Metabolomic analysis reveals spermatozoa and seminal plasma differences between Duroc and Liang guang Small-spotted pig

Abstract

The Liang guang Small-spotted pig is a well-known Chinese indigenous pig that is valued for its exceptional meat quality. However, the Liang guang Small-spotted pig has a lower semen storage capacity, shorter storage time and worse semen quality compared to Duroc. Pig sperm used for artificial insemination (AI) loses part of vitality and quality when being stored in commercial solutions. Serious vitality losses and short shelf life of the semen are particularly prominent in Liang guang Small-spotted pig. In this study, the metabolites in seminal plasma and spermatozoa of Duroc and Liang guang Small-spotted pigs were identified using UHPLC-Q-TOF/MS technology. The findings indicated forty distinct metabolites concentrating on energy metabolic substrates and antioxidant capacity in Liang guang Small-spotted pig and Duroc seminal plasma, including D-Fructose, succinate, 2-dehydro-3-deoxy-d-gluconate, alanine betaine, citrate, carnitine, acetylcarnitine and so on. Seventeen different metabolites were explored, with a focus on glycerophospholipid metabolism in Liang guang Small-spotted pig and Duroc spermatozoa, primarily including glycerol 3-phosphate, acetylcarnitine, phosphatidylcholine (PC) 16:0/16:0, palmitoyl sphingomyelin, acetylcholine, choline, glycerophosphocholine, betaine, L-carnitine, creatinine and others. This study reveals the metabolite profile of spermatozoa and seminal plasma among different pig breeds and might be valuable for understanding the mechanisms that lead to sperm storage capacity. Metabolites involved in energy metabolism, antioxidant capacity and glycerophospholipid metabolism might be key to the poor sperm storage capacity in Liang guang Small-spotted pig.

Keywords: Liang guang small-spotted pig; boar; metabolomic; seminal plasma; spermatozoa.

Figure 1

Assessment of semen quality parameters between Duroc and Liang guang Small-spotted pig. (A) Sperm total motility parameter was evaluated at days 1, 3, and 5 of semen preservation, respectively, (B) sperm progressive motility, (C) plasma membrane integrity, (D) acrosomal membrane integrity, (E) reactive oxygen species, and (F) mitochondrial membrane potential were assessed on day 1. Date are presented as the mean ± SEM, and different letters indicate significant differences; unmarked means the difference is not significant.

Figure 2

Presentation of the metabolomic differences.

Figure 3

OPLS-DA scores plots based on sperm and seminal plasma samples showing the correlations between the DSP, LSP, DS and LS group in both positive and negative modes (n = 5 per group). (A, B) Seminal plasma scores plots of (A) R2y = 99%, Q2 = 0.92 (positive). (B) R2y = 99%, Q2 = 0.76 (negative). (C, D) Sperm scores plots of (C) R2y = 99%, Q2 = 0.84 (positive). (D) R2y = 99%, Q2 = 0.85 (negative). (E–H) Corresponding statistical validation of OPLS-DA models by permutation analyses.

Figure 4

Metabolites identified in seminal plasma by OPLS-DA model VIP > 1. (A, B) Was detected in negative and positive ions mode respectively. (C) KEGG analysis of the differential metabolites in seminal plasma of Duroc and Liang guang Small-spotted pig. The bubble size indicates the influencing factor in the topological analysis; the bubble color represents the P-value of enrichment analysis.

Figure 5

Metabolites identified in sperm by OPLS-DA model VIP > 1. (A, B) Was detected in negative and positive ions mode, respectively. (C) KEGG analysis of the differential metabolites in sperm of Duroc and Liang guang Small-spotted pig. The bubble size indicates the influencing factor in the topological analysis; the bubble color represents the P-value of enrichment analysis.

Figure 6

KEGG network annotated using iPath 3. Blue and orange represent the pathway analysis of differential metabolites in seminal plasma and sperm, respectively.