Dr. R.S. AbhilashARYA THERES2024-08-072024-08-072023-05-16https://krishikosh.egranth.ac.in/handle/1/5810212995The present study was designed to assess the effect of supplementing follicular fluid exosomes on in vitro maturation (IVM), fertilisation, cleavage and to evaluate the effect of exosomes on heat-induced alterations in the developmental competence of bovine oocytes. Ovaries from crossbred cattle were collected and all visible surface follicles were aspirated and culture-grade oocytes were subjected to further study. A total of 523 oocytes selected for the study were randomly divided into four groups. Exosomes were isolated from bovine follicular fluid by differential ultracentrifugation and western blotting was performed to confirm the presence of exosomal marker CD63. Groups I (n=111) and II (n=247) oocytes were subjected to IVM at 38.5°C for 24 h and groups III (n=81) and IV (n=84) oocytes at 41°C (heat-stress) for the first 14 h followed by 38.5°C for the next 10 h. Group II and IV oocytes were supplemented with exosomes at the rate of 1 µL per 100 µL IVM medium. Following IVM of oocytes for 24 h, IVF was carried out by co-incubating with capacitated spermatozoa for 18 h and embryo culture was carried out subsequently. The maturation rate differed significantly (p ≤0.01) between the oocytes with and without exosome supplementation. The highest maturation rate was observed in group II (95.80 ± 1.67) followed by group IV (83.47 ± 0.39), group I (76.10 ± 0.95), and the lowest in group III (64.56 ± 2.59). Fertilisation was assessed after 48 h of sperm oocyte co-incubation and the cleavage rate based on the no: of matured oocytes kept for fertilisation were 42.50 ± 6.44, 45.56 ± 2.00, 19.30 ± 5.12 and 33.73 ± 2.11 in groups I, II, III and IV respectively. Cleavage rate based on the total number of oocytes kept for maturation in groups I, II, III, and IV were 32.47 ± 5.23, 43.66 ± 2.13, 12.47 ± 3.40 and 28.14 ± 1.64 per cent, respectively. Fertilization rate based on the number of matured oocytes were 49.58 ± 8.93, 56.01 ± 2.87, 22.47 ± 7.11 and 38.69 ± 3.97 per cent respectively in groups I, II, III and IV. Also, fertilization rates based on the total number of oocytes kept for maturation were 37.85 ± 7.01, 53.68 ± 3.02, 14.37 ± 4.32 and 32.26 ± 3.17 per cent respectively in groups I, II, III and IV. The results indicate that heat stress (group III) significantly declines the fertilisation rate (22.47 ± 7.11), and supplementation of exosomes(group IV) enhances the fertilisation rate (38.69 ± 3.97 ) suggesting that exosomes can alleviate the heat stress. Also, a significantly higher fertilisation rate in group II (56.01 ± 2.87) compared to group I (49.58 ± 8.93 ) suggests the positive effect of exosomes on fertilisation. From the present investigation, it can be concluded that follicular fluid exosome supplementation in maturation medium could enhance the fertilisation rate and alleviate heat-induced alterations in the developmental competence of bovine oocytes.EnglishThesis