Molecular, enzymatic responses and in vitro embryonic developmental competency of heat-shocked buffalo embryos co-cultured with granulosa cells monolayer.

The present study was designed to establish a suitable alternative approach to mitigate the adverse effect of high culture temperature on in vitro embryo development and the related molecular response in buffalo. Pre-cultured granulosa cells (GCs) were used as a monolayer during in vitro embryo culture until day 8 (day of fertilization = D0). Post fertilization, presumptive embryos were randomly assigned into two culture conditions: embryos cultured in the presence of GCs monolayer under normal culture temperature (N: 38.5 °C; GEN group) or heat shock (H: 40.5 °C; GEH group) and their counterpart groups of embryos cultured without GCs (EN and EH groups). Additionally, two groups of GCs monolayer were cultured without embryos up to day 8 under 38.5 °C (GN) or 40.5 °C (GH) for further spent culture media enzymatic analyses. Heat shock was administered for the first 2 h of culture then continued at 38.5 °C until day 8. The results indicated that under heat treatment, GCs enhanced (P ≤ 0.05) embryo cleavage and development (day 8) rates, which were comparable to the embryos cultured at 38.5 °C. On the molecular level, blastocysts of the GEH group showed similar expressions of metabolism-regulating genes (CPT2 and SlC2A1/GLUT1) and an antioxidant gene (SOD2) when compared to the blastocysts of the EN group. The relative expression of HSP90 was significantly up-regulated under heat shock and/or co-culture conditions. However, HSF1 expression was increased (P ≤ 0.05) in the GEH group. No statistical differences were observed among the study groups for the pluripotency gene NANOG, and stress resistance transcript NFE2L2. Regarding the enzymatic profile, the concentrations of SOD, total protein, and MDA were decreased (P ≤ 0.05) in the GEH group compared to the cultured GCs without embryos (GH group). In conclusion, GCs as a monolayer have a beneficial impact on alleviating heat stress at the zygote stage through the regulatory mechanisms of metabolic activity, defense system, and heat shock response genes.

Adaptive and Biological Responses of Buffalo Granulosa Cells Exposed to Heat Stress under In Vitro Condition.

The steroidogenesis capacity and adaptive response of follicular granulosa cells (GCs) to heat stress were assessed together with the underlying regulating molecular mechanisms in Egyptian buffalo. In vitro cultured GCs were exposed to heat stress treatments at 39.5, 40.5, or 41.5 °C for the final 24 h of the culture period (7 days), while the control group was kept under normal conditions (37 °C). Comparable viability was observed between the control and heat-treated GCs at 39.5 and 40.5 °C. A higher release of E2, P4 and IGF-1 was observed in the 40.5 °C group compared with the 39.5 or 41.5 °C groups. The total antioxidant capacity was higher in response to heat stress at 39.5 °C. At 40.5 °C, a significant upregulation pattern was found in the expression of the stress resistance transcripts (SOD2 and NFE2L2) and of CPT2. The relative abundance of ATP5F1A was significantly downregulated for all heat-treated groups compared to the control, while TNFα was downregulated in GCs at 39.5 °C. Expression analyses of stress-related miRNAs (miR-1246, miR-181a and miR-27b) exhibited a significant downregulation in the 40.5 °C group compared to the control, whereas miR-708 was upregulated in the 39.5 and 40.5 °C groups. In conclusion, buffalo GCs exhibited different adaptive responses, to the different heat stress conditions. The integration mechanism between the molecular and secretory actions of the GCs cultured at 40.5 °C might provide possible insights into the biological mechanism through which buffalo GCs react to heat stress.

Physiological and molecular aspects of heat-treated cultured granulosa cells of Egyptian buffalo (Bubalus bubalis).

The physiological and molecular responses of granulosa cells (GCs) from buffalo follicles were investigated when there were in vitro heat stress conditions imposed. The cultured GCs were heat-treated at 40.5 °C for 24, 48 or 72 h while GCs of the control group were not heat-treated (37 °C). There were no differences in viability between control and heat-treated groups. There was an upward trend in increase in E2 secretion as the duration of heat stress advanced, being greater (P ≤ 0.05) for the GCs on which heat stress was imposed for 72 as compared with 24 h. In contrast, P4 release was less (P ≤ 0.05) from GCs heat-treated for 48 h than those cultured for 24 h and GCs of the control group. The relative abundance of ATP5F1A and SOD2 mRNA transcripts was consistent throughout the period when there was imposing of heat stress to sustain mitochondrial function. The relative abundance of CPT2 transcript was less in heat-treated GCs than in GCs of the control group. There was a greater relative abundance of SREBP1 and TNF-α mRNA transcripts after 48 h of heat-treatment of GCs than GCs of the control group. In conclusion, the results from the current study indicate buffalo GCs cultured when there was imposing of heat stress maintained normal viability, steroidogenesis and transcriptional profile. The stability of antioxidant status and increased transcription of genes regulating cholesterol biosynthesis and stress resistance may be defense mechanisms of buffalo GCs against heat stress.

The effect of vitrification of immature bovine oocytes to the subsequent in vitro development and gene expression.

Immature bovine oocytes were vitrified using the cryotop method and their post-warming survivability and capability to undergo in vitro maturation, fertilization and subsequent embryonic development were evaluated. In addition throughout the embryonic 2-cell, 4-cell, morula and blastocyst stages, the expression of four developmentally important genes (Cx43, CDH1, DNMT1 and HSPA14) was analysed using the real-time polymerase chain reaction (PCR). Immature oocytes (n = 550) were randomly assigned to non-vitrified (fresh) or cryotop vitrification groups using ethylene glycol (EG) with 1,2 propanediol (PROH) or dimethylsulphoxide (DMSO). After warming, oocytes survivability, embryo cleavage and embryonic developmental rates were not statistically different between the two cryoprotectants groups. However, the DMSO group had a lower (P < 0.05) oocyte maturation rate compared with the fresh and PROH groups. For morula and blastocyst rates, the DMSO group achieved a lower (P < 0.05) morula rate compared with the fresh group, while at the blastocyst stage, there were no differences between fresh and both cryoprotectants groups. For molecular analysis, at the 4-cell stage, most studied genes showed an inconsistent pattern of expression either from the PROH or DMSO groups. Noteworthily, these differences were limited at the morula and blastocyst stages. In conclusion, the cryotop method is sufficient for vitrification of immature bovine oocytes, both for embryonic developmental competence and at the molecular level. Moreover, PROH showed some advantage over DMSO as a cryoprotectant.

In vitro oocyte fertilization and subsequent embryonic development after cryopreservation of bovine ovarian tissue, using an effective approach for oocyte collection.

This study was undertaken to assess dissection/puncture combined technique for collecting large number of oocytes from bovine ovaries and to determine the effect of ovarian tissue cryopreservation on the oocytes capability to undergo in vitro maturation, fertilization and subsequent embryonic development. Ovaries (n=31) of slaughtered cows were cut into small fragments using a scalpel blade and the ovarian tissues were randomly assigned to cryopreserved by slow freezing and vitrification and non cryopreserved (fresh) groups. Oocytes were collected from non-atretic follicles from fresh and post-thawing ovarian tissue by the puncture method. The advantage of this technique appeared through morphologically good quality cumulus-oocyte complex (COC) recovery rate from fresh tissue (31.7±2.0 oocytes/ovary). However, the cryopreservation affected the post thawing total and good quality COC recovery rates from slow freezing (26.6±2.0 and 23.5±2.3 oocytes/ovary, respectively) and vitrification groups (21.7±1.1 and 17.6±1.8 oocyte/ovary, respectively). The maturation rate resulted in significant differences between the fresh tissue (94.1±1.1%) and the two cryopreservation groups. Moreover, this rate was significantly higher in the slow freezing group (80.1±1.3%) than in the vitrification group (73.0±1.9%). No statistical differences were observed in the cleavage and the embryonic developmental rates between fresh tissue group and cryopreservation groups. Furthermore the number of embryos produced per animal was statistically higher for fresh tissues than for slow freezing and the vitrification groups (34.4±1.4, 27.8±3.1 and 22.0±0.7, respectively). In conclusion, dissection method followed by puncture of bovine ovaries greatly maximizes the number of good quality oocytes recovered, as well as the number of embryos obtained per animal. Ovarian tissue can be successfully cryopreserved by slow freezing and vitrification.