Study On The Effect Of L-citrulline Supplementation On Rams Semen Quality Based On Metabolome And Transcriptome

This study explored the effect of in-feed L-citrulline(L-Cit)supplementation toward rams reproduction performance and its underlying mechanisms,which provided theoretical support of further application in herd breeding.32 Turpan black rams were randomly divided into three test groups and one control group with 8 rams in each.The control group were fed with basic diet,and the experimental groups I,II and III were supplemented with 4 g/d/ram,8 g/d/ram and 12 g/d/ram of L-Cit respectively.The experiment lasted for 72 days.After the beginning of the experiment,semen was collected every 5days,and semen quality was measured every 10 days to determine the optimum dose of L-cit supplementation.Follow-up study was carried out in between control group and the group receiving optimum dose of L-Cit supplementary feeding(12g/d/ram).On day 71 post follow-up study,semen was collected to measure the integrity of sperm plasma membrane,acrosome integrity and mitochondrial membrane potential activity.On day 0 and day 71 post follow-up study,rams semen was collected by pseudo vaginal method before morning feeding,and the contents of sugars,amino acids and pyruvate in seminal plasma were measured by high flux targeting.On day 0 and day 72 post follow-up study,fasted blood samples were collected from jugular vein with plain tube for high-throughput non targeted metabolism study.On day 73 post follow-up study,three rams were choosen from control group and the experimental group and slaughtered respectively;the hypothalamus tissue and testicular reticulum tissue of the same part of the right testis were collected for transcriptome detection.After supplemented with L-Cit,the sperm density,number of rectilinear motile sperm,sperm motility and mitochondrial membrane potential of rams increased significantly(P < 0.01).Compared with the control group,the contents of xylose and pyruvate in seminal plasma of rams in the experimental group increased significantly(P < 0.01),and arabinose and maltose decreased significantly(P < 0.01);L-valine,L-threonine,L-arginine,L-tryptophan,L-isoleucine,L-lysine,L-methionine,L-aspartic acid and Nα-Acetyl-L-lysine in seminal plasma,sarcosine and L-homoserine increased significantly(P < 0.01).After non target detection of ram serum,there were 112 foreign bodies with poor metabolism in estrous period compared with anoestrous period.After KEGG analysis,the pyrimidine metabolic pathway increased significantly(P < 0.01);After feeding L-Cit for 72 days,there were 124 foreign bodies with poor metabolism.After KEGG analysis,arginine biosynthesis and mineral absorption pathway increased significantly(P < 0.01);After feeding L-Cit for 72 days,there were 27 foreign bodies with poor metabolism in the control group and the experimental group.After KEGG analysis,ABC transport,protein digestion and absorption,mineral absorption and aminoacyl t RNA biosynthesis pathway increased significantly(P < 0.01).The average of raw reads of ram hypothalamus sequencing was49,841,836.67,including 49,137,344.67 in the control group and 50,546,328.67 in the experimental group.A total of 961 expressed genes were detected by gene analysis,including 524 up-regulated genes and 437 down-regulated genes;After KEGG analysis,the protein digestion and absorption pathway increased significantly(P < 0.01);Protein network interaction analysis showed that there were 107 more than 5 associated genes in the hypothalamus,including 68 up-regulated genes and 39 down-regulated genes.The average number of raw reads of the original data obtained from testicular tissue sequencing was 50,318,616,including 46,891,972 in the control group and 53,745,260 in the experimental group.A total of 715 expressed genes were detected,including 260 up-regulated genes and 455 down-regulated genes;After KEGG analysis,glycolysis / gluconeogenesis and amino sugar and nucleotide sugar metabolic pathways increased significantly(P < 0.01);Protein network interaction analysis showed that there were 18 associated genes,13 up-regulated genes and 5 down-regulated genes.In conclusion,in-feed supplementation of L-Cit can improve the semen quality of rams.L-Cit can promote the metabolism of amino acids,protein,sugar and minerals in rams,and then regulate the reproductive performance of rams.RPL8,RPS20,RPL12,RPL21,RPS3 A,RPS5,RPS15 A in hypothalamus and LOC101115026 in testis may be important candidate genes regulating reproductive traits of rams.