Cryopreservation induces differential remodeling of the proteome in mammalian spermatozoa. How these proteome changes relate to the loss of sperm function during cryopreservation remains unsolved. The present study aimed to clarify this issue evaluating differential changes in the proteome of fresh and frozen-thawed pig spermatozoa retrieved from the cauda epididymis and the ejaculate of the same boars, with clear differences in cryotolerance. Spermatozoa were collected from 10 healthy, sexually mature, and fertile boars, and cryopreserved using a standard 0.5 mL-straw protocol. Total and progressive motility, viability, and mitochondria membrane potential were higher and membrane fluidity and reactive oxygen species generation lower in frozen-thawed (FT) epididymal than ejaculated spermatozoa. Quantitative proteomics of fresh and FT spermatozoa were analyzed using a LC-ESI-MS/MS-based Sequential Window Acquisition of All Theoretical Spectra approach. Cryopreservation quantitatively altered more proteins in ejaculated than cauda epididymal spermatozoa. Differential protein–protein networks highlighted a set of proteins quantitatively altered in ejaculated spermatozoa, directly involved in mitochondrial functionality which would explain why ejaculated spermatozoa deteriorate during cryopreservation.

Factors causing variability in ovarian follicle size among weaned sows are not well known. This field study aimed to disclose influencing factors and evaluate if the differences at weaning were established during lactation. Ovaries were scanned using transrectal ultrasound. The first experiment was conducted over a year with 191 randomly chosen sows that were hierarchically grouped (p < 0.001) according to ovarian follicle diameter reached at weaning: Small (0.20–0.30 cm; n = 37), medium (0.31–0.39 cm; n = 75), and large (0.40-1.00 cm; n = 69). Sows with small follicles showed a higher incidence of post-weaning anestrus (p < 0.01), longer wean-to-estrus/ovulation intervals (p < 0.01) and farrowing smaller litters (p < 0.05). Ovaries with small follicles were more common among sows weaned in summer–autumn than in winter–spring (p < 0.01) and among sows of lower parity (1–3) (p < 0.05). In the second experiment, with 40 sows randomly chosen at farrowing, the ovaries were scanned at 7, 14, and 21 d post-partum. Sows showed great variability in ovarian follicular size during lactation with a consistent relationship between the three measurement times (r = 0.84, p < 0.01). Follicle size was smaller in sows nursing in summer–autumn than in winter–spring (p < 0.05). In conclusion, early lactation dictates the great variability in ovarian follicular diameter at weaning shown by sows. Sows with smaller follicles at weaning had longer intervals for estrus and ovulation and smaller litters at farrowing and they were in greater numbers among sows weaned during the summer and fall and among those with fewer previous farrowing.

The objective of the present study was to evaluate the possible changes of oxytocin concentrations in saliva during and after ejaculate collection in breeding boars usually used in artificial insemination programs. Saliva samples of 33 boars were collected the day before ejaculate collection (DB), during the ejaculation time (T0) and two hours after ejaculate collection (T2). Free oxytocin and oxytocin linked to proteins concentrations were measured by two methods previously developed and validated for saliva of pigs. Younger boars, boars with higher libido intensity and boars of the Pietrain breed showed higher values of oxytocin in saliva during ejaculation than the day before. In addition, boars with higher libido showed higher concentrations two hours after ejaculate collection than during the day before. These changes were of higher magnitude and significance when oxytocin linked to proteins was measured. In conclusion, this study demonstrated for the first time that ejaculation influences the salivary oxytocin concentrations in breeding boars, although this influence varies according to age, libido and breed.

The seminal plasma (SP) modulates the female reproductive immune environment after mating, and microRNAs (miRNAs) could participate in the process. Considering that the boar ejaculate is built by fractions differing in SP-composition, this study evaluated whether exposure of mucosal explants of the sow internal genital tract (uterus, utero-tubal junction and isthmus) to different SP-fractions changed the profile of explant-secreted miRNAs. Mucosal explants retrieved from oestrus sows (n = 3) were in vitro exposed to: Medium 199 (M199, Control) or M199 supplemented (1:40 v/v) with SP from the sperm-rich fraction (SRF), the post-SRF or the entire recomposed ejaculate, for 16 h. After, the explants were cultured in M199 for 24 h to finally collect the media for miRNA analyses using GeneChip miRNA 4.0 Array (Affymetrix). Fifteen differentially expressed (False Discovery Rate (FDR) < 0.05 and Fold-change ≥ 2) miRNAs (11 down- versus 4 up-regulated) were identified (the most in the media of uterine explants incubated with SP from post-SRF). Bioinformatics analysis identified that predicted target genes of dysregulated miRNAs, mainly miR-34b, miR-205, miR-4776-3p and miR-574-5p, were involved in functions and pathways related to immune response. In conclusion, SP is able to elicit changes in the miRNAs profile secreted by female genital tract, ultimately depending SP-composition.

Genetic resource banks (GRB) preserve the genetic material of endangered, valuable individuals or genetically relevant breeds. Semen cryopreservation is a crucial technique to reach these goals. Thus, we aimed to assess the sperm parameters of semen doses from the native pig breed Gochu Asturcelta stored at the GRB of Principado de Asturias (GRB-PA, Gijón, Spain), focusing on intrinsic and extrinsic (boar, season) factors. Two straws per boar (n = 18, 8–71 months of age) were thawed, pooled, and assessed after 30 and 150 min at 37 °C by CASA (computer-assisted sperm analysis system; motility and kinematic parameters) and flow cytometry (viability, acrosomal status, mitochondrial activity, apoptosis, reactive oxygen species, and chromatin status). The effects of age, incubation, and season on post-thawing quality were determined using linear mixed-effects models. Parameters were on the range for commercial boar breeds, with chromatin status (SCSA: fragmentation and immaturity) being excellent. Incubation decreased sperm quality and functionality. The boar age did not have a significant effect (p > 0.05), but the between-boar variability was significant (p < 0.001). The season significantly affected many parameters (motility, kinematics, viability, acrosomal status, mitochondrial activity), especially after 150 min of incubation. In general, samples collected in spring and summer showed higher quality post-thawing, the lowest in winter. In conclusion, the sperm doses from the Gochu Asturcelta breed stored at the GRB-PA showed excellent chromatin status and acceptable characteristics after thawing. Therefore, boar and seasonal variability in this autochthonous breed could be relevant for cryobank management.

Glutathione S-transferases Mu 3 (GSTM3) is an essential antioxidant enzyme whose presence in sperm has recently been related to sperm cryotolerance, quality and fertility. However, its role in seminal plasma (SP) as a predictor of the same sperm parameters has never been investigated. Herein, cell biology and proteomic approaches were performed to explore the presence, origin and role of SP-GSTM3 as a sperm quality and in vivo fertility biomarker. GSTM3 in SP was quantified using a commercial Enzyme-Linked Immunosorbent Assay (ELISA) kit specific for Sus scrofa, whereas the presence of GSTM3 in testis, epididymis and accessory sex glands was assessed through immunoblotting analysis. Sperm quality and functionality parameters were evaluated in semen samples at 0 and 72 h of liquid-storage, whereas fertility parameters were recorded over a 12-months as farrowing rate and litter size. The presence and concentration of GSTM3 in SP was established for the first time in mammalian species, predominantly synthesized in the epididymis. The present study also evidenced a relationship between SP-GSTM3 and sperm morphology and suggested it is involved in epididymal maturation rather than in ejaculated sperm physiology. Finally, the data reported herein ruled out the role of this antioxidant enzyme as a quality and in vivo fertility biomarker of pig sperm.