Whole-Genome Resequencing of Ujimqin Sheep Identifies Genes Associated with Vertebral Number.

The number of vertebrae is a crucial economic trait that can significantly impact the carcass length and meat production in animals. However, our understanding of the quantitative trait loci (QTLs) and candidate genes associated with the vertebral number in sheep (Ovis aries) remains limited. To identify these candidate genes and QTLs, we collected 73 Ujimqin sheep with increased numbers of vertebrae (T13L7, T14L6, and T14L7) and 23 sheep with normal numbers of vertebrae (T13L6). Through high-throughput genome resequencing, we obtained a total of 24,130,801 effective single-nucleotide polymorphisms (SNPs). By conducting a selective-sweep analysis, we discovered that the most significantly selective region was located on chromosome 7. Within this region, we identified several genes, including VRTN, SYNDIG1L, LTBP2, and ABCD4, known to regulate the spinal development and morphology. Further, a genome-wide association study (GWAS) performed on sheep with increased and normal vertebral numbers confirmed that ABCD4 is a candidate gene for determining the number of vertebrae in sheep. Additionally, the most significant SNP on chromosome 7 was identified as a candidate QTL. Moreover, we detected two missense mutations in the ABCD4 gene; one of these mutations (Chr7: 89393414, C > T) at position 22 leads to the conversion of arginine (Arg) to glutamine (Gln), which is expected to negatively affect the protein's function. Notably, a transcriptome expression profile in mouse embryonic development revealed that ABCD4 is highly expressed during the critical period of vertebral formation (4.5-7.5 days). Our study highlights ABCD4 as a potential major gene influencing the number of vertebrae in Ujimqin sheep, with promising prospects for future genome-assisted breeding improvements in sheep.

Exploring the formation mechanism of short-tailed phenotypes in animals using mutant mice with the TBXT gene c.G334T developed by CRISPR/Cas9.

With the change in diet structure, individuals prefer to consume mutton with less fat. However, sheep tail has a lot of fat. We identified a breed of low-fat short-tailed sheep (i.e., Hulunbuir short-tailed sheep). It is necessary to develop an animal model that can promote research on the potential mechanisms of the short-tail phenotype in sheep, which results from the TBXT gene c.G334T mutation. To create animal models, we selected mice as experimental animals. Mouse embryos lacking the TBXT protein, which crucially regulates mouse embryonic development, cannot develop normally. We utilized CRISPR/Cas9 gene editing technology to generate site-specific mutation (c.G334T) in the TBXT gene of mice, and found that the mouse TBXT mutation (c.G334T) leads to a short-tail phenotype. Furthermore, we investigated the interaction between TBXT and Wnt signaling pathways. The expressions of TBXT, Axin2, Dkk1, Wnt3, Wnt3a, and Wnt5a were discovered to be significantly different between mutant embryos and wild embryos by obtaining mouse embryos at various developmental stages and examining the expression relationship between the TBXT and Wnt signaling pathway-related components in all of these embryos. Therefore, as a transcription factor, TBXT regulates the expression of the aforementioned Wnt signaling pathway components by forming a regulatory network for the normal development of mouse embryos. This study enriches the research on the functional role of the TBXT in the development of mouse embryos and the mechanism by which the short-tailed phenotype in sheep develops.

NELFA and BCL2 induce the 2C-like state in mouse embryonic stem cells in a chemically defined medium.

A minority of mouse embryonic stem cells (ESCs) display totipotent features resembling 2-cell stage embryos and are known as 2-cell-like (2C-like) cells. However, how ESCs transit into this 2C-like state remains largely unknown. Here, we report that the overexpression of negative elongation factor A (Nelfa), a maternally provided factor, enhances the conversion of ESCs into 2C-like cells in chemically defined conditions, while the deletion of endogenous Nelfa does not block this transition. We also demonstrate that Nelfa overexpression significantly enhances somatic cell reprogramming efficiency. Interestingly, we found that the co-overexpression of Nelfa and Bcl2 robustly activates the 2C-like state in ESCs and endows the cells with dual cell fate potential. We further demonstrate that Bcl2 overexpression upregulates endogenous Nelfa expression and can induce the 2C-like state in ESCs even in the absence of Nelfa. Our findings highlight the importance of BCL2 in the regulation of the 2C-like state and provide insights into the mechanism underlying the roles of Nelfa and Bcl2 in the establishment and regulation of the totipotent state in mouse ESCs.

Cyclic AMP mediates ovine cumulus-oocyte gap junctional function via balancing connexin 43 expression and phosphorylation.

Gap junction channels in cumulus-oocyte complexes (COCs) enable the transmission and communication of small molecular signals between adjacent cells, such as cAMP. However, the regulation of gap junction function (GJF) by cAMP and the underlying mechanisms involved are not fully clarified. This study investigated the effect of cAMP on connexin 43 (CX43) expression and GJF in ovine COCs using immunofluorescence, quantitative real-time PCR (qRT-PCR), western blotting, and GJF detection. The CX43 was only found in the cumulus cells (CCs) side of ovine COC. The intra-oocyte cAMP showed a significant increase at 30 min, while the intra-CC cAMP exhibited two peaks at 10 min and 1 h during in vitro maturation (IVM). Phosphorylated CX43 protein exhibited an immediate increase at 10 min, and CX43 protein displayed two peaks at 10 min and 1 h during IVM. The duration of pre-IVM exposure to forskolin and IBMX significantly enhanced phosphorylated and total CX43, as well as Gja1 and Creb genes, for 10 min; these effects were counteracted by Rp-cAMP. Both pre-IVM with forskolin and IBMX for 1 h and the GJF and CX43/p-CX43 ratio were elevated. The closure of gap junction channels caused by phosphorylated CX43 to prevent cAMP outflow from oocytes in early IVM of COC. Cyclic AMP upregulated phosphorylated and total CX43 via genomic and non-genomic pathways, but its functional regulation was dependent on the balance of the two proteins. This study provides a new insight into the regulatory mechanism between cAMP and GJF, which would improve IVM in animal and clinical research.

A Single-Cell Atlas of an Early Mongolian Sheep Embryo.

Cell types have been established during organogenesis based on early mouse embryos. However, our understanding of cell types and molecular mechanisms in the early embryo development of Mongolian sheep has been hampered. This study presents the first comprehensive single-cell transcriptomic characterization at E16 in Ujumqin sheep and Hulunbuir short-tailed sheep. Thirteen major cell types were identified at E16 in Ujumqin sheep, and eight major cell types were identified at E16 in Hulunbuir short-tailed sheep. Function enrichment analysis showed that several pathways were significantly enriched in the TGF-beta signaling pathway, the Hippo signaling pathway, the platelet activation pathway, the riboflavin metabolism pathway, the Wnt signaling pathway, regulation of the actin cytoskeleton, and the insulin signaling pathway in the notochord cluster. Glutathione metabolism, glyoxylate, and dicarboxylate metabolism, the citrate cycle, thyroid hormone synthesis, pyruvate metabolism, cysteine and methionine metabolism, thermogenesis, and the VEGF signaling pathway were significantly enriched in the spinal cord cluster. Steroid biosynthesis, riboflavin metabolism, the cell cycle, the Hippo signaling pathway, the Hedgehog signaling pathway, the FoxO signaling pathway, the JAK-STAT signaling pathway, and the Wnt signaling pathway were significantly enriched in the paraxial mesoderm cluster. The notochord cluster, spinal cord cluster, and paraxial mesoderm cluster were found to be highly associated with tail development. Pseudo-time analysis demonstrated that the mesenchyme can translate to the notochord in Ujumqin sheep. Molecular assays revealed that the Hippo signaling pathway was enriched in Ujumqin sheep. This comprehensive single-cell map revealed previously unrecognized signaling pathways that will further our understanding of the mechanism of short-tailed sheep formation.

Study on the changes of LHR, FSHR and AR with the development of testis cells in Hu sheep.

The process of testis development in mammals is accompanied by the proliferation and maturation of Sertoli, Leydig and germ cells. Spermatogenesis depends on hormone regulation, which must bind to a receptor to exert its biological effects. The changes in Hu sheep testis cell composition and FSHR, LHR and AR expression during different developmental stages are unclear (newborn, puberty and adulthood). To address this, using single-cell RNA sequencing, we analyzed testis cell composition and hormone receptor expression changes during three important developmental stages of Hu sheep. We observed significant changes in the composition of somatic and germ cells in different Hu sheep testis developmental stages. Furthermore, we analyzed the FSHR, LHR and AR distribution and expression changes at three important periods and verified them by qRT-PCR and immunofluorescence. Our results suggest that after birth, the proportion of germ cells increased gradually, peaking in adulthood; the proportion of Sertoli cells decreased gradually, reaching the lowest in adulthood; and the proportion of Leydig cells increased and then decreased, reaching the lowest in adulthood. In addition, FSHR, LHR and AR are mainly located in Sertoli, Leydig and germ cells. LHR and FSHR expression decreased with increasing age, while AR expression increased and then decreased with increasing age.

Equine ß-defensin 1 regulates cytokine expression and phagocytosis in S. aureus-infected mouse monocyte macrophages via the Paxillin-FAK-PI3K pathway.

ß-defensin-1 (BD-1) is a rich source of disulfide bonds and antibacterial peptides that exhibit direct bactericidal function. The expression of BD-1 is primarily induced by external stimulation and is known to correlate with TLR-mediated inflammation, suggesting its association with innate immune responses. Equine ß-defensin-1 (eBD-1) belongs to the BD-1 family. Our previous study demonstrated that eBD-1 enhances cytokine expression and promotes macrophage phagocytosis of S. aureus, although the underlying mechanism remains unknown. In this study, we utilized a PI-3K inhibitor (PKI-402) to treat eBD-1 -treated S. aureus-infected macrophages in vitro. Our results revealed that PKI-402 decreased the expression of eBD-1-promoted TNF-α, IL-6, CXCL10, CD40, RANTES, and p65 mRNA. To further investigate the relationship between eBD-1 and phagocytosis, we examined the expression of paxillin and FcγRIII (CD16 receptor) using western blot and immunofluorescence techniques. Our findings demonstrated that eBD-1 enhanced CD16 and paxillin expression in S. aureus -infected macrophages. Considering the correlation between paxillin expression and focal adhesion kinase (FAK), we transfected FAK siRNA into macrophages and evaluated paxillin expression using western blot analysis. Additionally, we quantified the number of S. aureus phagocytosed by macrophages. The results indicated a reduction in both paxillin expression and the number of S. aureus phagocytosed by macrophages upon FAK siRNA treatment. Our study showed the eBD-1 promotes cytokine mRNA expression in S. aureus-infected macrophages regulated by PI-3K-NF-κB pathway, and it increases macrophage phagocytosis of S. aureus associated with the FAK-paxillin signaling pathway.

Study of spermatogenic and Sertoli cells in the Hu sheep testes at different developmental stages.

Spermatogenesis is a highly organized process by which undifferentiated spermatogonia self-renew and differentiate into spermatocytes and spermatids. The entire developmental process from spermatogonia to sperm occurs within the seminiferous tubules. Spermatogenesis is supported by the close interaction of germ cells with Sertoli cells. In this study, testicular tissues were collected from Hu sheep at 8 timepoints after birth: 0, 30, 90, 180, 270, 360, 540, and 720 days. Immunofluorescence staining and histological analysis were used to explore the development of male germ cells and Sertoli cells in the Hu sheep testes at these timepoints. The changes in seminiferous tubule diameter and male germ cells in the Hu sheep testes at these different developmental stages were analyzed. Then, specific molecular markers were used to study the proliferation and differentiation of spermatogonia, the timepoint of spermatocyte appearance, and the maturation and proliferation of Sertoli cells in the seminiferous tubules. Finally, the formation of the blood-testes barrier was studied using antibodies against the main components of the blood-testes barrier, ß-catenin, and ZO-1. These findings not only increased the understanding of the development of the Hu sheep testes, but also laid a solid theoretical foundation for Hu sheep breeding.

Reprogramming efficiency and pluripotency of mule iPSCs over its parents†.

The mule is the interspecific hybrid of horse and donkey and has hybrid vigor in muscular endurance, disease resistance, and longevity over its parents. Here, we examined adult fibroblasts of mule (MAFs) compared with the cells from their parents (donkey adult fibroblasts and horse adult fibroblasts) (each species has repeated three independent individuals) in proliferation, apoptosis, and glycolysis and found significant differences. We subsequently derived mule, donkey, and horse doxycycline (Dox)-independent induced pluripotent stem cells (miPSCs, diPSCs, and hiPSCs) from three independent individuals of each species and found that the reprogramming efficiency of MAFs was significantly higher than that of cells of donkey and horse. miPSCs, diPSCs, and hiPSCs all expressed the high levels of crucial endogenous pluripotency genes such as POU class 5 homeobox 1 (POU5F1, OCT4), SRY-box 2 (SOX2), and Nanog homeobox (NANOG) and propagated robustly in single-cell passaging. miPSCs exhibited faster proliferation and higher pluripotency and differentiation than diPSCs and hiPSCs, which were reflected in co-cultures and separate-cultures, teratoma formation, and chimera contribution. The establishment of miPSCs provides a unique research material for the investigation of "heterosis" and perhaps is more significant to study hybrid gamete formation.

Effects of the CDC10 (Septin 7) Gene on the Proliferation and Differentiation of Bovine Intramuscular Preadipocyte and 3T3-L1 Cells.

Intramuscular fat content and marbling affecting meat quality are important economic traits in beef cattle. CDC10 (cell division cycle 10 or Septin 7), a member of the septin family involved in cellular proliferation, was considered as a functional and positional candidate gene for beef marbling. In a previous study, we revealed that the expression levels of CDC10 were also positively correlated with marbling scores in Japanese Black cattle. However, the regulatory mechanism of the CDC10 gene on IMF deposition in cattle remains unclear. In the present study, flow cytometry, EdU proliferation assays, and Oil Red O staining results showed that overexpression of CDC10 could promote the differentiation of bovine intramuscular preadipocyte (BIMP) and 3T3-L1 cells, whereas knockdown of CDC10 resulted in the opposite consequences. Furthermore, quantitative PCR and Western blotting results showed that overexpression of CDC10 could promote the expression levels of adipogenic marker genes PPARγ and C/EBPα at both mRNA and protein levels in BIMP and 3T3-L1 cells, whereas knockdown of CDC10 resulted in the opposite consequences. Our results provide new insights into the regulatory roles of CDC10 in adipocytes in animals.

In vitro oxidation and digestibility profiles of iron-loaded whey protein isolate/gum Arabic complexes with different morphologies.

This study was designed to investigate the promotion of oxidation of lipids in oil-in-water (o/w) emulsions and digestive properties of the bionic dynamic gastrointestinal system of whey protein isolate (WPI) and gum arabic (GA) complexes loaded with iron ions, which were fabricated previously and shown as WPI/GAFe3+ nanoparticles (WGS) and WPI/GAFe3+ fibers (WGF). Compared with emulsions containing Fe3+ and GA-loaded complex (GAFe3+), WGS and WGF greatly improved the oxidative stability of lipids along with the reduced lipid oxidation products and volatile compounds, attributed to the encapsulation of iron ions. During the bionic dynamic gastrointestinal digestion, the iron ion release of WGF was significantly higher than that of WGS, probably due to different assembled internal structures. Accordingly, two proposed WPI/GAFe3+ complexes with different morphologies are expected to be developed as novel stable iron fortifiers with delayed lipid oxidation and controlled iron-ion release in food emulsions.

Effects of novel variants in BMP15 gene on litter size in Mongolia and Ujimqin sheep breeds.

Reproductive traits, such as ovulation rate and litter size, are important factors influencing the sheep industry. The bone morphogenetic protein 15 (BMP15) is a major gene affecting the reproductive traits in sheep, and multiple mutations in BMP15 gene could affect the ovulation rate and litter size in many sheep breeds, showing high breed specificity. However, identification of novel variations and seeking breed-specific markers associated with litter size in other sheep breeds are still important. In this study, we sequenced the BMP15 gene of Mongolia sheep, and 12 novel variants were detected by direct sequencing and whole-genome resequencing. Among them, the g.50985975 G > A polymorphism in intron and synonymous c.755 T > C (Leu252Pro) in exon 2 of BMP15 were significantly associated with the litter sizes of Mongolia ewes (P < 0.01 and P < 0.01, respectively), as well as the g.50988478C > A and g.50987863G > A in the promoter region of BMP15 were significantly associated with the litter sizes of Ujimqin ewes (P < 0.05 and P < 0.01, respectively). The c.755 T > C mutation is predicted to change the tertiary structure of BMP15. Our findings may provide potentially useful genetic markers for increasing litter size in sheep.

Equus ß-Defensin-1 Regulates Innate IMMUNE Response in S. aureus-Infected Mouse Monocyte Macrophage.

Beta-defensin-1 (BD-1) is among the class of antibacterial peptides that are rich in disulfide bonds, have direct antibacterial activity and showed enhanced expression following external stimulation. However, existing research studies only treated BD-1 to cell models without stimulation from pathogen-associated molecular patterns (PAMPs), which will further influence our understanding of the role of BD-1. In this study, we map the tissue distribution of Equus BD-1 (i.e., eBD-1, ass BD-1, and mule BD-1) and compare their expression levels in various tissues. We further characterize the three kinds of Equus BD-1 by analyzing their full-length cDNA. We showed that eBD-1, ass BD-1, and mule BD-1 have an identical (100%) open reading frame (ORF). The ORF encoding OEBD-1 expressed the ORF in the E. coli Top10 expression system. This expression system was combined with an S. aureus-infected J774A.1 macrophage cell line to determine the influence on innate immune mediator expression. Using this expression model system, it was determined that the OEBD-1 protein enhanced IL-6 and TNF-α secretion. It can also promote TLR2, IL-1ß, CCL2, CCL7, CXCL10 and NF-κB p65 mRNA expression. Moreover, OEBD-1 upregulates phosphorylation of ATK, Syk and IκB-α. In addition, OEBD-1 enhances the macrophage's ability to phagocytose S. aureus. In conclusion, Equus BD-1 was shown to play an essential role in macrophage-involved innate immune responses in an in vitro system.

Dosage Compensation of the X Chromosome during Sheep Testis Development Revealed by Single-Cell RNA Sequencing.

Dosage compensation is a mechanism first proposed by Susumu Ohno, whereby X inactivation balances X gene output between males (XY) and females (XX), while X upregulation balances X genes with autosomal gene output. These mechanisms have been actively studied in Drosophila and mice, but research regarding them lags behind in domestic species. It is unclear how the X chromosome is regulated in the sheep male germline. To address this, using single-cell RNA sequencing, we analyzed testes in three important developmental stages of sheep. We observed that the total RNA per cell from X and autosomes peaked in SSCs and spermatogonia and was then reduced in early spermatocytes. Furthermore, we counted the detected reads per gene in each cell type for X and autosomes. In cells experiencing dose compensation, close proximity to MSL (male-specific lethal), which is regulated the active X chromosome and was observed. Our results suggest that there is no dose compensation in the pre-meiotic germ cells of sheep testes and, in addition, MSL1 and MSL2 are expressed in early germ cells and involved in regulating mammalian X-chromosome inactivation and activation.

Reprogramming barriers in bovine cells nuclear transfer revealed by single-cell RNA-seq analysis.

Many progresses have recently been achieved in animal somatic cell nuclear transfer (SCNT). However, embryos derived from SCNT rarely result in live births. Single-cell RNA sequencing (scRNA-seq) can be used to investigate the development details of SCNT embryos. Here, bovine fibroblasts and three factors bovine iPSCs (3F biPSCs) were used as donors for bovine nuclear transfer, and the single blastomere transcriptome was analysed by scRNA-seq. Compared to in vitro fertilization (IVF) embryos, SCNT embryos exhibited many defects. Abnormally expressed genes were found at each stage of embryos, which enriched in metabolism, and epigenetic modification. The DEGs of the adjacent stage in SCNT embryos did not follow the temporal expression pattern similar to that of IVF embryos. Particularly, SCNT 8-cell stage embryos showed failures in some gene activation, including ZSCAN4, and defects in protein association networks which cored as POLR2K, GRO1, and ANKRD1. Some important signalling pathways also showed incomplete activation at SCNT zygote to morula stage. Interestingly, 3F biPSCNT embryos exhibited more dysregulated genes than SCNT embryos at zygote and 2-cell stage, including genes in KDM family. Pseudotime analysis of 3F biPSCNT embryos showed the different developmental fate from SCNT and IVF embryos. These findings suggested partial reprogrammed 3F biPS cells as donors for bovine nuclear transfer hindered the reprogramming of nuclear transfer embryos. Our studies revealed the abnormal gene expression and pathway activation of SCNT embryos, which could increase our understanding of the development of SCNT embryos and give hints to improve the efficiency of nuclear transfer.

In vitro digestibility and fermentability profiles of wheat starch modified by chlorogenic acid.

This study was designed to investigate the effect of chlorogenic acid (CA) on starch digestibility and fermentability in vitro. Compared with wheat starch (WS), WS-CA complexes exhibited a looser porous gel matrix, and higher solubility and swelling power with the addition of different proportion of CA. The WS-CA complexes significantly reduced the digestive rate of the gelatinized WS, and increased the proportion of resistant starch (RS) ranging from 31.70 % to 69.63 % much higher than that in the gelatinized WS (26.34 %). The residual WS-CA complexes after 24 h of fermentation with human feces induced the production of short-chain fatty acid, as well as the proliferation of gut microbiota such as genera Megamonas and Parabacteroides positively associated with the improvement of human health. The results suggest that complex of starch and CA could be a promising method for developing starchy foods with lower starch hydrolysis and promoting the growth of probiotics.

The Effect of Ghrelin on the Maturation of Sheep Oocytes and Early Embryonic Development In Vitro.

In vitro maturation (IVM) of sheep oocytes and early embryonic development are of great scientific importance for the study of reproductive development in sheep. Ghrelin is an important hormone that regulates the secretion of the growth hormone (GH). In this study, different gradients of ghrelin (0, 100, 200, and 300 ng/mL) were added to the IVM system of sheep oocytes to observe their cell morphology, and Hosesth 33342 staining was used to determine the time taken for oocytes to reach different developmental stages. We found 200 ng/mL ghrelin to be the optimal concentration. The RNA-seq analysis showed that many signaling pathways were significantly altered by ghrelin. Cell cycle, Wnt, and oxidative phosphorylation were activated; the P53 was inhibited. These pathways together regulate the maturation of oocytes and early embryonic development in vitro. The effects of the addition of ghrelin were verified by the expression of GLUT1 in early embryonic development. The results suggest that adding ghrelin shortens the duration of the IVM of sheep oocytes and hinders early embryonic development. This study provides new insights into the effects of exogenous ghrelin on sheep oocyte maturation and early embryonic development in vitro.

Advances in Bioactivity of MicroRNAs of Plant-Derived Exosome-Like Nanoparticles and Milk-Derived Extracellular Vesicles.

MicroRNA (miRNA) is a class of small noncoding RNA involved in physiological and pathological processes via the regulation of gene expression. Naked miRNAs are unstable and liable to degradation by RNases. Exosome-like nanoparticles (ELNs) secreted by plants and extracellular vesicles (EVs) found in milk are abundant in miRNAs, which can be carried by ELNs and EVs to target cells to exert their bioactivities. In this review, we describe the current understanding of miRNAs in plant ELNs and milk EVs, summarize their important roles in regulation of inflammation, intestinal barrier, tumors, and infantile immunological functions, and also discuss the adverse effect of EV miRNAs on human health. Additionally, we prospect recent challenges centered around ELN and EV miRNAs for interventional applications and provide insights of grain-derived ELNs and miRNAs interventional use in human health. Overall, plant ELNs and milk EVs can transfer miRNAs to mitigate the pathological status of recipient cells by mediating the expression of target genes but may also exert some side effects. More studies are required to elucidate the in-depth understanding of potential interventional effects of ELN and EV miRNAs on human health.

Development and Application of a High-Resolution Melting Analysis with Unlabeled Probes for the Screening of Short-Tailed Sheep TBXT Heterozygotes.

The short-tailed phenotype has long been considered one of the best traits for population genetic improvement in sheep breeding. In short-tailed sheep, not only is tail fat eliminated but also the pubic area is exposed due to the lack of a tail covering, giving them an advantage in reproduction. Recent studies have shown that two linked mutations in sheep TBXT at nucleotides 333 and 334 are associated with the short-tailed phenotype. In the population of short-tailed sheep, several heterozygous mutants of this gene are found. In our research, we used high-resolution melting (HRM) to identify homozygous and heterozygous genotypes in a flock of short-tailed sheep and compared the results with those of Sanger sequencing, which were identical. This demonstrates that our established HRM method, a rapid and inexpensive genotyping method, can be used to identify homozygous and heterozygous individuals in short-tailed sheep flocks.

Effect of lactoferrin on ram sperm motility after cryopreservation.

OBJECTIVE: The objective of this study was to analyse the differentially abundant proteins caused by freeze-thawing of ram sperm and explore candidate proteins of interest for their ability to improve ram sperm cryopreservation outcomes in vitro. METHODS: Sperm were from three mature Dorper. Fresh and frozen sperm proteins were extracted, and the differentially abundant proteins were analysed by mass spectrometry. Among these proteins, lactoferrin (LTF) was selected to be added before cryopreservation. Next, sperm samples were diluted in Tris extender, with the addition of 0, 10, 100, 500, and 1,000 µg/mL of LTF. After thawing, sperm quality was evaluated by motility, plasma membrane integrity, mitochondrial activity and reactive oxygen species (ROS). RESULTS: Cryopreservation significantly altered the abundance of 40 proteins; the abundance of 16 proteins was increased, while that of 24 proteins was decreased. Next, LTF was added to Tris extender applied to ram sperm. The results showed that sperm motility and plasma membrane integrity were significantly improved (p<0.05) by supplementation with 10 µg/mL LTF compared to those in the control group. There was no significant difference in mitochondrial activity between the 0 µg/mL group and other groups (p>0.05). Supplementation of the cryoprotective extender with 10 µg/mL LTF led to decreased ROS levels compared with those in the control and other groups (p<0.05). CONCLUSION: The LTF is an important protein during cryopreservation, and the addition of 10 µg/mL LTF to a cryoprotective extender can significantly improve the function of frozen ram sperm.