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 kinaseα/β(GSK3α/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 GSK3α/β-hexokinase pathway;this technique could be used to facilitate the efficient production of sexed dairy goat embryos.

Fa Ren;Huaming Xi;Yijie Ren;Yu Li;Fei Wen;Ming Xian;Mengjie Zhao;Dawei Zhu;Liqiang Wang;Anmin Lei;Jianhong Hu

Key Laboratory of Animal Genetics,Breeding and Reproduction of Shaanxi Province,College of Animal Science and Technology,Northwest A&F University,No.22 Xinong Road,Yangling,Shaanxi 712100,People's Republic of China;College of Veterinary Medicine,Northwest A&F University,Yangling 712100,Shaanxi,China

畜牧与生物技术杂志（英文版）

[1]Fa Ren;Huaming Xi;Yijie Ren;Yu Li;Fei Wen;Ming Xian;Mengjie Zhao;Dawei Zhu;Liqiang Wang;Anmin Lei;Jianhong Hu-.TLR7/8 signalling affects X-sperm motility via the GSK3α/β-hexokinase pathway for the efficient production of sexed dairy goat embryos)[J].畜牧与生物技术杂志（英文版）,2022(02):401-417

sexed,doe,sexing,ejaculate,fertilisation,superovulatory

TLR7,signalling,affects,motility,via,GSK3,hexokinase,pathway,efficient,production,dairy,embryos,Background,Goat,milk,very,similar,human,terms,its,abundant,nutrients,ease,digestion,To,derive,greater,economic,benefit,farmers,require,more,offspring,does,however,buck,approximately,At,present,artificial,insemination,after,separation,flow,cytometry,primary,means,controlling,livestock,However,not,been,successfully,utilised,aimed,goats,Results,In,this,study,novel,simple,technology,that,activates,toll,like,receptor,thereby,inhibiting,was,investigated,Our,results,showed,coding,chromosome,expressed,spermatids,testis,measured,from,cross,sections,epididymis,respectively,Importantly,located,tail,Upon,activation,phosphorylated,forms,glycogen,synthase,nuclear,kappa,were,detected,causing,reduced,mitochondrial,activity,ATP,levels,High,segregated,upper,layer,lower,Following,vitro,activated,XX,females,subsequently,used,vivo,response,nine,collected,uterus,two,conceived,Eight,these,one,XY,Conclusions,reveals,mechanism,technique,could,facilitate

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