharma (2014) (89-92%) using French mini straw and higher than Mishra et al. (2012) (81.35%) using straw. The post thaw recovery rate was noticed immediately after warming/thawing the oocytes which were placed in hemi-straw in serial dilutions of sucrose. The recovery rate of oocytes for different concentrations of cryoprotectants were placed in Table 1. There was no significant difference in post-thaw recovery rate among different treatment groups. The recovery rate of vitrified thawed oocytes has been reported to vary from 80-100% in different species (Fuku et al., 1992 and Nowshari et al., 1994) which was in agreement with the present study. The loss of oocytes oocur due to sticking of oocytes on inner wall of straw adhering to rough surface or oocyte disintegration due to improper vitrification. Furthermore, it was demonstrated that warming is also a critical step of procedure and that the efficiency of vitrification may be improved by utilizing higher concentration of sucrose and multistep dilution approach. Step wise dilution might be helpful to reduce osmotic injury of vitrified oocytes after thawing.

Morphological survival rate of vitrified mature oocytes post-thawing

The morphological survival rate of oocytes for different concentrations of cryoprotectants were shown in Table 1. Highest survival rate was noticed in 30% EG (90.55±0.79 per cent) while lowest was seen in 10% DMSO (77.56±1.59 per cent). In the current study, the morphological survival rate in 20% EG+20% DMSO (88.45±1.95 per cent) was in agreement to the findings of Attanasio et al. (2007) (83.6-92.8%) using cryotop, Sharma and Loganathasamy (2007) (92.1%), Gautam et al. (2008) (96%) using straw and higher than findings of Luna et al. (2001) (72.6%), Morato et al. 2008 (60.3%- 60.6%) using cryotop and OPS and Mishra et al. (2012) (74.28%) using straw. The survival rate noticed in 30%EG (90.55±0.79 per cent) was correlated with the findings of Boonkusol et al. (2007) (89.3%), Gasparrini et al. (2007) (95.8%) and Gautam et al. (2008) (85%) using 35% EG and 40% EG, respectively.

Damage of vitrified mature oocytes post-thawing

The proportion of damage was significantly higher in 10% DMSO (22.44±1.59 per cent) than 20% (14.40±2.93 per cent), 30% EG (9.44±.79 per cent) and 20% EG+20% DMSO (10.60±2.19 per cent) (Fig 1). Mishra et al. (2012) reported proportion of morphologically survived and damaged oocytes as 74.28% and 25.71%, respectively using 20% EG+20% DMSO which were lower and higher, respectively than the present study.

Fertilization rate and embryonic development of vitrified mature oocytes

The cleavage/fertilization rate in the present study was significantly higher in 30% EG (41.65±0.53 per cent), 20% EG+20% DMSO (43.29±0.65 per cent) and 30% DMSO (39.75±0.69 per cent) than 20% EG (30.95±0.81 per cent), 10% EG (27.71±0.56 per cent), 20% DMSO (33.22±0.52 per cent), 10% DMSO (25.38±0.60 per cent) and 15% EG+15% DMSO (38.01±1.24 per cent). Among all the treatment groups, cleavage rate was significantly less in 10% DMSO (25.38±0.60 per cent). The cleavage rate in 30% EG (41.65±0.53 per cent) was similar to the findings of Sripunya et al., 2010 (41.65%) using SSV, Ocampo et al., 2014 (40.0%) using 40% EG. In the present study, the cleavage rate in 20 % EG +20% DMSO (43.29±0.65 per cent) was correlated with Attanasio et al., 2007 (36.3-55.3%), Morato et al., 2008 (31.5-46.1%). In the current study, the cleavage rate in 15% EG+15% DMSO (38.01±1.24 per cent) was correlated with the reports of Zhou et al., 2010 (35.2%). After culturing, different stages of embryonic development Fig 2 like cleaved oocytes, 2 cell, 4 cell, 8 cell and morula stage embryos were shown in Table 2.

Fig 2: In vitro embryonic development of oocytes in vitrified group.


Table 2: Effect of vitrification on in vitro fertilization and embryonic development of vitrified mature oocytes using different concentrations of cryoprotectants.



Fertilization rate and embryonic development of buffalo oocytes in control (Non-Vitrified) group

The fertilization rate (50.34±0.82 per cent) recorded in this group was like observations made by Madan et al., 1994 (54.4%), Samad et al., 1998 (55.38%), Hegab et al. (2009) (50%) using TALP medium, Sadeesh et al. (2014) (44.1± 2.9-64.8±3.8 per cent) using TALP and BO medium. This is higher than reports of Hammam et al. (2010) (11-24%), Mehmood et al. (2011) (34%) using sperm TALP medium, Khandoker et al. (2012) (23.28-30.52%) using BO medium, Singh et al. (2012) (20.16%) using modified TALP medium, Rahman et al. (2015) (19.63±3.11 and 29.52±1.98 per cent) and Ruhil and Purohit (2015) (16.4%). In the present study, swim up method in modified SOF medium was found to be the best method for preparation of buffalo bull spermatozoa, as determined by significantly higher recovery of motile spermatozoa. On contrary, the present fertilization rate recorded was lower than the findings of Abdoon et al. (2001) (63.0±0.5%), Tatham et al. (2001) (67.2%), Patil and Totey (2003) (78.4%), Neglia et al. (2003) (65%), Jamil et al. (2007) (63.75±2.81%) using Ca++ tyrodes medium, Mehmood et al. (2007) (75%) using hypotaurine, epinephrine and Kadoom et al. (2014) (64.5%) using IVF-TALP medium. The reduced embryonic development in the present study may be due to high sensitivity of buffalo oocytes and embryos to oxidative stress because of their high lipid content. Increased oxidative stress appears to be a major factor impairing in vitro mammalian development.

CONCLUSION

The mature oocytes are more resistant to cryopreservation. This might be because of the cytoskeleton of the first meiotic division in immature oocytes is particularly susceptible to damage. Mature oocytes display a more flexible cytoskeleton, which may be one reason that they are less subjected to cryo damage (Allworth and Albertini, 1993). The results of the present study showed that concentrated solutions of permeating cryoprotectants like EG and DMSO were required for successful cryopreservation of oocytes when rapid cooling and warming rates are used which was supported by Arav (2014). Therefore, it was suggested that cryopreservation of buffalo oocytes can be best achieved with 30% EG, 30% DMSO and 20% EG+20% DMSO with least damage to the oocytes.
 

CONFLICT OF INTEREST

The authors have no conflict of interest.
 

REFERENCES


  1. Abdoon, A.S.S, Kandil, O.M, Otoi, T and Suzuki, T. (2001). Influence of oocyte quality, culture media and gonadotropins on cleavage rate and development of in vitro fertilized buffalo embryos. Animal Reprod. Science. 65: 215-223.

  2. Allworth, A.E. and Albertini, D.F. (1993) Meiotic maturation in cultured bovine oocytes is accompanied by remodeling of the cumulus  cell cytoskeleton. Developmental Biology. 158: 101-112.

  3. Arav, A. (2014) Cryopreservation of oocytes and embryos. Theriogenology. 81: 96-102.

  4. Attanasio, L., De Rosa, A., Boccia, L., Mariotti, E., Zicarelli, L. and Gasparrini, B. (2007) Effects of warming procedures on the survivability of in vitro matured buffalo (Bubalus bubalis) oocytes vitrified by Cryotop. Italian J. of Animal Science. 6(2): 735-738.

  5. Boonkusol, D., Faisaikarm, T., Dinnyes, A. and Kitiyanant, Y. (2007) Effects of vitrification  procedures on subsequent  development and ultrastructure of in vitro matured swamp buffalo (Bubalus   bubalis) oocytes. Reprod. Fertility and Dev. 19(2): 383-391.

  6. Dolakasaria, D., Dutta, D.J., Dev, H. and Raj, H. (2013) Developmental competence of post thaw vitrified bovine oocytes. International J. of Advanced Research. 1(9): 825-830.

  7. Dua D., Alam, A., Chauhan, M.S., Palta, P. and Singh, M.K. (2021). Effect of Vitrification on Folliculogenesis-Related Genes in Ovarian Follicles of Bubalus bubalis. Indian J. of Animal Research. 55(8): 873-878. doi:10.18805/ijar.B-4245.

  8. Dutta, D.J., Dev, H. and Raj, H. (2013) In vitro blastocyst development of post-thaw vitrified bovine oocytes. Vet. World. 6(10): 730-733.

  9. Fuku, E., Kojima, T., Shioya, T.Y., Marcus, F.G.J. and Downey, B.R. (1992) In vitro fertilization and development of frozen- thawed bovine oocytes. Cryobiology. 29: 485-492.

  10. Gautam, S.K., Verma, V., Palta, P., Chauhan, M.S. and Manik, R.S. (2008) Effect of type of cryoprotectant on morphology and developmental competence of in vitro matured buffalo (Bubalus bubalis) oocytes subjected to slow freezing or vitrification. Reprod. Fertility Dev. 20: 490-496.

  11. Gasparrini, B., Attanasio, L., De Rosa, A., Monaco, E., Di Palo, R. and Campanile, G. (2007) Cryopreservation of in vitro matured buffalo (Bubalus bubalis) oocytes by minimum volumes vitrification methods. Animal Reprod. Science. 98: 335-342.

  12. Hammam, A.M., Whisnant, C.S., Elias, A, Zaabel, S. M., Hegab, A.O. and Naga, A.E.M. (2010). Effect of media, sera and hormones on in vitro maturation and fertilization of water buffalos (Bubalus bubalis). J. of Animal and Veterinary Advances. 9: 27-31.

  13. Hegab, A.O., Montasser, A.E., Hammam, A.M., Naga, A.E.M.A. and Zaabel, S.M. (2009). Improving in vitro maturation and cleavage rates of buffalo oocytes. Animal Reprod. 6(2): 416-421.

  14. Jamil, H., Samad, H.A., Qureshi, Z.I., Rehman, N. and Lodhi, L.A. (2007). Effect of bull and sperm preparation method on in vitro fertilization of buffalo oocytes. Pakistan Veterinary J. 27(1): 29-34.

  15. Kadoom, A.K., Khalek, A.E.A., Shamiah, S.H.M., Sharawy, M.E.E.L and Razek, A.E.L. (2014). In vitro maturation, fertilization and development of prepubertal and mature buffalo oocytes. Egyptian J. of Animal Production. 51(2): 65-69.

  16. Khandoker, M.A.M.Y., Reza, M.M.T., Asad, L.Y., Saha, S., Apu, A.S. and Hoque, S.A.M. (2012). In vitro maturation of buffalo oocytes and fertilization by cattle spermatozoa. Bangladesh J. of Animal Science. 41(1): 6-12.

  17. Leibo, S.P. (1980) Water permeability and its activation energy of fertilized and unfertilized mouse ova. The J. of Membrane Biology. 53: 179-188.

  18. Loganathasamy. and Sharma, G.T. (2014). Effect of meiotic stages during in vitro maturation on the post thaw recovery of buffalo oocytes. Veterinary Science Research J. 5(1): 33-37.

  19. Madan, M.L., Chauhan, M.S., Singla, S.K. and Manik, R.S. (1994). Pregnancies established from water buffalo (Bubalus bubalis) blastocysts derived from in vitro matured in vitro fertilized oocytes and co-cultured with cumulus and oviductal cells. Theriogenology. 42: 591-600.

  20. Mehmood, A., Anwar, M. and Naqvi, S.S.M. (2007). Capacitation of frozen thawed buffalo bull (Bubalus bubalis) spermatozoa with higher heparin concentration. Reprod. in Domestic Animals. 42: 376-379.

  21. Mehmood, A., Muhammad, A. andrabi, S.M.H., Afzal, M. and Naqvi, S.M.S. (2011). In vitro maturation and fertilization of buffalo oocytes: The effect of recovery and maturation methods. Turkish J. of Veterinary and Animal Science.35(6): 381-386.

  22. Men, H., Monson, R.L. and Rutledge, J.J. (2002). Effect of meiotic stages and maturation protocols on bovine oocyte’s resistance to cryopreservation. Theriogenology. 55: 1095-1103.

  23. Mishra, A., Chandra, V. and Sharma, T.G. (2012). Effect of Vitrification on in vitro matured buffalo oocytes. Buffalo Bulletin. 31(2): 81-83.

  24. Morato, R., Izquierdo, D., Paramio, M.T. and Mogas, T. (2008). Cryotops versus open-pulled straws (OPS) as carriers for the cryopreservation of bovine oocytes: Effects on spindle and chromosome configuration and embryo development. Cryobiology. 57: 137-141.

  25. Neglia, G., Gasparrini, B., Di Brienza, C.V., Di Palo, R., Campanile, G., Presicce, G.A. and Zicarelli, L. (2003). Bovine and buffalo. Theriogenology. 59: 1123-1130.

  26. Niemann, H. (1991). Cryopreservation of ova and embryos from livestock: Current status and research needs. Theriogenology. 35: 109-124.

  27. Nowshari, M.A., Nayudu, P.L. and Hodges, J.K. (1994). Effect of cryoprotectant concentration, equilibration time and thawing procedure on survival and development of rapid frozen-thawed mature mouse oocytes. Theriogenology. 42: 1193-1204.

  28. Ocampo, M.B., Ocampo, L.C. and Soriano, R.P. (2014). Vitrification of swamp buffalo ocytes. J. of Agricultural Technology. 10(1): 67-78.

  29. Parrish, J.J., Krogenaes, J.L.A. and Parrish, S.J.L. (1995). Effect of bovine sperm separation by either swim-up or Percoll method on success of in vitro fertilization and early embryonic development. Theriogenology. 44: 859-869.

  30. Patil, S. and Totey, S. (2003). Developmental failure of hybrid embryos generated by in vitro fertilization of water buffalo (Bubalus bubalis) oocyte with bovine spermatozoa. Molecular Reprod and Deve. 64: 360-368.

  31. Pitroda, M.H., Khillare, K.P., Amle, M.B., Meshram, M.D., Mali, A.B., M.N. Rangnekar, M.N. and Zawar, S. (2024). Cleavage Rate of in vitro Matured Oocytes Fertilized with Conventional Semen in Buffaloes. Indian J. of Animal Research. 58(5):  736-745. doi:10.18805/IJAR.B-4428.

  32. Rall, W.F. (1992). Cryopreservation of oocytes and embryos: methods and applications. Animal Reprod. Science. 28: 237-245.

  33. Rahman, A.N.M.I., Khandoker, M.A.M.Y., Asad, L., Saha, S., Paul, R.C. and Debnath, S. (2015). In vitro Maturation and Fertilization of buffalo oocytes cultured in media supplemented with bovine serum albumin. Iranian J. of Applied Animal Science.5(3): 545-551.

  34. Ruhil, S. and Purohit, G.N. (2015). In vitro fertilization of buffalo oocytes matured in vitro in three different media. Indian j. of Animal  Reproduction. 36(2): 10-13.

  35. Sadeesh, E.M., Shah, F., Balhara, A.K., Thirumaran, S.M.K., Yadav, S. and Yadav, P.S. (2014). Effect of growth factor and antioxidant on in vitro maturation of oocytes and cleavage rates of in vitro produced Indian buffalo (Bubalus bubalis) embryos. Veterinary Archive. 84: 459-474.

  36. Samad, H.A., Khan, I.Q. Rehman, N.U. and Ahmad, N. (1998). The recovery, in vitro maturation and fertilization of Nili-Ravi buffalo follicular oocytes. Asian Australian J. of Animal Science. 11: 491-497.

  37. Sharma, G.T. and Loganathasamy, K. (2007). Effect of meiotic stages during in vitro maturation on the survival of vitrified-warmed buffalo oocytes. Veterinary Research Communication. 31(7): 881-93.

  38. Singh, R.P., Shah, R.G. and Tank, P.H. (2012). Influence of different quality of buffalo oocytes on In-vitro maturation and fertilization. Indian J. of Animal Reprod. 33(1): 9-13.

  39. SPSS, (2005). SPSS Base applications Guide Version 16.0, Chicago, IL, USA.

  40. Sripunya, N., Somfai, T., Inaba, Y., Nagai, T., Imai, K. and Parnpai, R. (2010). A comparison of cryotop and solid surface vitrification methods for the cryopreservation of in vitro matured bovine oocytes. J. of Reprod. and Dev. 56(1): 176-181.

  41. Tatham, B.G., Feehan, T. and Pashen, R. (2001). Buffalo and cattle hybrid embryo development is decreased by caffeine treatment. Theriogenology. 59: 709-717.

  42. Vandervorst, M., Vanderzwalmen, P. and Standaart, V. (2001). Blastocyst transfer after vitrification in a hemi-straw system. Human Reprod. 16: 153-154.
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