Fractionated Seminal Plasma of Boar Ejaculates Analyzed by LC–MS/MS: Its Effects on Post-Thaw Semen Quality

Abstract

This study aimed to characterize the protein composition of fractionated seminal plasma (SP) by liquid chromatography mass spectrometry (LC–MS/MS) analysis and investigate its effects on survival of frozen-thaw (FT) boar spermatozoa following storage. Seminal plasma (SP) was fractionated by gel filtration chromatography to give two fractions, SP1 with more than 40 kDa (>40 kDa) and SP2 with less than 40 kDa (<40 kDa). SP1 and SP2 were subjected to LC–MS/MS and bioinformatics analysis. Following cryopreservation, FT boar semen (n = 7) was thawed in Beltsville Thawing Solution (BTS), BTS + SP1 or BTS + SP2, stored at different periods and subjected to post-thaw (PT) quality assessment. A total of 52 and 22 abundant proteins were detected in SP1 and SP2, respectively. FN1, ANGPTL1, and KIF15 proteins were more abundance in SP1, whereas a high abundance of spermadhesins (PSP-I and PSP-II) was detected in SP2. Proteins of the fractionated SP were involved in various biological processes, such as cell motility and signal transduction. The dominant pathway of SP1 proteins was the apelin signaling pathway (GNA13, MEF2D, SPHK2, and MEF2C), whereas a pathway related to lysosome (CTSH, CTSB, and NPC2) was mainly represented by SP2 proteins. In most of the boars, significantly higher motility characteristics, membrane integrity, and viability were observed in FT spermatozoa exposed to SP1 or SP2 compared with BTS. The results of our study confirm that a combination of several proteins from the fractionated SP exerted beneficial effects on the sperm membrane, resulting in improved quality characteristics following PT storage.

Should All Fractions of the Boar Ejaculate Be Prepared for Insemination Rather Than Using the Sperm Rich Only?

“…Although studied in other species, the impact of female responses to seminal plasma in the offspring’s health has not been evaluated in porcine yet. In our case, the type of seminal doses not only differed between them in protein and metabolite composition (as indicated by other studies; [ 13 , 14 , 40 ]), but also in the percentage of plasma. However, when the piglets born were evaluated for growth and blood analyses, no differences were found between the three experimental groups, indicating a possible lack of influence by the composition and/or percentage of seminal plasma present in the semen doses used.…”

“…The extender used in our study is recommended for medium-term conservation, which can aid in conserving the sperm quality when different seminal plasma proportions and fraction origin are used. Furthermore, the seminal plasma proteins not only vary between fractions of the ejaculates [ 13 , 14 ], but there are also inter-breed [ 32 ] and inter-male variations [ 33 ]. In our case, no male effect was observed.…”

“…However, currently, boar studs are including semi-automatic ejaculate collectors [ 10 , 11 , 12 ] instead of the traditional manual gloved-hand method, where the entire ejaculate is collected. It is known that boar seminal plasma composition varies depending on the fraction [ 2 , 13 , 14 ], which influences sperm conservation. However, other factors in addition to seminal plasma composition could impact semen conservation, such as seminal plasma proportion [ 8 , 15 ] or sperm concentration [ 16 , 17 ].…”

Presence and localization of apelin and its cognate receptor in canine testes using immunohistochemical and RT-PCR techniques

“…These observations suggest that apelin is involved in spermatogenesis of dog, as well as other adipokines reported in mammalian, via paracrine or endocrine manner mechanism (Thomas et al 2013 ; Kurowska et al 2018 ). Recently it was observed that the apelin signaling pathway was the most dominant in the seminal plasma of boar (Fraser et al 2021 ). This observation could allow us to hypothesize that, if its presence in seminal plasma is confirmed also in the dog, apelin produced by the testes could represent one of the components of the seminal plasma with potential roles in key reproductive events (Martinez et al 2020 ).…”

Effects of rumen-protected fat on changes of metabolites and reproductive genes in testes of Malin rams

“…The testes' tissues were crushed into a fine powder using a pestle and mortar with liquid nitrogen. The testes tissue samples were prepared as described in previous studies (Chen et al, 2015;Fraser et al, 2021), with slight modifications. Briefly, approximately 150 mg of each pulverized sample was homogenized in 450 μl methanol in two steps to make a final volume of 900 μl to precipitate the proteins.…”