IN VITRO EMBRYO PRODUCTION FROM VITRIFIED MATURE BOVINE OOCYTES

Abstract

Hany Mohamed Amin Laithy. In vitro Embryo Production from Vitrified Mature Bovine Oocytes. Unpublished Ph.D. Thesis, Department of Animal Production, Faculty of Agriculture, Ain Shams University, 2016. Cryopreservation of animal oocytes will permit germplasm of valuable or unique females to be preserved for extended times. The objective of this research was to derive a procedure to cryopreserve bovine oocytes by vitrification to be used as recipients for somatic cell nuclear transfer (SCNT). The present study was conducted to investigate the impact of different cryoprotectants like Ethylene glycol (EG), Propanediol (PROH) and Dimethyl sufoxide (DMSO) alone or thier combinations, cryodevices, Straw, open pulled straw (OPS), solid surface vitrification (SSV), and L-carnitine supplementation in oocytes maturation medium on morphology, survival, fertilization rate and developmental competence of mature bovine oocytes using vitrification. The vitrification of bovine oocytes was performed by six experiments. The first, second and third experiment were conducted using (10, 20 and 40%) concentration of EG, (10, 20 and 40%) DMSO, (2.5, 5 and 10%) of PROH. The fourth experiment was conducted using combinations of the best results from the first three experiments as follow (5%PROH + 20%EG), (5%PROH + 20%DMSO), (20%EG + 20%DMSO), (5%PROH + 20%EG + 20%DMSO). The fifth experiment was conducted using different cryodevices (OPS-Straw-SSV) on vitrfication with best results from fourth experiment. The sixth experiment was conducted using L-carnitine supplementation in maturation medium for control and treated groups at different concentrations (0.1,0.3,0.5 mM/ml), with best cryoprotectant from fourth experiment and best cryodevices from fifth experiment. It was found that combination of cryoprotectants (20% EG + 20% DMSO), proved to be more efficient than using other combinations or Propanediol (PROH), Dimethyl sulfoxide (DMSO) and ethylene glycol (EG) alone. The morphological damages were founded to be less (10.09%) using combination of (20% EG + 20% DMSO) compared with 5% PROH or 20% DMSO alone which were (34.14%) and (27.27%), respectively. The survival rate was found (92.45%) compared with 20% EG or 20% DMSO which was (91.54% and 90.63%), respectively. The fertilization rate was significantly (P<0.05) higher using combination (20%EG + 20% DMSO) (57.14) compared with PROH, DMSO and EG which recorded (40.0%, 44.83%, 44.61%) respectively. The developmental rate cleavage, morula to the blastocyst stage were significantly (P<0.05) higher (10.03%, 8.73%, 5.98%) using combinations of (20% EG + 20% DMSO) than using 5% PROH or 20% DMSO alone. PROH and DMSO when used alone found to induce adverse effect on the morphology, fertilization rate and developmental competence of bovine oocytes. However, combination of 20% EG + 20% DMSO found better to maintain the integrity and internal structure of bovine oocytes by vitrification. Using cryodevice (OPS, SSV techniques) was significantly (P<0.05) higher viability rate (94.73% and 95.23%), fertilization rate (61.11% and 67.50%), and cleavage rate (24.32 and 31.25%) than 0.25ml semen straw technique. It was found that 0.3 concentration of L-Carnitine resulted in significantly higher normal morphology, viability and fertilization rate (70.18%, 92.11% and 62.86%) respectively than control and (0.1,0.5) concentration. Also supplementation of maturation medium of immature cow oocytes with different concentrations of L.C (0.1, 0.3 and 0.5 mM /ml) for 24 h., in the presence of 0.3 mM LC, resulted in an increase of percentages of cleavage rate (40.48%), morula (19.05%) and blastocyst rate (11.90%) compared with control and other treatments.