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Zearalenone (ZEA), which is produced by several fusarium mycotoxins, is found in animal feed and food products, and can exert estrogen-like activity. Melatonin (MT) is emerging as a supplement that can fight the toxic effects of mycotoxins. With a variety of physiological functions that play crucial roles in the development of animal germ cells and embryos, melatonin regulates circadian rhythms and has an anti-inflammatory and anti-oxidative role. This study investigated the protective effects of melatonin against ZEA in porcine early embryonic development. Our results showed that ZEA adversely affected this development, while melatonin supplementation ameliorated the toxic effects. ZEA exposure increased oxidative stress and impaired mitochondrial function, which may affect blastocyst formation. Moreover, we found that ZEA exposure promotes apoptosis, DNA damage, and autophagy in porcine blastocysts. The toxic effects of ZEA on early embryos may be the result of oxidative stress-mediated early apoptosis, while melatonin treatment significantly improved these phenotypes in ZEA-exposed porcine early embryos. Taken together, our results indicate that melatonin has a protective effect on defects caused by ZEA during early porcine embryonic development.

The present study aimed to investigate the effect of vitrification temperature (VT) and cryoprotective agent concentrations (CPAs) on the mRNA transcriptome of bovine mature oocytes after vitrifying at immature stage. Cumulus oocyte complexes (COCs) were randomly divided into the following five groups: fresh oocytes (control), oocytes vitrified in liquid helium (LHe; -269 °C) with 5.6 M CPAs (LHe 5.6 M), oocytes vitrified in LHe with 6.6 M CPAs (LHe 6.6 M), oocytes vitrified in liquid nitrogen (LN; -196 °C) with 5.6 M CPAs (LN 5.6 M), and oocytes vitrified in LN with 6.6 M CPAs (LN 6.6 M). We performed two experiments in this study. In experiment 1, after vitrification and thawing, oocytes of vitrified and control groups were subjected to in vitro maturation (IVM), in vitro fertilization (IVF) and in vitro culture (IVC). The rates of normal morphology, maturation, cleavage, and blastocyst formation in LHe 5.6 M were higher than those in LN 5.6 M (P 0.05). The rates of normal morphology and cleavage in LHe 6.6 M were higher than those in LN 6.6 M (P 0.05). However, the maturation and blastocyst rates were similar (P 0.05) between LHe 6.6 M and LN 6.6 M. The blastocyst rate of 13.31% in LHe 5.6 M was the highest among all vitrified groups (P 0.05). In experiment 2, the mRNA transcriptome of each sample was analyzed by Smart-Seq4, and the differentially expressed genes (DEGs) were detected by edgeR (P ≤ 0.05; fold-change ≥ 2). A total of 505 DEGs (342 upregulated and 163 downregulated genes) were detected in LHe 5.6 M; 609 DEGs (493 upregulated and 116 downregulated genes) were detected in LHe 6.6 M; 218 DEGs (101 upregulated and 117 downregulated genes) were determined in LN 5.6 M; and 221 DEGs (104 upregulated and 117 downregulated genes) were detected in LN 6.6 M. LHe vitrification affected the mRNA transcriptome of bovine mature oocytes after vitrifying at immature stage mainly by upregulating gene expression. Decreased CPAs (5.6 M) reduced the effect of vitrification on mRNA transcriptome when LHe vitrification was used. Among the DEGs closely related to bovine oocytes, the genes possibly related to VT were ND2, MPV17L2, PIF1, LPIN1, IMP3, BRD1, DCTN3, DERA, ATP7B, NEK5, HVCN1, and MARK2. The gene that may be associated with CPAs is CC2D2A. Genes that may be affected by VT and CPAs included PGK1, SLC7A3, FITM2, NPM3, ISCU, CWC15, and PSAP.

We evaluated the effects of different vitrification temperatures (VTs) and cryoprotective agent concentrations (CPAs) on the viability and expressions of long non-coding RNA (lncRNA) in bovine oocytes following vitrification at the germinal vesicle (GV) stage. Our findings provide a theoretical support for improvement of the cryopreservation technology of bovine immature oocytes (BIOs). Bovine cumulus oocyte complexes (COCs) were collected and randomized into five groups: fresh oocytes (control), oocytes vitrified in liquid helium (LHe; -269 °C) with 5.6 M CPAs (LHe 5.6 M), oocytes vitrified in LHe with 6.6 M CPAs (LHe 6.6 M), oocytes vitrified in liquid nitrogen (LN; -196 °C) with 5.6 M CPAs (LN 5.6 M), and oocytes vitrified in LN with 6.6 M CPAs (LN 6.6 M). Of the four vitrification groups, the LHe 5.6 M group exhibited the highest blastocyst rate (13.22%), followed by the LHe 6.6 M group (10.19%) and LN 6.6 M group (9.77%), while the LN 5.6 M group had the lowest blastocyst rate (1.87%). Then, lncRNA expressions in the five groups were profiled. A total of 18,271 lncRNAs were identified, of which 2,158 were differentially expressed lncRNAs (DELs) in the vitrified groups, compared to the fresh group (P 0.05; fold-change2). Co-location (cis) and co-expression (trans) prediction revealed 14 differentially expressed target genes (DETGs), which corresponded to 17 DELs. Based on grouping data and expression profiles of the DELs, we demonstrated that different VTs (-269 °C vs. -196 °C) can affect the expressions of MSTRG.12295.5, MSTRG.37123.1, MSTRG.37930.2, MSTRG.40464.9, MSTRG.8869.3 and MSTRG.26680.6. Expressions of these lncRNAs were affected by CPAs only in the condition of vitrification with LHe (-269 °C). Expressions of MSTRG.35129.6 were associated with exposures to both VTs and CPAs; while expressions of MSTRG.3578.3, MSTRG.40576.3, MSTRG.6723.5, MSTRG.32862.4, MSTRG.1184.4, MSTRG.33110.3, MSTRG.40454.2, MSTRG.41073.2, MSTRG.44732.4 and MSTRG.6729.3 might be related to vitrification. Co-expression analysis showed that MSTRG.12295.5, MSTRG.37930.2, MSTRG.40454.2, MSTRG.8869.3 and MSTRG.6723.5 expressions affect oocyte development after vitrification by regulating target gene expressions. Taken together, improvement of the developmental ability of BIOs after LHe vitrification maybe attributed to changes in expressions of some lncRNAs. Our findings elucidate on the molecular mechanisms underlying the development of BIOs under different VTs and CPAs.

Aging oocytes undergo various molecular, cellular, and biochemical changes. Aging of oocytes results in reduced embryo developmental capacity and blastocyst quality, which is thought to be caused partly by the accumulation of reactive oxygen species (ROS). This study aimed to determine the effect of l -carnitine (LC) on the development of embryos formed from aged oocytes in vitro. The development and quality of the blastocysts in the LC-treated group were significantly higher than those in the untreated aged group after in vitro fertilization (IVF). In addition, after LC treatment, the level of intracellular ROS in aged group significantly decreased, and glutathione (GSH) levels significantly increased compared with those in the untreated aged group. There was no significant difference in the mitochondrial membrane potential among the three groups. Moreover, ROS could induce autophagy and LC3 antibody was widely used as a marker for detecting autophagy. The fluorescence intensity of LC3 in the aged group was significantly higher than that of LC3 in the LC-treated group. Furthermore, Real-time reverse transcriptase-polymerase chain reaction showed that the mRNA levels of antioxidation genes GPX4 and SOD1 were significantly higher in embryos from LC-treated group than in those from the untreated aged group. In summary, our results indicated that LC can improve the developmental capacity of embryos from aging oocytes in vitro by reducing oxidative stress.

This study aimed to investigate the developmental potential of and the ultrastructural changes and gene expression differences resulting from liquid helium (LHe; -269 °C) vitrification in immature bovine oocytes. Immature oocytes were randomly divided into three groups: fresh oocytes (control, negative control), oocytes vitrified in liquid nitrogen (LN group, positive control), and oocytes vitrified in LHe (LHe group). In experiment 1, the rates of normal morphology, maturation, cleavage, and blastocyst in the LHe group were higher than those in the LN group (87.1% vs. 80.5%, 51% vs. 48%, 41.7% vs. 36.8%, and 13% vs. 8.5%, respectively; P 0.05), and the rates of development in the control group (100%, 73.2%, 62%, and 39.8%) were higher than those in the treated groups (P 0.05). In experiment 2, oocytes displayed various degrees of injury at the ultrastructural level after vitrification, but more severe degeneration was observed in the LN group, such as formation of several lipid droplets, swelling of mitochondria, and absence of cortical granules. Compared with the LN group, fewer lipid droplets, relatively intact mitochondria, and clustered cortical granules were distributed in the cytoplasm of oocytes in the LHe group. In experiment 3, the mRNA expression levels of p53, CDC20, Eg5, and Npm2 were investigated by real-time quantitative polymerase chain reaction. Expression levels of the kinesin Eg5 and the apoptotic gene p53 expression levels were higher in the LN group compared with the control and LHe groups (P 0.05). CDC20 and Npm2 expression did not differ significantly between the LN and LHe groups (P0.05), the CDC20 expression in the LN and LHe groups were lower than control group (P0.05), the Npm2 expression in LHe group was lower than control group (P0.05), but there was no significant difference between the LN and control groups (P0.05). In conclusion, LHe vitrification decreased the negative effect of cryoinjury on the ultrastructure of some organelles and the expression of some related genes, thereby improving the viability of immature bovine oocytes compared to LN vitrification.

The objectives of this study were to compare the effectiveness of liquid helium (LHe) and liquid nitrogen (LN2) as cryogenic liquid for vitrification of bovine immature oocytes with open-pulled straw (OPS) system and determine the optimal cryoprotectant concentration of LHe vitrification. Cumulus oocyte complexes were divided into three groups, namely, untreated group (control), LN2 vitrified with OPS group, and LHe vitrified with OPS group. Oocyte survival was assessed by morphology, nuclear maturation, and developmental capability. Results indicated that the rates of normal morphology, maturation, cleavage, and blastocyst (89.3%, 52.8%, 42.7%, and 10.1%, respectively) in the LHe-vitrified group were all higher than those (79.3%, 43.4%, 34.1%, and 4.7%) in the LN2-vitrified group (P0.05) although the corresponding rates in both treated groups decreased compared with the control group (100%, 75.0%, 64.9%, and 40.8%; P0.05). Normal calves were obtained after the transfer of blastocysts derived from LHe- and LN2-vitrified oocytes. The effects of the different vitrification solutions (EDS30, EDS35, EDS40, EDS45, and EDS50) in LHe vitrification for bovine immature oocytes vitrification were examined. No difference was found in the rates of morphologically normal oocytes among the EDS30 (87.9%), EDS35 (90.1%), EDS40 (89.4%), and EDS45 (87.2%) groups (P0.05). The maturation rate of the EDS35 group (65.0%) was higher than those of the EDS30 (51.3%), EDS40 (50.1%), EDS45 (52.1%), and EDS50 groups (36.9%; P0.05). No significant differences were observed in the cleavage and blastocyst rates between the EDS35 (49.0% and 12.1%) and EDS40 (41.7% and 10.2%) groups. However, the cleavage and blastocyst rates in the EDS35 group were higher (P0.05) than those of the EDS30 (36.2% and 6.8%), EDS45 (35.9% and 5.8%), and EDS50 (16.6% and 2.2%) groups. In conclusion, LHe can be used as a cryogenic liquid for vitrification of bovine immature oocytes, and it is more efficient than LN2-vitrified oocytes in terms of blastocyst production. EDS35 was the optimal cryoprotectant agent combination for LHe vitrification in this study.