Comprehensive Integrated Analyses of Proteins and Metabolites in Equine Seminal Plasma (Horses and Donkeys)

Abstract

Background: The reproductive ability of equine species is a critical component of equine breeding programs, with sperm quality serving as a primary determinant of reproductive success. In this study, we perform an integrative analysis of proteomics and metabolomics in seminal plasma to identify proteins and metabolites associated with sperm quality and reproductive ability in equine species.

Methods: We utilized the CEROS instrument to assess the morphology and motility of sperm samples from three horses and three donkeys. Additionally, we statistically analyzed the mating frequency and pregnancy rates in both species. Meanwhile, the 4D-DIA high-throughput proteomic and metabolomic profiling of seminal plasma samples from horses and donkeys revealed a complex landscape of proteins and metabolites.

Results: Our findings reveal a certain degree of correlation between seminal plasma proteins and metabolites and sperm quality, as well as overall fertility. Notably, we found that the proteins B3GAT3, XYLT2, CHST14, HS2ST1, GLCE, and HSPG2 in the glycosaminoglycan biosynthesis signaling pathway; the metabolites D-glucose, 4-phosphopantetheine, and 4-hydroxyphenylpyruvic acid in the tyrosine metabolism, starch, and source metabolisms; and pantothenate CoA biosynthesis metabolism present unique characteristics in the seminal plasma of equine species.

Conclusions: This comprehensive approach provides new insights into the molecular mechanisms underlying sperm quality and has identified potential proteins and metabolites that could be used to indicate reproduction ability. The findings from this study could be instrumental in developing novel strategies to enhance equine breeding practices and reproductive management. Future research will focus on exploring their potential for clinical application in the equine industry.

Keywords: donkey; horse; metabolomics; proteomics; seminal plasma; sperm.

Scheme 1

A schematic of the experimental design and workflow. The semen of horses and donkeys was collected and separated to obtain semen plasma, which was used for proteomic and metabolomic sequencing and joint analysis. The remaining semen was utilized to assess sperm and pregnancy statistics, and the results of the seminal plasma sequencing and the sperm quality experiments were subsequently correlated. AI: artificial insemination.

Figure 1

A proteomic analysis of horse and donkey seminal plasma. (a) A PCA plot showing the distinct clusters of proteins within the seminal plasma of horses and donkeys. The blue triangles represent samples of horses, and the red triangles represent samples of donkeys. (b) UpSet plot revealing the common and specific abundances of proteins in the seminal plasma of horses and donkeys. (c) A volcano plot demonstrating the differentially abundant proteins in the seminal plasma of horses and donkeys, where the donkey proteins and horse proteins are represented by blue and red dots, respectively. (d) A bubble chart showing the top 10 KEGG signaling pathways enriched with differentially abundant proteins in the seminal plasma of horses and donkeys. (e) A stacked bar chart showing the percentages of B3GAT3, XYLT2, CHST14, GLCE, and HS2ST1.

Figure 2

A metabolomics analysis of horse and donkey seminal plasma. (a) Counts of metabolites identified in positive and negative ion modes. (b) The proportions of identified metabolites within each chemical classification. (c) The OPLS–DA of metabolomic data for seminal plasma of donkeys and horses in positive ion mode. (d) A diagram of model verification in positive ion mode. (e) A volcano plot of differentially abundant metabolites between the seminal plasma of a donkey and a horse in positive ion mode. (f) A heatmap of metabolites between the seminal plasma of a donkey and a horse in positive ion mode. The blue dashed box represents the data in the positive mode. (g) The enriched KEGG pathways of differentially abundant metabolites between the seminal plasma of donkeys and horses. (h) MetPA analysis of key metabolites. (i) The abundances of D-glucose in the seminal plasma of horses and donkeys. (j) The abundances of 4-phosphopantetheine in the seminal plasma of horses and donkeys. (k) The abundances of 4-hydroxyphenylpyruvic acid in the seminal plasma of horses and donkeys. The data are presented as the means ± SDs. p < 0.05 was considered to indicate a statistically significant difference.

Figure 3

Integrated proteomics and metabolomics analysis. (a) A Venn diagram depicting the quantity of pathways analyzed through the joint analysis of proteomics and metabolomics. (b) A sunburst diagram revealing the intensity of correlation between differential proteins and metabolites. (c) A PPI network in donkey and horse seminal plasma. (d) The correlation between the sperm motility of horses and the key proteins in horse seminal plasma. (e) The correlation between the sperm motility of donkeys and the key proteins in donkey seminal plasma. (***) indicate a highly statistically significant difference (p < 0.001).

Figure 4

Integrative proteomics and metabolomics analyses of seminal plasma revealed the sperm quality and reproductive capacity of equines. Upon combined proteomic and metabolomic analysis, we discerned several seminal plasma proteins and metabolites intimately associated with sperm quality and fertility in equine species. The green arrow indicates that horse seminal plasma proteins are positively correlated with sperm motility; the red arrow indicates that horse seminal plasma proteins are negatively correlated with sperm motility; the purple arrow indicates that donkey seminal plasma proteins are negatively correlated with sperm motility.