Comparative Analysis of Proteomic Characteristics in Seminal Plasma Between Horses and Donkeys

Abstract

Horses and donkeys, as integral members of the equine family, exhibit distinct reproductive capabilities and characteristics. Seminal plasma, the fluid component of semen, contains a variety of proteins that play critical roles in sperm function and fertility. This study aimed to systematically compare the protein profiles in the seminal plasma of horses and donkeys, thereby elucidating the molecular differences between these two species. The study utilized 4D-DIA proteomics technology to analyze seminal plasma from horses and donkeys and further validated key proteins through Western blot. Our findings revealed significant variations in seminal plasma protein composition between horses and donkeys. We identified 2380 and 2385 proteins in the seminal plasma of horses and donkeys. Among these proteins, 59 are solely present in the seminal plasma of horses, and 64 uniquely exsit in that of donkeys, respectively. These insights enhance our understanding of the biological mechanisms underlying the reproductive distinctions between these equine species. Moreover, the identified species specific proteins may be essential for thier sperm quality and function, which holds practical value for breeding programs and investigations.

Keywords: 4D-DIA; donkey; equid; horse; pregnancy rate; proteomic profile; seminal plasma.

Figure 1

Schematic of the experimental design and workflow. Horse and donkey semen were collected, with a portion utilized for assessing sperm quality and artificial insemination, and the remainder was subjected to centrifugation to isolate seminal plasma for subsequent protein sequencing. The identification of key proteins was confirmed through Western blotting.

Figure 2

Evaluation of horse and donkey sperm quality and gestation. (a) CASA features of horse sperm. (b) CASA features of donkey sperm. (c) Pregnancy outcomes of artificial insemination in horses. Different colors correspond to different horses, with solid squares denoting horses that conceived after artificial insemination and hollow squares denoting horses that did not conceive. (d) Pregnancy outcomes of artificial insemination in donkeys. Different colors correspond to different donkeys, with solid squares denoting donkeys that conceived after artificial insemination, whereas hollow squares denote donkeys that did not conceive. (e) Pregnancy rates of horses and donkeys. The data are expressed as the means ± SDs. p < 0.05 indicates a statistically significant difference. “ns” represents not significant. H1: Horse1, H2: Horse2, H3: Horse3; D1: Donkey1, D2: Donkey2, D3: Donkey3.

Figure 3

Seminal plasma proteome of horses and donkeys. (a) PCA of seminal plasma proteomes between horses and donkeys. (b) Correlation circle diagram of seminal plasma proteomes between horses and donkeys. (c) UpSet plot of seminal plasma between horses and donkeys. (d) CDC of seminal plasma proteomes between horses and donkeys.

Figure 4

Presentation of seminal plasma CEPs in horses and donkeys. (a) The highly expressed TOP10 protein in the seminal plasma of horses and donkeys connects the same proteins on both sides in a straight line. Left: horse; right: donkey. (b) The relative abundance of 7 CEPs in the seminal plasma of horses and donkeys. (c) KLK1E2 and SPINK2 protein expression in the seminal plasma of horses and donkeys was determined via proteomic sequencing data. (d) PPIs of KLK1E2 and SPINK2 in Equus species. The data are expressed as the means ± SDs. “ns” represents not significant. H1: Horse1, H2: Horse2, H3: Horse3; D1: Donkey1, D2: Donkey2, D3: Donkey3.

Figure 5

Classification of seminal plasma SEPs in horses and donkeys. (a) A total of 59 SEPs in horse seminal plasma, with the different-sized circles indicating the expression levels of proteins. (b) The expression levels of the top 10 SEPs in horse seminal plasma. (c) GPX5 protein expression in horse and donkey seminal plasma was determined by Western blotting. (d) Quantification of the relative intensity of GPX5/TUBB. (e) A total of 64 SEPs in donkey seminal plasma, with the different-sized circles indicating the expression levels of proteins. (f) Expression levels of the top 10 SEPs in donkey seminal plasma. (g) SERPINE2 protein expression in horse and donkey seminal plasma was determined by Western blotting. (h) Quantification of the relative intensity of SERPINE2/TUBB. TUBB protein expression was employed as an internal reference for normalization. p < 0.05 indicates a statistically significant difference.

Figure 6

Comparison of seminal plasma DEPs in horses and donkeys. (a) Volcano plot of horse and donkey seminal plasma DEPs, with downregulated and upregulated proteins represented by green and purple dots, respectively. (b) Enrichment circle diagram displaying the primary classification of the top 10 KEGG signaling pathways in horse and donkey seminal plasma. (c) Bubble chart displaying the three-level classification of the top 10 KEGG signaling pathways in horse and donkey seminal plasma. (d) The expression of the top 10 DEPs in horse and donkey seminal plasma. (e) FOLR2 protein expression in horse and donkey seminal plasma was determined by Western blotting. (f) Quantification of the relative intensity of FOLR2/TUBB. (g) ACACA protein expression in horse and donkey seminal plasma was determined by Western blotting. (h) Quantification of the relative intensity of ACACA/TUBB. TUBB protein expression was employed as an internal reference for normalization. p < 0.05 indicates a statistically significant difference. H1: Horse1, H2: Horse2, H3: Horse3; D1: Donkey1, D2: Donkey2, D3: Donkey3.

Figure 7

Correlation between horse and donkey seminal plasma protein and sperm quality. (a) The PPI network of horse and donkey seminal plasma CEPs, SEPs, and DEPs. H-SEPs: horse seminal plasma SEPs. D-SEPs: donkey seminal plasma SEPs. (b) The correlation between horse seminal plasma protein and normal sperm morphology and motility. (c) The correlation between donkey seminal plasma protein and normal sperm morphology and motility. The scale (0.5 to −0.5) in color indicates whether the correlation is positive (purple) or negative (green). A single asterisk (*) indicates a statistical difference (p < 0.05); a double asterisk (**) indicates a significant statistical difference (p < 0.01).

Figure 8

Protein characteristics in horse and donkey seminal plasma. The orange text represents CEPs in horse and donkey seminal plasma, the black text represents DEPs in horse and donkey seminal plasma, the green text represents SEPs in horse seminal plasma, and the purple text represents SEPs in donkey seminal plasma. The red frame highlights the identification of protein markers in the seminal plasma of horses and donkeys, as revealed by this study.