Embryonic Development and Gene Expression in Oocytes Cultured In Vitro in Supplemented Pre‐Maturation and Maturation Media

Abstract

This study aimed at assessing the effect of the addition of brain-derived neurotrophic factor (10 ng/ml BDNF) and/or cysteamine (100 μm CYS) during pre-maturation and BDNF, CYS or leptin (10 ng/ml LEP) during maturation culture in vitro on embryo development and oocyte gene expression in cattle. Oocytes were obtained by the aspiration of 2- to 8-mm follicles from slaughtered cows. In Experiment 1, oocytes were pre-matured for 24 h with 10 μm butyrolactone I in the presence or not of BDNF and/or CYS followed by in vitro maturation (IVM), fertilization (IVF) and culture (IVC). In Experiment 2, oocytes were submitted to IVM with BDNF, CYS or LEP or no supplements followed by IVF and IVC. In Experiment 3, oocytes were pre-matured with BDNF and CYS followed by IVM or only in vitro matured with BDNF. Samples for quantitative PCR (qPCR) were collected after pre-maturation (BGV) and after IVM of pre-matured oocytes (BMII) or immediately after follicle aspiration (immature control = GV) and IVM (matured control = MII). Embryo production was not affected by the inclusion of the different factors either during pre-maturation or maturation culture (∼ 43% blastocysts, p>0.05). Transcripts analysis showed that most genes (NLRP5, ZAR1, GPX1, KEAP1, SPHK2, HSP70 and PSMP1) were downregulated (p<0.05) after IVM irrespective of being previously pre-matured. The relative abundance of BAX, BCL2, IGFBP3 and ARFRP1 transcripts was unaffected by pre-maturation or maturation (p>0.05). In conclusion, supplementation of in vitro pre-maturation (BDNF and/or CYS) or maturation media (BDNF, CYS or LEP) did not improve embryo development. Gene expression was not affected by pre-maturation treatment, but some genes were downregulated after maturation, probably related to selective and differential degradation.

Developmental competence of different quality bovine oocytes retrieved through ovum pick-up following in vitro maturation and fertilization

“…The decreased expression of ZAR1 in Group 2 COCs might be the reason for their arrest at the zygotic stage and lack of syngamy between the maternal and paternal pronuclei (Wu et al, 2003), hence poorer cleavage rate and subsequent arrest at 8-to 16-cell stage, as observed in our study. The decrease in expression of HSF1 and ZAR1 after IVM, as observed in our study, is in agreement with previous studies conducted in bovine species (Adona et al, 2010;Romar et al, 2011). The increased expression of proapoptotic genes (BAX and BID) in Group 2 COCs functions as the indicator of their lower survival and developmental ability.…”

The Influence of Morphology, Follicle Size and Bcl‐2 and Bax Transcripts on the Developmental Competence of Bovine Oocytes

“…The in vitro production of embryos is an important biotechnological technique in the field of animal reproduction, widely used in research, but also in commercial settings. Notwithstanding its widespread use, the in vitro production of embryos associated with follicular aspiration guided by ultrasound (ovum pickup) (Pontes et al 2012) or ovaries taken from abattoirs, an abundant source of oocytes (Dey et al 2012), still suffers certain limitations due to the relatively low production of embryos (Rizos et al 2008;Adona et al 2011).…”

Using cysteine/cystine to overcome oxidative stress in goat oocytes and embryos cultured in vitro

“…Thus, the GSH content of oocytes protects the zygote and early embryos from oxidative damage prior to genomic activation (7). The addition of cysteine to oocyte culture media has been shown to increase intracellular levels of GSH in buffaloes (8,9), pigs (10) and goats (11). However, the effects of supplementation with a specific concentration of cysteine depend on the dose, the species and the maturation medium (2).…”