Effect of Ca Ionophore On Blastocyst Production Following Intracytoplasmic Sperm Injection in Caprine Oocytes

Abstract

The aim of the present investigation was to study the effect of calcium ionophore activation on blastocyst production following intracytoplasmic sperm injection (ICSI) in in vitro-matured Caprine oocytes. A total of 470 in vitro-matured oocytes were selected and randomly divided in to three groups. Cumulus oocyte complexes (COCs) recovered by slicing the Caprine ovaries were matured in TCM199 supplemented with 10% foetal bovine serum (FBS) + 10% follicular fluid + FSH (5 μg/ml) + LH (10 μg/ml) + estradiol (1 μg/ml) + EGF (10 ng/ml) + BSA (3 mg/ml) for 27 h in humidified atmosphere at 38.5°C with 5% CO2 in CO2 incubator. After 27 h of culture, selected COCs (n = 470) were separated from cumulus cells by treating with 0.1% hyaluronidase enzyme and passing repeatedly through a fine pipette and randomly divided into three groups. In group 1, (n = 168) matured oocytes were injected with injection micropipette without sperm as control. In group 2, (n = 152) capacitated spermatozoa were injected into cytoplasm of in vitro-matured oocytes through injection micropipette. In group 3, (n = 150) capacitated spermatozoa were injected into cytoplasm of in vitro-matured oocytes through injection micropipette and then activated with 5 μm Ca ionophore for 5 min. The oocytes of all groups were then culture in RVCL media for embryo development. The cleavage rate was observed after 48-72 h of injection. The cleavage rate and blastocyst production in group 1, 2 and 3 were 0.00 and 0.00, 18.42 and 3.57 and 61.33% and 16.30%, respectively. The result indicated that mechanical activation failed to induce cleavage in in vitro-matured Caprine oocytes, whereas chemical activation of intracytoplasmic sperm-injected in vitro-matured Caprine oocytes showed significantly higher cleavage rate and blastocyst production as compare to non-activated oocytes.

Folate receptor‐1 is vital for developmental competence of goat embryos

“…Despite effective knockdown, 5.87% of embryos was able to develop into blastocysts, possibly due to the presence of maternally contributed FOLR1 which may eventually exhausted until blastocyst stage. Our results are in line with a previous study (Strandgaard et al, 2017), which demonstrated the compromised ability of mouse embryos to develop to blastocyst stage after TA B L E 1 Effect of FOLR1 knockdown on the rate of blastocyst production injection (Kharche et al, 2016;Magata et al, 2019;Menéndez-Blanco et al, 2020;Shirazi et al, 2018) and cloning by somatic cell nuclear transfer (Gorczyca et al, 2021;Hernández Martínez et al, 2020;Samiec et al, 2019;Skrzyszowska & Samiec, 2020Wiater et al, 2021).…”

“…The future studies at our laboratory may decipher some of these questions in order to thoroughly elucidate the importance of FOLR1 as a pivotal biomarker suitable for prediction of augmented susceptibility of embryonic genomes to efficient epigenetic reprogramming of their transcriptional activities. The latter could enable the embryologists to estimate the increased developmental competences and molecular quality of caprine and other mammalian species embryos produced by such modern assisted reproductive technologies as microsurgical IVF by intracytoplasmic sperm injection (Kharche et al., 2016; Magata et al., 2019; Menéndez‐Blanco et al., 2020; Shirazi et al., 2018) and cloning by somatic cell nuclear transfer (Gorczyca et al., 2021; Hernández Martínez et al., 2020; Samiec et al., 2019; Skrzyszowska & Samiec, 2020, 2021; Wiater et al., 2021).…”

Effect of hypoxia on production of caprine chimeric blastocyst

“…Tetraploid fertilized embryos production: Collection of ovaries, recovery of oocytes and in vitro maturation (IVM) was done as per the method of Kharche et al (2016). In vitro fertilization (IVF) was carried out as per the method described by Kharche et al (2011) with slight modifications.…”

Assessment of different stages of parthenogenetic embryos for production of embryonic stem cell like colonies

“…Collection of ovaries, recovery of oocytes and in vitro maturation (IVM) was done as per the method of Kharche et al (2016). Briefly, after 27 h of in vitro maturation, denuded oocytes were then chemically activated with 5 µM calcium ionophore for 5 to 7 min followed by 4 h culture in 2 mM DMAP.…”