Mito-TEMPO Improves the Meiosis Resumption and Mitochondrial Function of Vitrified Sheep Oocytes via the Recovery of Respiratory Chain Activity.

Vitrification is a crucial method for preserving animal germ cells. Considering the increased oxidative stress and organelle damage incurred, it is still necessary to make the process more efficient for oocytes. As the energy source of oocytes, mitochondria are the most abundant organelle in oocytes and play a crucial role in their maturation. Here, we found that Mito-TEMPO, a mitochondria-targeted antioxidant, could efficaciously improve the oxidative stress injury of vitrified oocytes by recovering mitochondrial function via the mitochondrial respiratory chain. It was observed that Mito-TEMPO not only improves oocyte viability and meiosis but also maintains spindle structure. A subsequent study indicated that Mito-TEMPO effectively rescued mitochondrial dysfunction and attenuated vitrification-induced oxidative stress. Further investigation revealed that Mito-TEMPO regulates vitrified oocytes' intracellular Ca2+ homeostasis and ATP content and provides strong antioxidant properties. Additionally, an analysis of the transcriptome at the single-cell level revealed that the respiratory chain mediates the beneficial effect of Mito-TEMPO on vitrified oocytes. Overall, our findings indicate that supplementing oocytes with Mito-TEMPO is an effective method to shield them from the damage caused by vitrification. In addition, the beneficial effects of Mito-TEMPO on vitrified sheep oocytes could inspire further investigations of the principles underlying oocyte cryobiology in other animals.

Metabolomic and Transcriptomic Changes Underlying the Effects of L-Citrulline Supplementation on Ram Semen Quality.

This study examined the effects of L-Cit supplementation on ram semen quality through metabolomics and transcriptomics. A total of 16 rams were randomly categorized into two groups. The control group was fed a basic diet, whereas the experimental group received feed supplemented with 12 g/d of L-Cit. Semen and blood were collected from the rams on days 0 and 72 to measure sugar, pyruvate, amino acid, and nontargeted metabolite contents. Additionally, hypothalamic and testicular tissues were collected for a transcriptomic analysis. We found 27 differential metabolites between the control and experimental groups, of which 21 were downregulated (p < 0.05) and 6 were upregulated (p < 0.05). Compared with the control group, xylose and pyruvate contents in seminal plasma increased by 43.86% and 162.71%, respectively (p < 0.01). Additionally, the levels of 11 amino acids showed a significant increase in seminal plasma (p < 0.01). Furthermore, 961 and 715 differentially expressed genes were detected in the hypothalamic and testicular tissues, respectively. The pathways of significant enrichment in the hypothalamus and testes were protein digestion, absorption, glycolysis/gluconeogenesis, and amino as well as nucleotide sugar metabolisms. In the present study, L-Cit improved protein synthesis and blood metabolism, consequently increasing the contents of most amino acids in ram seminal plasma. Specifically, the hypothalamus controlled the expression of glycolysis/gluconeogenesis-related genes in the testes through its metabolites released into the serum, thereby providing energy for sperm production, which led to a decrease in the sugar content of seminal plasma.

Effect of L-citrulline supplementation on sperm characteristics and hormonal and antioxidant levels in blood and seminal plasma of rams.

With the aim of providing a theoretical basis for the application of L-citrulline (L-Cit) in animal husbandry, the effects of L-Cit on reproductive hormone levels, antioxidant capacity and semen quality of rams were studied by feeding them varying doses of L-Cit. A total of 32 rams were randomly divided into four groups with eight rams each. After all rams were trained to donate sperm normally, the control group was fed a basic diet, whereas the experimental groups I, II and III were provided with feed supplemented with 4, 8 and 12 g/d of L-Cit respectively. The experiment was conducted for 70 days, during which blood samples were collected from the jugular vein on days 0, 15, 30, 45 and 60, and semen samples were collected on days 0, 20, 40 and 60. In the same group, 100 µl of semen was used to test for quality, The rest of the semen sample and blood samples were centrifuged at 600 g for 15 min, and the supernatant and serum, respectively, were used to determine the levels reproductive hormones and antioxidant indices. Ram semen samples were also collected on day 70 and used to study sperm plasma membrane, substitution and mitochondrial membrane potential. Compared with the control group, the groups receiving L-Cit showed an increase in sperm concentration and number of linear motile sperm (p < .01); a decrease in the number of dead sperm (p < .01); an increase in sperm viability, particularly in groups II and III (p < .01); and an increase in sperm mitochondrial membrane potential (p < .01). Moreover, groups I, II and III showed significantly higher levels of serum gonadotropin-releasing hormone (GnRH), glutathione peroxidase (GSH-Px) and nitric oxide (NO) (p < .01). Luteinizing hormone (LH) and follicle-stimulating hormone (FSH) levels increased in groups I (p < .05), II (p < .05) and III (p < .01), whereas testosterone (T), catalase (CAT) and superoxide dismutase (SOD) levels increased in groups I and II (p < .01). Serum total antioxidant capacity (T-A) increased (p < .05), whereas both hydroxyl radical (·OH) and peroxy radical ( O 2 · - ) levels decreased (p < .01). Compared with the control, all groups had significantly higher SOD and GSH-Px in their seminal plasma (p < .01), and groups I, II (p < .05 for both) and III (p < .01) had higher levels of GnRH and FSH. LH, CAT and NO levels increased in group I (p < .05), II and III (p < .01 for both); malondialdehyde levels decreased in groups I, II (p < .05 for both) and group III (p < .01); and O 2 · - levels decreased in groups I, II and III (p < .01). Under our experimental conditions, GnRH, FSH, LH, T, CAT, SOD, T-A, GSH-PX and NO levels in the serum and seminal plasma of rams receiving L-Cit increased, whereas Oestradiol (E2 ), O 2 · - and ·OH levels in the seminal plasma decreased; this improved the semen quality of rams supplemented with L-Cit. Moreover, supplementation with 12 g/d gave the best results.

Identifying the heat resistant genes by multi-tissue transcriptome sequencing analysis in Turpan Black sheep.

Heat stress not only affects the physical condition but also affects reproductive performance in sheep. A thorough understanding of the molecular and physiological mechanisms underlying heat stress would certainly improve livestock productivity and provide genetic evaluation ways for heat resistant breeds selection. In this study, 85 Turpan Black sheep, a breed exhibited excellent heat resistance from long-term artificial selection, and 85 heat sensitive Kazakh sheep in Turpan basin were tested for physiological and reproductive performance from July to August in summer. The results showed that the estrus rate was significantly higher in Turpan Black sheep (P < 0.05), while the heart rate and respiratory rate of Turpan Black sheep are significantly lower than that of Kazakh sheep (P < 0.05). Furthermore, to clarify genes participated in heat stress response, the pituitary, ovarian and hepatic tissues from three Turpan Black sheep and three Kazakh sheep were subjected to RNA-seq. The results indicated that 32, 49 and 69 genes were up-regulated, and 39, 60 and 145 genes were down-regulated in pituitary, ovarian and hepatic tissues in Turpan Black sheep compared with that of the Kazakh sheep, respectively. KEGG and gene set enrichment analysis showed that the differentially expressed genes were mainly involved in signal transduction pathways. In particular, the differentially expressed genes in hepar were enriched in the energy metabolism pathway, while the differentially expressed genes in ovarian tissue were enriched in the ovarium steroidogenesis pathway. In conclusion, our results implied that the pituitary-ovary axis might include hepar as downstream targeted organism in heat resistant regulation. Under heat stress, the signals released from pituitary would impact steroidogenesis in ovary, and further alter energy metabolism in hepar. As we know, this is the first comparative study to investigate the gene expression in multi-tissue in sheep under heat stress.