Developmental competence of ovum pick up derived Sahiwal oocytes in maturation media supplemented with cysteamine and melatonin

Description

Antioxidants, cysteamine, and melatonin have an important role in mitochondrial membrane potential (ΔΨM), in vitro nuclear maturation, and the developmental competence of oocytes. A comprehensive study was planned to investigate the effect of cysteamine 50 µM and melatonin 10-9 mol L-1 as antioxidants on ΔΨM, in vitro nuclear maturation, and developmental competence of ovum pick-up (OPU) derived Sahiwal oocytes. Culturable grade OPU-derived Sahiwal oocytes were divided into three in vitro maturation groups cultured in TCM-199 supplemented with cysteamine 50 µM, melatonin 10-9 mol L-1, and TCM-199 alone, for assessing nuclear maturation by Lamin/ DAPI and developmental competence of oocytes. ΔΨM was assessed by JC-1 staining in the pre-maturation group and post-maturation cysteamine, melatonin, and control groups. Red to green ratio of fluorescence intensity on JC-1 staining was higher (p < 0.05) in melatonin (1.19±0.04) and cysteamine (1.09±0.04) supplementation groups as compared to control (0.81±0.10) and pre-maturation (0.71±0.03) groups. ΔΨM improved post-maturation in all the treatment and control groups compared to the pre-maturation group (0.71±0.03). Melatonin supplementation improved (p < 0.05) M-II stage oocytes (6.5±0.65, 68.13 percent) as compared to cysteamine supplemented (5.25±0.25, 55.63 percent) and control (4.75±0.25, 50.63 percent) groups. The COC expansion rate was higher in the antioxidant-supplemented group. Fertilization rate, cleavage rate, and blastocyst rate were higher (p < 0.05) in the melatonin-supplemented group (92.31, 59.17, and 20.56 percent) as compared to cysteamine supplemented (82.96, 41.48 and 11.39 percent) and control (75.28, 27.59 and 5.19 percent) groups, respectively. In conclusion, cysteamine and melatonin supplementation as antioxidants in the in vitro maturation media improved (p < 0.05) ΔΨM. Significant improvement in MII stage oocytes, cleavage, and blastocyst rate in OPU-derived Sahiwal cattle oocytes by supplementation of melatonin to the IVM medium compared to cysteamine supplemented and control groups. Melatonin improved both cytoplasmic and nuclear maturation thereby improving the developmental competence of OPU-derived Sahiwal oocytes. Cysteamine supplementation improved the cytoplasmic maturation and not the nuclear maturation and developmental competence of OPU derived Sahiwal oocytes.

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Ovum pick up and IVM 36 OPU sessions were performed under epidural anesthesia using an ultrasound machine and vacuum pump. Follicles were aspirated using a sterile needle, and follicular fluid was collected in a 50 ml tube. The fluid was then shifted to the laboratory for isolation, evaluation, and processing of oocytes, ensuring proper handling to minimize temperature variation. Oocyte searching Animal follicular contents were filtered and poured into a 90mm petridish for oocyte searching. Culturable COCs were divided into two portions: 10% before maturation stained with JC-1 stain, and 90% subjected to in vitro maturation. In vitro Maturation (IVM) In vitro Maturation (IVM) media was prepared and maintained in a CO2 incubator for 2 hours before OPU. The media included TCM 199, FSH, LH, FCS, and Gentamicin, with supplementations in groups. IVM oocytes were cultured and stained with JC-1 and Lamin/DAPI. In vitro Fertilization (IVF) In vitro fertilization of matured Sahiwal oocytes using frozen-thawed semen from Sahiwal bulls was conducted using OPU derived oocytes and sperm, with the resulting sperm and oocytes co-incubated in a CO2 incubator. In vitro Culture (IVC) of Zygotes The study involved in vitro culture of zygotes using 500 µl of IVC media. After 18 hours of co-incubation, presumptive zygotes were washed three times with 70 µl drops of IVC, removed, and placed in a CO2 incubator. The number of cleaved structures was counted and detached. Processing of COCs for JC-1 staining The study involved denuding mature oocytes in droplets of 0.5 percent hyaluronidase, estimating mitochondrial membrane potential using the JC-1 assay kit, and incubating them with 2 μM of JC-1 stain for 30 minutes. Samples were observed under a microscope and camera, and JC-1 monomers were detected using green fluorescence. Mitochondrial activity was evaluated by comparing red/green fluorescence intensity. The procedure was conducted in dark. Processing of COCs for Lamin/DAPI staining: In vitro matured oocytes were denuded in 0.5 % hyaluronidase, fixed in 4% paraformaldehyde solution, and subjected to immuno-cytochemical staining to detect nuclear envelope and chromatin configurations. Oocytes were permeabilized, blocked, and placed in a 1:300 mouse anti-lamin A/C antibody and 1:200 secondary antibody. Mounted oocytes were observed under a Zeiss Axiolab epifluorescence microscope and Axiocam 202 mono camera. The oocytes were classified into Germinal vesicle (GV), Germinal vesicle break down (GVBD), Metaphase-I (M-I), and Metaphase-II stages. Statistical analysis Nuclear maturation, fertilization and embryonic development of oocytes were analyzed using ANOVA by SPSS 26 software. Fixed factor were groups variable factors were parameters viz. percent red oocytes, red/green ratio, percent M-II oocytes, COCs expansion, cleavage, blastocyst rates.

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