Effect of antifreeze protein I in the freezing solution on in vivo-derived sheep embryos.

This study evaluated the effects of different antifreeze protein type I (AFP I) concentrations added to a slow freezing solution in sheep in vivo-derived embryos. Good-quality embryos were allocated into: AFP-free (CONT); 0.1 µg/mL of AFP I (AFP0.1); or 0.5 µg/mL of AFP I (AFP0.5). After thawing, embryos were in vitro cultured (IVC) for 48 h. At 24 h and 48 h of IVC, dead cells and apoptosis, mitochondrial activity, intracellular reactive oxygen species (ROS), and glutathione (GSH) evaluations were performed. At 24 h, evaluated embryos were submitted to RT-qPCR for metabolism (SIRT2, PRDX1, OCT4, CDX2) and quality (AQP3, CDH1, HSP70, BAX, BCL2) genes. The in vitro survival rate was 56% (22/39) for CONT, 60% (32/53) for AFP0.1, and 53% (23/43) for AFP0.5 (p > 0.05). A tendency (p = 0.09) for a higher blastocyst hatching rate was noted in AFP0.1 (62%) compared to AFP0.5 (33%), and both groups were similar to CONT (50%). An increased (p < 0.05) mitochondrial activity at 24 h was observed in AFP0.1 compared to CONT. No differences (p > 0.05) were observed in oxidative stress homeostasis and viability between treatments. A downregulation (p < 0.05) of CDH1 in AFP0.1 and a downregulation of AQP3 in AFP0.5 were observed in comparison to the other groups. An upregulation (p < 0.05) was detected in HSP70 and BCL2 on AFP0.5 compared to AFP0.1 group. The addition of AFP I in slow freezing solution can benefit cryopreserved sheep in vivo-derived embryos, without affecting embryonic survival.

The coasting time affects the quality of cumulus-oocyte complexes in superstimulated ewes.

We hypothesized that the coasting time may be beneficial to the quality of cumulus-oocyte complexes recovered from live ewes, as reported in cattle. The present study assessed the effect of coasting times on the quantity and quality of cumulus-oocyte complexes (COCs) in sheep. All ewes were subjected to the "Day 0 protocol", followed by an ovarian stimulation (80 mg of pFSH in three decreasing doses), varying only the coasting time [12 (G12), 36 (G36), or 60 h (G60]. In Experiment 1, data regarding follicular population was assessed. In Experiment 2, the COC quality was checked by their morphology, brilliant cresyl blue (BCB) test, evaluation of chromatin condensation pattern, and oocyte diameter. In Experiment 3, genes related to oocyte developmental competence were evaluated in BCB + COCs. The oocytes in the G60 group had more (P < 0.05) large follicles than the other groups and oocytes with a greater diameter than the G12. Oocyte morphology was similar (P > 0.05) among groups, as well as the BCB + COCs quantity. The G60-oocytes presented a better (P < 0.05) configuration of chromatin condensation compared with the other groups and a greater (P < 0.05) gene expression of BMP15, MATER, ZAR1, and PTGS2 compared with G12, and PTGS2 and HAS2 compared with G36 group. In conclusion, 60 h of coasting time positively affects the quality of COCs recovered after subjecting ewes to the "Day 0 protocol" and ovarian superstimulation. Implementing the appropriate coasting time to a given protocol can positively impact the in vitro embryo production outcomes in sheep.

Antifreeze proteins for low-temperature preservation in reproductive medicine: A systematic review over the last three decades.

Antifreeze proteins (AFPs) are synthesized by diverse non-mammalian species, allowing them to survive in severely cold environments. Since the 1990s, the scientific literature reports their use for low-temperature preservation of germplasm. The aim of this systematic review was to compile available scientific evidence regarding the use of AFP for low-temperature preservation of several reproductive specimens. Internet databases were consulted using the terms: "antifreeze protein" OR "AFP" OR "antifreeze glycoprotein" OR "AFGP" OR "ice-binding protein" OR "IBP" OR "thermal hysteresis protein" AND "cryopreservation". From 56 articles, 87 experiments testing AFPs in low-temperature preservation of gametes, embryos or reproductive tissues/cells were fully analyzed and outcomes were annotated. A positive outcome was considered as a statistically significant improvement on any parameter evaluated after low-temperature preservation with AFP, whereas a negative outcome included worsening of any evaluated parameter, in comparison to untreated groups or groups treated with a lower concentration of AFP. The findings indicated that research on the use of AFP as a cryoprotectant for reproductive specimens has increased markedly over the past decade. Some experiments reported both positive and negative results, which depended, on AFP concentration in the preservation media. Variation in the outcomes associated with species was also observed. Among the 66 experiments conducted in mammals, 77.3% resulted in positive, and 28.8% in negative outcomes after the use of AFP. In fishes, positive and negative outcomes were observed in 71.4% and 33.3% of 21 experiments, respectively. Most positive outcomes included preserving cell post-warming survival. The beneficial effect of AFP supports its use in cryobiological approaches used in human and veterinary medicines and animal protein industry. Moreover, combination of different AFP types, or AFP with antioxidants, or even the use of AFP-biosimilar, comprise some promising approaches to be further explored in cryopreservation.

Reproductive Seasonality Affects In Vitro Embryo Production Outcomes in Adult Goats.

Reproductive seasonality may have a considerable influence on the efficiency of assisted reproductive technologies in seasonal species. This study evaluated the effect of season on cleavage, blastocyst rates and quality of in vitro produced (IVP) goat embryos. In total, 2348 cumulus-oocyte complexes (COCs) were recovered from slaughterhouse ovaries and subjected to the same IVP system throughout 1.5 years (49 replicates). The odds ratio (OR) among seasons was calculated from values of cleavage and blastocyst rates in each season. Cleavage rate was lower (p < 0.05) in spring (anestrus), in comparison with either autumn (peak of breeding season) or summer, while the winter had intermediate values. Furthermore, lower OR of cleavage was observed in spring. Blastocyst formation rate (from initial number of COCs) was higher (p < 0.05) in autumn (52 ± 2.5%) when compared with the other seasons (combined rates: 40 ± 1.9%). Moreover, its OR was higher (p < 0.05) in autumn compared to all other seasons and impaired in the spring compared to winter (OR: 0.54) and summer (OR: 0.48). Embryo hatchability and blastocyst cell number were similar (p > 0.05) among seasons. In conclusion, the breeding season leads to improved oocyte developmental competence, resulting in higher cleavage and blastocyst yield, whereas embryo quality remained similar throughout the years.

In vitro production of small ruminant embryos: latest improvements and further research.

This review presents the latest advances in and main obstacles to the application of invitro embryo production (IVEP) systems in small ruminants. This biotechnology is an extremely important tool for genetic improvement for livestock and is essential for the establishment of other biotechnologies, such as cloning and transgenesis. At present, the IVEP market is almost non-existent for small ruminants, in contrast with the trends observed in cattle. This is probably related to the lower added value of small ruminants, lower commercial demand and fewer qualified professionals interested in this area. Moreover, there are fewer research groups working on small ruminant IVEP than those working with cattle and pigs. The heterogeneity of oocytes collected from growing follicles in live females or from ovaries collected from abattoirs remains a challenge for IVEP dissemination in goats and sheep. Of note, although the logistics of oocyte collection from live small ruminant females are more complex than in the bovine, in general the IVEP outcomes, in terms of blastocyst production, are similar. We anticipate that after appropriate training and repeatable results, the commercial demand for small ruminant invitro -produced embryos may increase.

Embryo development is impaired in goats that are treated for hydrometra and subsequently subjected to superovulation.

BACKGROUND: Reproductive efficiency after hydrometra (HD) treatment is usually unsatisfactory. METHODS: To identify mechanisms involved in low reproductive efficiency of HD-treated goats, pluriparous dairy goats treated for HD (n=10, HD) or with no reproductive disorders (n=11, control: CONT) were induced to oestrus and superovulated. Goats were mated with fertile bucks and seven days after oestrus, non-surgical embryo recovery was performed. Embryos were evaluated and gene expression was performed. RESULTS: There were no differences (P>0.05) in sexual behaviour parameters, superovulation response, mean number of retrieved structures and viable embryos between groups; although embryo recovery rate was higher (P=0.01) in CONT group. Structures in delayed stage (8-16 cells) were more frequent (P<0.05) in HD (29 vs 1 per cent) goats, as well as the percentage of advanced embryos was greater (P<0.05) for CONT (59.3 vs 33.3 per cent) goats. However, the expression of genes related to apoptosis (BAX and Bcl-2), trophectoderm differentiation (CDX2) and pluripotency maintenance (NANOG) was not affected (P>0.05) in embryos that reached the morulae and blastocyst stages. CONCLUSION: Although the HD embryos that developed to morula and blastocyst stages showed no change in the expression of genes related to their quality and implantation capacity, overall, embryo development was impaired in HD-treated goats.