RESUMEN
Oocyte cryopreservation is not yet considered a reliable technique since it can reduce the quality and survival of oocytes in several species. This study determined the effect of different concentrations of antifreeze protein I (AFP I) on the vitrification solution of immature cat oocytes. For this, oocytes were randomly distributed in three groups and vitrified with 0 µg/mL (G0, 0 µM); 0.5 µg/mL (G0.5, 0.15 µM), or 1 µg/mL (G1, 0.3 µM) of AFP I. After thawing, oocytes were evaluated for morphological quality, and compared to a fresh group (FG) regarding actin integrity, mitochondrial activity and mass, reactive oxygen species (ROS) and glutathione (GSH) levels, nuclear maturation, expression of GDF9, BMP15, ZAR-1, PRDX1, SIRT1, and SIRT3 genes (normalized by ACTB and YWHAZ genes), and ultrastructure. G0.5 and G1 presented a higher proportion of COCs graded as I and while G0 had a significantly lower quality. G1 had a higher percentage of intact actin in COCs than G0 and G0.5 (P < 0.05). There was no difference (P > 0.05) in the mitochondrial activity between FG and G1 and they were both higher (P < 0.05) than G0 and G0.5. G1 had a significantly lower (P < 0.05) mitochondrial mass than FG and G0, and there was no difference among FG, G0, and G0.5. G1 had higher ROS than all groups (P < 0.05), and there was no difference in GSH levels among the vitrified groups (P > 0.05). For nuclear maturation, there was no difference between G1 and G0.5 (P > 0.05), but these were both higher (P < 0.05) than G0 and lower (P < 0.05) compared to FG. Regarding gene expression, in G0 and G0.5, most genes were downregulated compared to FG, except for SIRT1 and SIRT3 in G0 and SIRT3 in G0.5. In addition, G1 kept the expression more similar to FG. Regardless of concentration, AFP I supplementation in vitrification solution of immature cat oocytes improved maturation rates, morphological quality, and actin integrity and did not impact GSH levels. In the highest concentration tested (1 µg/mL), AFP maintained the mitochondrial activity, reduced mitochondrial mass, increased ROS levels, and had the gene expression more similar to FG. Altogether these data show that AFP supplementation during vitrification seems to mitigate some of the negative impact of cryopreservation improving the integrity and cryosurvival of cat oocytes.
Asunto(s)
Criopreservación , Oocitos , Vitrificación , Animales , Criopreservación/veterinaria , Criopreservación/métodos , Gatos , Oocitos/efectos de los fármacos , Vitrificación/efectos de los fármacos , Femenino , Crioprotectores/farmacología , Proteínas Anticongelantes/farmacología , Proteínas Anticongelantes/genética , Especies Reactivas de Oxígeno/metabolismo , Mitocondrias/efectos de los fármacos , Glutatión/farmacología , Glutatión/metabolismoRESUMEN
There is still no consensus regarding the role of lipid modulators during in vitro embryo production. Thus, we investigated how lipid reducers during the in vitro maturation of oocytes (IVM) or in vitro culture (IVC) of embryos impact their cryotolerance. A literature search was performed using three databases, recovering 43 articles for the systematic review, comprising 75 experiments (13 performed in IVM, 62 in IVC) and testing 13 substances. In 39 % of the experiments, an increase in oocyte and/or embryo survival after cryopreservation was reported, in contrast to 48 % exhibiting no effect, 5 % causing negative effects, and 8 % influencing in a dose-dependent manner. Of the 75 experiments extracted during IVM and IVC, 41 quantified the lipid content. Of those that reduced lipid content (n = 26), 50 % increased cryotolerance, 34 % had no effect, 8 % harmed oocyte/embryo survival, and 8 % had different results depending on the concentration used. Moreover, 28 out of the 43 studies were analyzed under a meta-analytical approach at the IVC stage in cattle. There was an improvement in the cryotolerance of bovine embryos when the lipid content was reduced. Forskolin, l-carnitine, and phenazine ethosulfate positively affected cryotolerance, while conjugated linoleic acid had no effect and impaired embryonic development. Moreover, fetal bovine serum has a positive impact on cryotolerance. SOF and CR1aa IVC media improved cryotolerance, while mSOF showed no effect. In conclusion, lipid modulators did not unanimously improve cryotolerance, especially when used in IVM, but presented positive effects on cryotolerance during IVC when reaching lipid reduction.
Asunto(s)
Criopreservación , Técnicas de Cultivo de Embriones , Animales , Criopreservación/veterinaria , Criopreservación/métodos , Técnicas de Cultivo de Embriones/veterinaria , Lípidos/química , Técnicas de Maduración In Vitro de los Oocitos/veterinaria , Técnicas de Maduración In Vitro de los Oocitos/métodos , Fertilización In Vitro/veterinaria , Bovinos/embriología , Metabolismo de los Lípidos , Embrión de Mamíferos/fisiologíaRESUMEN
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.