Nanda, TrilokYosef Deneke Belachew2017-08-182017-08-182007http://krishikosh.egranth.ac.in/handle/1/5810028907Availability of developmentally competent oocytes, especially cryopreserved ones, is critical for in vitro embryo production and application of related biothecniques. Reportedly, matured oocytes are better vitrified than their immature counterparts. The objective of the present study was to assess the effect of BSA in place of FCS as maturation media supplement on vitrification of in vitro matured buffalo oocytes, to study the effect of different concentrations of ethylene glycol as a CPA on the vitrification of buffalo oocytes and their post-thaw potential for in vitro fertilization. Oocytes were aspirated from abattoir ovarian follicles of 2-8mm diameter. Oocytes were matured in TCM-199 supplemented with hCG, PMSG and containing either 0.4% BSA (group-I) or 10% FCS (group-II). Matured oocytes, denuded by vortexing, were equilibrated for 2 min each either in 2.5M ethylene glycol (EG) or 3.6M EG and vitrified either in 5M EG or 7.2M EG, before loading in 0.25ml French straws which were then plunged directly in liquid nitrogen. After at least one week of preservation, thawing was performed with a serial dilution of EG in 0.5M, 0.25M, 0.125M sucrose solutions. Post-thaw viability of oocytes was assessed either by morphological appearance of uniformly spread cytoplasm within intact zona, or staining of dead oocytes with trypan blue or by ultimate ability of the thawed oocytes to undergo in vitro fertilization as determined by cleavage. Post-thaw percentage of morphologically normal oocytes was higher in group-I oocytes vitrified in 7.2M EG (76.8%, 146/190) than in group-II oocytes (61.6% 98/ 159). A significantly higher percentage of morphologically normal oocytes were also recovered when oocytes were vitrified in 5 M EG. Namely, 81.8% for group-I (123/151) and 41.4% (79/191) for group-II, respectively.Thirty four in 7.2M EG vitrified warmed oocytes were randomly selected from group-I and 30 from group-II and were subjected to trypan blue vital staining, of which 24 (70.6%) from group-I and 6 (20%) from group-II were found to be viable. The same high survivability was also observed in group-I oocytes (64.6%, 31/48) than group-II oocytes (11.8%, 4/34), when oocytes were vitrified in 5M EG. Viability assessment by post-thaw IVF revealed that only 7 of 68 oocytes randomly selected in group-II (10.3%) got fertilized, in comparison to 22/112 oocytes (19.8%) in group-I as 7.2M EG was used for vitrification. Whereas 8.0% of group-I oocytes (6/75) and 4.4% group-II oocytes (2/45) got fertilized when the EG concentration was 5 M. These results suggested that for successful vitrification, BSA supplementation has a positive influence on post-thaw survival and maintenance of developmental competence of in vitro matured buffalo oocytes, in comparison to FCS supplement. Possible reason may include hardening of zona pellucida in FCS matured oocytes, which may affect permeation of cryoprotective agent into the oocytes for successful vitrification and also affect ability of sperm penetration in post-thaw oocyte. The overall low percentage of morphologically normal, viable oocytes and the low post-thaw IVF rate in oocytes vitrified in 5 M EG may be due to improper dehydration of the oocytes during vitrification.enThesis