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Subject: Veterinary Physiology × Author: BAISHYA, DIPANNITA × End date: 2019 × Type: Thesis ×
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    ThesisItemOpen Access
    OPTIMIZATION OF CULTURE MEDIA FOR IN-VITRO BOVINE EMBRYO DEVELOPMENT: GROWTH FACTORS AND SERUM INFLUENCES
    (College of Veterinary Science, Assam Agricultural University, Khanapara, Guwahati, 2019-07) BAISHYA, DIPANNITA; Bora, Arundhati
    The present experiment was conducted to study the effect of certain growth factors (EGF, IGF-1and their combination) and serum influences on possible potentialization of culture media for in vitro cattle embryo development. In experiment I, 224 nos. of cattle ovaries were collected. The mean number ovarian follicles recovered per type-I ovary were 5.30 which was significantly higher (P≤0.001) than the corresponding values 3.27 of the type-II ovaries. The mean recovery of cumulus oocyte complexes per type-I ovary was 3.41 and the corresponding value was 1.67 in type-II ovaries. Two different types of maturation and culture media viz: SBMM (Serum Basic Maturation Media) containing modifiedTCM-199+ serum (10%,Fetal Bovine Serum)+ Sodium pyruvate + glutamine + gentamicin + pFSH + hMG inj+ E2 (estradiol), SFBMM(Serum Free Basic Maturation Media) containing modified TCM-199 + PVP + BSA + Sodium pyruvate+ L-glutamine+ p FSH+ gentamicin + hMG inj+ E2 (estradiol), SBCM (Serum Basic culture media): mCR2aa stock +10%FBS+ Gentamicin, SFBCM (Serum Free Basic Culture Media) containing mCR2aa stock+ BSA-V+ PVP+ Gentamicin were used for in vitro maturation and in vitro culture of the oocytes. EGF (30ng) and IGF-1 (100ng) were added in maturation media as well as embryo culture media singly and in combination in both the groups of media. Frozen bull semen straws of proven fertility were used and prepared for in vitro capacitation by density gradient method using B.O. media. The results revealed a significant (P<0.05) increase in maturation rate in serum supplemented media than serum free media (75.43± 3.25 vs 64.20 ±3.77) based on cumulus cells expansion. The cleavage percentages of serum supplemented culture media was found to be significantly higher (P<0.05) than serum free culture media (70.33±3.21 vs 55.81±4.33). In experiment -2: A total of 318 nos. of ovaries were collected with a recovery of 65 per cent culturable oocytes, representing 6.5 COCs per ovary. Growth factors EGF (30ng/ml), IGF-1 (100ng/ml) and their combination (EGF+IGF-1) were used in serum basic and serum free basic maturation and culture media for the study. There was no significant difference in respect of maturation, fertilization and embryonic development between EGF supplemented, IGF-1 supplemented and their combination (EGF+IGF-1) in serum and serum free basic culture media. However, when compared with the results of serum free basic maturation media supplemented with 30ng EGF and serum basic maturation media without EGF, the mean in vitro maturation percentage based on extrusion of polar body were found to be significantly higher (P<0.05) in supplemented media than the serum basic maturation media (70.00±14.49 vs 54.17±7.19). Similarly, when comparison was made with IGF-1 supplemented serum free basic maturation media with serum basic maturation media without IGF-1, the in vitro maturation percentage based on cumulus cells was found to be significantly higher (P<0.05) than serum basic maturation media (67.27±6.33 vs 75.43±3.25). However, in case of serum free basic maturation media supplemented with 30ng EGF+100ng IGF-1, the mean in vitro maturation percentage based on extrusion of polar body was significantly higher (P<0.05) than serum basic maturation media (80.00±10.33vs 54.17±7.19). The efficacy of EGF supplemented serum free basic culture media and serum basic culture media in respect of cleavage and early embryonic development was comparable at cleavage (2-cell) and blastocyst stage, while significantly higher (P<0.05) values were observed in 4 cell (57.14±4.83 vs 47.25±4.86), 8 cell (45.71± 4.83vs 31.87± 4.99) 16 cell (37.14±4.72 vs 20.88±3.21) and morula stage (27.62±4.36 vs 7.69±4.32) in EGF supplemented serum free culture media than serum basic culture media. Similarly, when the efficacy of IGF-1 supplemented serum free basic culture media were observed, no significant difference was obtained in 2-cell, 4-cell and blastocyst stages. On the contrary, the serum supplemented media showed significantly higher (P<0.05) 8-cell (45.71±4.86 vs 31.87±4.99), 16-cell (37.14± 4.72 vs 20.88±3.21) and morula stage (27.62±4.36 vs7.69±4.32) than serum basic culture media. EGF+IGF-1 supplemented serum free basic culture media when compared with serum basic culture media, significantly higher (P<0.05) values were found in respect to 2-cell (79.63± 2.35 vs 70.33±3.21), 4-cell (65.00±4.83 vs 47.25±3.23), 8 cell (56.00±3.42vs 31.87±4.99) 16 cell (48.00±4.72 vs 20.88±3.21) morula (37.00±2.13 vs 7.69±4.32) and blastocyst stage (10.23±2.08 vs 4.40±3.11). From the above findings, it can be concluded that addition of EGF and IGF-1 in combination in serum free basic maturation media has better stimulatory effect on nuclear maturation of oocytes in comparison to EGF and IGF- 1 supplementation individually. EGF and IGF-1 in combination in Serum Free culture media significantly increased blastocyste rates when compared with serum based culture media.