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.

Melatonin improves the vitrification of sheep morulae by modulating transcriptome.

Embryo vitrification technology is widely used in livestock production, but freezing injury has been a key factor hindering the efficiency of embryo production. There is an urgent need to further analyze the molecular mechanism of embryo damage by the vitrification process. In the study, morulae were collected from Hu sheep uterine horns after superovulation and sperm transfusion. Morulae were Cryotop vitrified and warmed. Nine morulae were in the vitrified control group (frozen), and seven morulae were vitrified and warmed with 10-5 M melatonin (melatonin). Eleven non-frozen morulae were used as controls (fresh). After warming, each embryo was sequenced separately for library construction and gene expression analysis. p < 0.05 was used to differentiate differentially expressed genes (DEG). The results showed that differentiated differentially expressed genes (DEG) in vitrified morulae were mainly enriched in protein kinase activity, adhesion processes, calcium signaling pathways and Wnt, PI3K/AKT, Ras, ErbB, and MAPK signaling pathways compared to controls. Importantly, melatonin treatment upregulated the expression of key pathways that increase the resistance of morulae against vitrification induced damage. These pathways include kinase activity pathway, ErbB, and PI3K/Akt signaling pathway. It is worth mentioning that melatonin upregulates the expression of XPA, which is a key transcription factor for DNA repair. In conclusion, vitrification affected the transcriptome of in vivo-derived Hu sheep morulae, and melatonin had a protective effect on the vitrification process. For the first time, the transcriptome profiles caused by vitrification and melatonin in sheep morulae were analyzed in single embryo level. These data obtained from the single embryo level provide an important molecular mechanism for further optimizing the cryopreservation of embryos or other cells.

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.

Presence of leptin and its receptor in the ram reproductive system and in vitro effect of leptin on sperm quality.

Leptin is a 16 kDa hormone encoded by obese (OB) gene in adipocytes. This molecule not only regulates energy metabolism but also plays a role in the reproduction of mammals. Leptin and its receptor (OBR) have been found in male reproductive systems of human, bovine, equine and pig. The effects of leptin on sperm quality vary widely from different research findings. However, the presence of leptin and its receptor in the ram reproductive system and the in vitro effect of leptin on sperm quality have not reported yet. In the present study, we found that the OB was highly expressed in primary and secondary spermatocytes of the testes, OBR was highly expressed in secondary spermatocytes of the testes. The expressions of OB were in stereocilia of epididymis and in columnar cells of epididymal caput and cauda, the expressions of OBR were in columnar cells of epididymis and in stereocilia of epididymal corpus and cauda. The presence of both OB and OBR in testes, epididymis and sperm were confirmed through RT-PCR, immunolocalization and Western blot analyses. The RT-qPCR results indicated OB and OBR had higher expression levels in epididymal sperm than that of the ejaculated sperm in rams. When sperm were treated with 5 ng/mL leptin, the progressive motility (P < 0.01), straight-line velocity (VSL) (P < 0.05), average path velocity (VAP) (P < 0.05), membrane mitochondrial potential (MMP) (P < 0.01) and viability (P < 0.05) significantly increased, while DNA fragmentation index (DFI) and reactive oxygen species (ROS) significantly decreased compared to the control (P < 0.01), and the other semen parameters such as acrosome integrity and acrosome reaction rate had no significant changes between groups (P > 0.05). In conclusion, this is probably the first report describing localization of leptin and its receptors in the reproductive system of rams and their effects on sperm quality parameters. Our findings suggest that 5 ng/mL leptin treatment enhanced sperm motility, viability and MMP, and decrease DFI and ROS without obvious influence on the acrosome reaction in ram sperm. The potential mechanisms may be related to leptin's ability to reduce the oxidative stress and apoptosis of sperms and improve their mitochondrial function and energy supply, therefore, to maintain the physiological homeostasis of the sperm.

Melatonin promotes the growth and development of lambs by increasing growth hormone and testosterone, targeting on apoptosis signaling pathway and intestinal microflora.

Melatonin is an indole-like neuroendocrine hormone. A large number of studies have shown that melatonin can improve production performance of ewes, but it is not clear in lambs. In this study, the growth and development of the 2-month-old lambs implanted with melatonin were monitored for 60 days. The results showed that the growth rate of body weight and body skew length of lambs with melatonin treatment were significantly improved compared to the controls. The similar results were also observed in red blood cell count, hematocrit, red blood cell volume distribution width, the levels of growth hormone, testosterone, immunoglobulin A, immunoglobulin M and albumin. In addition, the cross sectional area of muscle fibers and adipose cells of lambs with melatonin implantation were also significantly increased compared to the controls (P<0.05). To further explore the potential mechanisms, the muscle and adipose tissue were selected for transcriptome sequencing. KEGG enrichment results showed that melatonin regulated the expression of genes related to apoptotic signaling pathway in muscle and adipocytes. Since the intestinal microbiota are involved in the nutritional balance and animal growth, the 16SrRNA sequencing related to the intestinal microbiota was also performed. The data indicated that the structural differences of fecal microflora mainly occur in the pathways of Cardiovascular disease, Excretory system and Signaling molecules and interaction. In brief, melatonin promotes the growth and development of lambs. The potential mechanisms may be that melatonin increased the growth hormone and testosterone mediated apoptosis signaling pathway and regulated intestinal microbial flora. Our results provide valuable information for melatonin to improve the production of sheep husbandry in the future.

The Mechanism of Heat Stress Resistance During Spermatogenesis in Turpan Black Sheep.

Heat stress can affect the reproductive function of livestock and cause harm to animal production, which can seriously damage the economic interests of livestock producers. Therefore, it is important to explore the effect of heat stress on reproductive function to improve livestock production. In this study, the experimental animals Turpan black sheep and Suffolk sheep were selected as controls, each with 10 sheep, and the reproductive physiological performance was measured in Turpan, China from April to August when there was no heat stress to strong heat stress. The results showed that the sperm density, vitality, and kinematic parameters of Suffolk sheep were significantly lower than that in Turpan black sheep (p < 0.01) after heat stress, while the sperm acrosome malfunctions and DNA damage were significantly higher in Suffolk sheep (p < 0.01). In addition, the endogenous levels of reproductive hormones and oxidative stress indicators in the blood of Turpan black sheep were stable before and after heat stress treatment, while Suffolk sheep showed different degrees of fluctuations. There was no significant difference in testicular histomorphology between the two after heat stress treatment. However, Suffolk sheep showed a significantly decreased number of spermatocytes after heat stress treatment (p < 0.05). It was found that during meiosis, the proportion of cells in the meiotic zygotene stage of Suffolk sheep was significantly higher than that of Turpan black sheep. To investigate the mechanism of normal spermatogenesis in Turpan black sheep under heat stress, we performed RNA-Seq analysis on the testis. The results showed that there were 3,559 differential genes in Turpan black sheep before and after heat stress, with 2,118 up-regulated genes and 1,441 down-regulated genes. The enrichment analysis of GO and KEGG showed that the differential genes are mainly involved in cellular component organization or biogenesis, cell cycle process, mitotic cell cycle process, meiotic cell cycle process, double-strand break repair and Rap1 signaling pathway, Ras signaling pathway, Cell cycle, signaling pathways regulating pluripotency of stem cells Oocyte meiosis. Genes related to spermatogenesis, SYCP2, TDRD9, BRDT, CEP120, BRCA1, etc. were significantly up-regulated in Turpan black sheep after heat stress. In summary, our results showed that the up-regulation of genes involved in spermatogenesis protects the normal production of sperm in Turpan black sheep under HS, thereby achieving normal reproductive function.Our research systematically elucidated the mechanism of heat stress resistance during spermatogenesis in Turpan black sheep and provided potential possibilities for the subsequent breeding of new heat-resistant breeds.

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.

Melatonin improves the efficiency of super-ovulation and timed artificial insemination in sheep.

It has been well proved that melatonin participates in the regulation of the seasonal reproduction of ewes. However, the effects of short term treatment of melatonin on ewe's ovulation are still to be clarified. In this study, the effects of melatonin on the number of embryos harvested from superovulation, and the pregnant rate in recipients after embryo transferred have been investigated. Hu sheep with synchronous estrus treatment were given melatonin subcutaneously injection (0, 5, and 10 mg/ewe, respectively). It was found that the estrogen level in the group of 5 mg melatonin was significantly higher than that of other two groups at the time of sperm insemination (p < 0.05). The pregnant rate and number of lambs in the group of 5 mg melatonin treatment was also significantly higher than that of the rests of the groups (P < 0.05). In another study, 31 Suffolk ewes as donors and 103 small-tailed han sheep ewes as recipients were used to produce pronuclear embryo and embryo transfer. Melatonin (5 mg) was given to the donors during estrus. The results showed that, the number of pronuclear embryos and the pregnancy rate were also significantly higher in melatonin group than that in the control group. In addition, 28 donors and 44 recipient ewes were used to produce morula/blastocyst and embryo transferring. Melatonin (5 mg) was given during estrus. The total number of embryos harvested (7.40 ± 1.25/ewe vs. 3.96 ± 0.73/ewe, P < 0.05) and the pregnant rate (72.3 ± 4.6% vs. 54.7 ± 4.0%, P < 0.05) and number of lambs were also increased in melatonin group compared to the control group. Collectively, the results have suggested that melatonin treatment 36 hours after CIDR withdrawal could promote the number and quality of embryos in vivo condition and increased the pregnant rate and number of lambs.