Can the addition of Interleukin-13 affect the cryosurvival of bovine embryos?

In this study, we investigated the impact of incorporating Interleukin-13 (IL-13) into the embryonic culture medium and its influence on cryotolerance and cellular viability of vitrified bovine embryos. Two distinct time points for IL-13 supplementation were explored: during the final hours of culture prior to cryopreservation and during the period of recultivation following cryopreservation and warming. Cryosurvival rates, total cell count, and cell viability were assessed using the TUNEL technique to determine the apoptotic percentage. Re-expansion and hatching rates did not show differences among all groups (P > 0.05), and the total cell number was comparable between the treated and control groups (P > 0.05). However, the group that received IL-13 before vitrification exhibited a higher apoptotic percentage (P < 0.05). This suggests that the anti-inflammatory effect of IL-13 may have impacted the embryo's defense capacity against the stress induced by cryopreservation, leading to an increased percentage of apoptosis, although it did not influence the developmental resumption capability.

Effect of IL-10 and TNF-α on the competence and cryosurvival of in vitro produced Bos indicus embryos.

In vitro-produced embryos are constantly exposed to stressful conditions that can lead to the activation of the apoptotic pathway. The nuclear Kappa B factor (NF-κB) is an inflammatory mediator that induces the expression of tumor necrosis factor (TNF-α), a pro-inflammatory cytokine, while interleukin-10 (IL-10), an anti-inflammatory cytokine, inhibits NF-κB activity. This study aimed to investigate the effects of IL-10 and TNF-α on the competence and cryosurvival of in vitro-produced bovine embryos. Embryos were produced in vitro using standard protocols, and Grade I blastocysts were vitrified using the Cryotop method. Non-vitrified and vitrified blastocysts were subjected to the TUNEL assay. In Experiment I, on day 6.5 (156 h post-insemination), the embryos were treated with PBS (control), 50 ng/mL of IL-10, or a combination of 25 ng/mL of TNF-α and 50 ng/mL of IL-10. Embryonic development and apoptotic rates were monitored. In Experiment II, the same groups were set up, with the addition of a group treated with 25 ng/mL of TNF-α alone. Grade I blastocysts were vitrified 5 h after treatment, and cryosurvival was monitored at until 48 h post-warming. The apoptosis rate and total cell number were investigated in the vitrified-hatched blastocysts. IL-10 alone did not affect developmental competence or cryosurvival (P > 0.05). The IL-10-treated embryos, when exposed in combination with TNF-α, presented a detrimental effect (P < 0.05) in the embryonic development of non-vitrified embryos. However, vitrified blastocysts had no negative effect (P > 0.05). The TNF-α treatment reduced (P < 0.05) the re-expansion rate at 6 h post-warming and increased (P < 0.05) the apoptosis rate in vitrified hatched blastocysts, whereas no effect (P > 0.05) of the treatments was detected in the hatching rate and total cell number post-warming. In conclusion, TNF-α has a detrimental effect on embryonic developmental competence and cryosurvival by compromising the development of non-vitrified embryos and apoptotic-related events of vitrified blastocysts, whereas IL-10, when in combination with TNF-α, appears to attenuate the detrimental effects of TNF-α.

Contribution of lipids to the organelle differential profile of in vitro-produced bovine embryos.

Each living organism is unique because of the lipid identity of its organelles. The diverse distribution of these molecules also contributes to the role of each organelle in cellular activity. The lipid profiles of whole embryos are well documented in the literature. However, this approach can often lead to the loss of relevant information at the subcellular and consequently, metabolic levels, hindering a deeper understanding of key physiological processes during preimplantation development. Therefore, we aimed to characterize four organelles in vitro-produced bovine embryos: lipid droplets (LD), endoplasmic reticulum (ER), mitochondria (MIT), and nuclear membrane (NUC), and evaluate the contribution of the lipid species to each organelle evaluated. Expanded blastocysts were subjected to cell organelle isolation. Thereafter, lipid extraction from cell organelles and lipid analysis using the Multiple Reaction Monitoring (MRM) profiling method were performed. The LD and ER displayed a greater number of lipids (Phosphatidylcholine - PC, Ceramide - Cer, and Sphingomielin - SM) with high signal-to-noise intensities. This result is due to the high rate of biosynthesis, lipid distribution, and ability to store and recycle lipid species of these organelles. The NUC had a more distinct lipid profile than the other three organelles, with high relative intensities of PC, SM, and triacylglycerols (TG), which is consistent with its high nuclear activity. MIT had an intermediate profile that was close to that of LD and ER, which aligns with its autonomous metabolism for some classes of phospholipids (PL). Our study revealed the lipid composition of each organelle studied, and the roles of these lipids could be associated with the characteristic organellar activity. Our findings highlight the lipid species and classes that are relevant for the homeostasis and function of each associated organelle and provide tentative biomarkers for the determination of in vitro embryonic development and quality.

A new direct transfer protocol for cryopreserved IVF embryos.

The global demand for in vitro-produced (IVP) embryos of determined sex has greatly increased over the last decade. Efficient protocols for the direct transfer of IVP embryos are lacking. This study aimed to compare the pregnancy rates for fresh, vitrified, or frozen/directly transferred IVP dairy cow embryos. Oocytes (n = 3171) recovered by ovum pickup (n = 112) from Girolando (Holstein-Gir) females (n = 36) were selected and submitted to IVM for 24 hours at 38.5 °C with 5% CO2 in air with saturated humidity. In vitro fertilization was performed with the thawed, sexed semen from 5 Holstein bulls. After IVF, presumptive zygotes were denuded and cultured for 7 days under the same IVM and IVF conditions of temperature and humidity, except with 5% CO2 and 5% O2. Grade I blastocysts were randomly assigned for either the transferred fresh, vitrified/thawing, or frozen/directly embryo transfer into previously synchronized recipient females. Conception rates were analyzed by binomial logistic regression, and a probability level of P < 0.05 was considered significant. The conception rates were 51.35 ± 1.87% (133/259) for the fresh embryos, 35.89 ± 3.87% (84/234) for the vitrified embryos, and 40.19 ± 4.65% (125/311) for the frozen directly transferred embryos. These data demonstrate that IVP embryos with sexed semen could be directly transferred into recipient cows with similar conception rates to vitrified embryos. The comparison found that the use of frozen embryos in direct transfer provides easier logistics and a more practical approach for the transfer of IVP embryos on dairy farms.