Investigation On The Freezability Of Inner Mongolia Cashmere Goat Semen Through Multilayer Omics Joint Analysis-based Approaches

Inner Mongolia cashmere goat is important local germplasm resource in China.Extensive efforts into the promotion of frozen semen production of excellent breeding bucks can improve the utilization efficiency of breeding male animals,accelerate the breeding process,and bring higher economic benefits for production.However,the frozen-thawed semen of Inner Mongolia cashmere goat decreased the viability of sperm after thawing and estrus conception rate,and showed significant individual differences in freezability.To establish an efficient semen cryopreservation system for Inner Mongolia cashmere goat,this study screened semen of Inner Mongolia cashmere goats for freezability.Specifically,by using a unified freezing and thawing procedure,collected semen are divided into high freezability group(HF)and low freezability group(LF),and the effect of sperm and seminal plasma on the semen freezability,was evaluated.Proteome-metabonomics analysis on seminal plasma of Inner Mongolia cashmere goat in HF and LF was conducted to identify potentially critical factors affecting sperm viability after thawing.Additionally,lipidomics analysis was used to characterize the lipid differences between thawed semen in HF and LF after seminal plasma was removed.The key findings are as follows:(1)Inner Mongolia cashmere goat semen exhibited individual differences in freezability,after freezing and thawing,with HF showing significantly higher sperm motility parameters and structural integrity,compare to those in LF group.The difference in sperm itself and the source and composition of seminal plasma can impact semen freezability plays an essential role in the freezability,property of semen,and impacted by the source and composition of seminal plasma.(2)By using TMT-labeled quantitative proteomics analysis,115 proteins were significantly differentially expressed between the seminal plasma of the HF and LF,with 44upregulated proteins in HF group seminal plasma,and 71 upregulated proteins in LF.GO functions of above differential proteins are closely related to 22 biological processes,14cellular components,and 10 molecular functions.Through KEGG pathway analysis,the 115differential proteins involve 91 pathways.Differentially expressed proteins UQCRC1,DNAH1,APOA1,ADAM32,HTT,CADM1,STIP1 are potential biomarkers for freezability,of Inner Mongolia cashmere goat semen.(3)Non-targeted metabonomics analysis was utilized to detect the differences between the seminal plasma of HF and LF group from the metabolic level.Under the positive and negative ionization modes,the seminal plasma of the HF and LF group were well distinguished,showing significant metabolic differences.In the positive ion mode,23different metabolites between the HF group and the LF group were identified,and 18different metabolites between the two groups were identified in the negative ion mode.These distinctive metabolites involve 17 metabolic pathways.The top 15 different metabolites ranked by VIP score are(from highest to lowest)citrate,Pro-Arg,ketoisocaproic acid,acetylcarnitine,L-glutamine,hypoxanthine,L-pyroglutamic acid,20-hydroxyl-prostaglandin F2a,15-keto-prostaglandin E1,L-leucine,tyramine,pantothenic acid,oleic acid,L-phenylalanine,and dihydrothymine,which could be used as biomarker candidates for freezability,semen of Inner Mongolia cashmere goats.(4)Interestingly,the differentially expressed proteins indicated by proteomics analysis and the distinctive metabolites suggested through metabonomics analysis both projected to the KEGG pathway,with 11 pathways involved.The differential proteins and differential metabolites are remarkably enriched in the mineral absorption pathway,containing the differential proteins LOC102172488 and FTH1(Ferritin Heavy Chain 1),and the differential metabolites L-glutamine,L-phenylalanine,and L-leucine therein.The differential protein TF(Transferrin)has high sequence identity with LOC102172488.Both TF and FTH1 are concentrated in the mineral absorption pathways.Both TF and FTH1 were expressed in HF and LF group sperm,however,the expression of TF protein in the LF group was significantly higher than that in the HF group,and the FTH1 protein level in the HF sperm was significantly greater than that in the LF group.The detection of mineral ions in HF an LF group seminal plasma showed that the Fe²⁺,Zn²⁺,Ca²⁺,Mg²⁺,Cr³⁺in HF seminal plasma was significantly more concentrated than that of LF group,suggesting that the transport of oxidation and storage of Fe could affect the freezability of sperm of Inner Mongolia cashmere goat semen,and the concentration of Fe²⁺,Zn²⁺,Ca²⁺,Mg²⁺,Cr³⁺in seminal plasma are potentially related to the freezability,of semen.(5)Non-targeted lipidomics approach was utilized to analyze the lipid characteristics of sperm in HF and LF after removing seminal plasma freez-thawed.The sperm of the HF group and the LF group can be distinguished well under either positive or negative ionization mode,indicating the existence of significant differences in lipid characteristics between the two,with an identification of 1133 lipid molecules,belonging to 29 lipid subclasses.Among these identified lipid molecules,13 of them showed significant differences,of which 3belonged to phosphatidylcholine(PC)subclass and 10 belonged to triglyceride(TG)subclass.The analysis indicated that the PC and TG molecules on the sperm membrane may play important roles in the sperm freezability,of Inner Mongolia cashmere goats.This study investigated the regulation of the semen freezability of Inner Mongolian cashmere goats,and analyzed the critical factors in seminal plasma and sperm and effects thereof on freezability by using multi-omics joint approaches.The results of this study obtained a better understanding of the semen freezability of Inner Mongolia cashmere goats,and contributed to a theoretical foundation for improving the quality of semen after thawing and establishing a high-qualified cryopreservation system for Inner Mongolia cashmere goat semen.