Background: Goat milk is very similar to human milk in terms of its abundant nutrients and ease of digestion. To derive greater economic benefit, farmers require more female offspring (does); however, the buck-to-doe offspring sex ratio is approximately 50%. At present, artificial insemination after the separation of X/Y sperm using flow cytometry is the primary means of controlling the sex of livestock offspring. However, flow cytometry has not been successfully utilised for the separation of X/Y sperm aimed at sexing control in dairy goats.Results: In this study, a novel, simple goat sperm sexing technology that activates the toll-like receptor 7/8 (TLR7/8), thereby inhibiting X-sperm motility, was investigated. Our results showed that the TLR7/8 coding goat X-chromosome was expressed in approximately 50% of round spermatids in the testis and sperm, as measured from cross-sections of the epididymis and ejaculate, respectively. Importantly, TLR7/8 was located at the tail of the X-sperm. Upon TLR7/8 activation, phosphorylated forms of glycogen synthase kinase α/β (GSK3 α/β) and nuclear factor kappa-B (NF-κB) were detected in the X-sperm, causing reduced mitochondrial activity, ATP levels, and sperm motility. High-motility Y-sperm segregated to the upper layer and the low-motility X-sperm, to the lower layer. Following in vitro fertilisation using the TLR7/8-activated sperm from the lower layer, 80.52 ± 6.75% of the embryos were XX females. The TLR7/8-activated sperm were subsequently used for in vivo embryo production via the superovulatory response; nine embryos were collected from the uterus of two does that conceived. Eight of these were XX embryos, and one was an XY embryo.Conclusions: Our study reveals a novel TLR7/8 signalling mechanism that affects X-sperm motility via the GSK3 α/β-hexokinase pathway; this technique could be used to facilitate the efficient production of sexed dairy goat embryos.

Summary: Oocyte-specific linker histone, H1foo, is localized on the oocyte chromosomes during the process of meiotic maturation, and is essential for mouse oocyte maturation. Bovine H1foo has been identified, and its expression profile throughout oocyte maturation and early embryo development has been established. However, it has not been confirmed if H1foo is indispensable during bovine oocyte maturation. Effective siRNAs against H1foo were screened in HeLa cells, and then siRNA was microinjected into bovine oocytes to down-regulate H1foo expression. H1foo overexpression was achieved via mRNA injection. Reverse transcription polymerase chain reaction (RT-PCR) results indicated that H1foo was up-regulated by 200% and down-regulated by 70%. Based on the first polar body extrusion (PB1E) rate, H1foo overexpression apparently promoted meiotic progression. The knockdown of H1foo significantly impaired bovine oocyte maturation compared with H1foo overexpression and control groups (H1foo overexpression = 88.7%, H1foo siRNA = 41.2%, control = 71.2%;P< 0.05). This decrease can be rescued by co-injection of a modified H1foo mRNA that has escaped from the siRNA target. However, the H1e (somatic linker histone) overexpression had no effect on PB1E rate when compared with the control group. Therefore we concluded that H1foo is essential for bovine oocyte maturation and its overexpression stimulates the process.

The significant morphological differences and abundant germplasm resources of Chinese indigenous dog breeds can be attributed to the diverse geographical environment, including plateaus, mountains, and a long history of raising dogs. The combination of both natural and artificial selection during the past several thousand years has led to hundreds of dog breeds with distinct morphological traits and environmental adaptations. China is one of the earliest countries to domesticate dogs and there are more than 50 ancient indigenous dog breeds. In this study, the run of homozygosity (ROH) and proportion of the autosomal genome covered by ROHs (F_ROH) were calculated for 10 dog breeds that are the most representative Chinese indigenous dogs based on 170K SNP microarray. The results of F_ROH showed that the Chuandong hound dogs (HCSSC) have the highest level of inbreeding among the tested breeds. The inbreeding in HCSSC occurred more recently than the Liangshan dogs (SCLSQ) dogs because of more numbers of long ROHs in HCSSC dogs, and the former also have higher inbreeding degree. In addition, there are significant differences in the inbreeding degree among different subpopulations of the same breed, such as the Thin dogs from Shaanxi and Shandong province. To explore genome-wide selection signatures among different breeds, including coat color, ear shape, and altitude adaptability, we performed genome selection analyses of F_ST and cross population extended haplotype homozygosity (XP-EHH). For the coat color, the F_ST analysis between Xiasi dogs (XSGZ) and HCSSC dogs was performed and identified multiple genes involved in coat color, hair follicle, and bone development, including MC1R, KITLG, SOX5, RSPO2, and TBX15. For the plateau adaptability, we performed F_ST and XP-EHH analyses between dogs from Tibet (Tibetan Mastiffs and Nyingchi dogs) and plain regions (Guangxi Biwei dogs GXBWQ and Guandong Sharpei dogs). The results showed the EPAS1 gene in dogs from Tibet undergo strong selection. Multiple genes identified for selection signals based on different usage of dogs. Furthermore, the results of ear shape analyses showed that MSRB3 was likely to be the main gene causing the drop ear of domestic dogs. Our study provides new insights into further understanding of Chinese indigenous dogs.

α‐Ketoglutarate (α‐KG) is a metabolite in the tricarboxylic acid cycle. It has a strong antioxidant function and can effectively prevent oxidative damage. Previous studies have shown that α‐KG exists in porcine follicles, and its content gradually increases as the follicles grow and mature. However, the potential mechanism of supplementation of α‐KG on porcine oocytes during in vitro maturation (IVM) has not yet been reported. The purpose of this study was to explore the effect of α‐KG on the early embryonic development of pigs and the mechanisms underlying these effects. We found that α‐KG can enhance the development of early pig embryos. Adding 20 μM α‐KG to the in vitro culture medium significantly increased the rate of blastocyst formation and the total cell number. Compared with to that of the control group, apoptosis in blastocysts of the supplement group was significantly reduced. α‐KG reduced the production of reactive oxygen species and glutathione levels in cells. α‐KG not only improved the activity of mitochondria but also inhibited the occurrence of apoptosis. After supplementation with α‐KG, pig embryo pluripotency‐related genes (OCT4, NANOG, and SOX2) and antiapoptotic genes (Bcl2) were upregulated. In terms of mechanism, α‐KG activates the Nrf2/ARE signaling pathway to regulate the expression of antioxidant‐related targets, thus combating oxidative stress during the in vitro culture of oocytes. Activated Nrf2 promotes the transcription of Bcl2 genes and inhibits cell apoptosis. These results indicate that α‐KG supplements have a beneficial effect on IVM by regulating oxidative stress during the IVM of porcine oocytes and can be used as a potential antioxidant for IVM of porcine oocytes.