Characterization of freezability-associated metabolites in boar semen

Sperm cryopreservation is widely used to protect genetic resources and improve utilization efficiency of semen in husbandry and biomedicine fields [1,2]. However, the conception and litter size of sow inseminated with boar frozen semen cannot generally reach the comparable levels to that of conventional cooled semen [3,4]. It is reported that the quality of frozen sperm is frequently affected by the origin of semen from different animal individuals [5,6]. Accumulating evidence indicates that sperm freezability, namely competence to withstand freeze-thawing process, highly differs between boars or across ejaculates within boars [7]. The pre-selection of good freezability semen before cryopreservation could promote the practical application of boar frozen sperm. Thus, it is necessary to screen reliable biomarkers of poor and good freezability in boar semen.

Traditional sperm functional parameters, such as acrosome and membrane integrity, mitochondrial membrane potential, DNA damage, and ATP levels, were not able to accurately distinguish poor and good freezability of boar semen [8,9]. An in-depth understanding of molecular markers associated with sperm freezability is a prerequisite to develop reliable strategies for predicting boar semen cryotolerance. Recent progresses in OMICS technologies and bioinformatics approaches allow us to identify freezability markers in boar semen. It was reported that boar sperm and seminal plasma comprise freezability-associated molecular markers at different OMICS levels [7,10,11]. Genetic analysis revealed that sixteen genomic markers for boar semen freezability were identified using amplified restriction fragment length polymorphism technology [12]. Polymorphisms in SCLT1, MAP3K20, MS4A2, ROBO1, STK35, and IFT27 genes expressed in boar spermatozoa were positively associated with post-thaw semen quality and could be considered as freezability markers [13,14]. It is well known that spermatozoa are transcriptionally quiescent germ cells. Transcirptome analysis showed that inflammation and apoptosis-related genes were overexpressed in poor freezability boar sperm and these transcripts could be thus potential markers for predicting the poor freezability of boar semen [15]. Proteomic profiling demonstrated that acrosin [16], fibronectin (FN1) [17], heat shock protein (HSP90AA1) [18], voltage-dependent anion channel 2 (VDAC2) [19], N-acetyl-b-hexosaminidase (b-HEX) [20], triosephosphate isomerase (TPI) [21], and niemann-pick C2 protein (NPC2) [22] were identified as freezability markers for boar sperm. Epitranscriptome analysis indicated that cryopreservation altered N6-methyladenosine (m6A) RNA methylation levels in boar sperm, suggesting the possibility that m6A modification is a freezability marker [23]. Of note, subtle changes at genomic, transcriptiomic, proteomic, and epitransciptomic levels could be eventually manifested at the metabolomics.

Previous studies indicated that metabolites supplementation in extender or diet improved the quality of liquid-stored semen [24,25]. Recent evidence showed that metabolic products, such as carbohydrates [26,27], lipids [28], amino acids [29], nucleotides [30], minerals [31,32], and vitamins [33,34], are involved in regulating the quality of frozen-thawed sperm. It was reported that seminal plasma from commercial boars displays differential metabolome between poor and good freezability semen and d-aspartic acid, N-acetyl-l-glutamate (NAG), and inosine could be potential markers for sperm freezability [35]. A recent study showed that a number of metabolites in sperm cells during holding time were identified as markers for predicting semen freezability [36]. However, integrated metabolomic analyses of boar sperm and seminal plasma between poor and good freezability from native pig breed semen remain to be known.

In the present study, metabolomic profile of boar sperm and seminal plasma from Chinese native pigs was performed to screen potential metabolic markers of sperm freezability. These findings will promote extension and application of frozen-thawed semen in swine industry.

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