Reproductive physiology of the boar: What defines the potential fertility of an ejaculate?

Despite decades of research and handling of semen for use in artificial insemination (AI) and other assisted reproductive technologies, 5-10% of selected boar sires are still considered sub-fertile, escaping current assessment methods for sperm quality and resilience to preservation. As end-product, the ejaculate (emitted spermatozoa sequentially exposed to the composite seminal plasma, the SP) ought to define the homeostasis of the testes, the epididymis, and the accessory sexual glands. Yet, linking findings in the ejaculate to sperm production biology and fertility is suboptimal. The present essay critically reviews how the ejaculate of a fertile boar can help us to diagnose both reproductive health and resilience to semen handling, focusing on methods -available and under development- to identify suitable biomarkers for cryotolerance and fertility. Bulk SP, semen proteins and microRNAs (miRNAs) have, albeit linked to sperm function and fertility after AI, failed to enhance reproductive outcomes at commercial level, perhaps for just being components of a complex functional pathway. Hence, focus is now on the interaction sperm-SP, comparing in vivo with ex vivo, and regarding nano-sized lipid bilayer seminal extracellular vesicles (sEVs) as priority. sEVs transport fragile molecules (lipids, proteins, nucleic acids) which, shielded from degradation, mediate cell-to-cell communication with spermatozoa and the female internal genital tract. Such interaction modulates essential reproductive processes, from sperm homeostasis to immunological female tolerance. sEVs can be harvested, characterized, stored, and manipulated, e.g. can be used for andrological diagnosis, selection of breeders, and alternatively be used as additives to improve cryosurvival and fertility.

Seminal extracellular vesicles alter porcine in vitro fertilization outcome by modulating sperm metabolism.

Porcine seminal plasma (SP) is loaded with a heterogeneous population of extracellular vesicles (sEVs) that modulate several reproductive-related processes. This study investigated the effect of two sEV subsets, small (S-sEVs) and large (L-sEVs), on porcine in vitro fertilization (IVF). The sEVs were isolated from nine SP pools (five ejaculates/pool) using a size-exclusion chromatography-based procedure and characterized for quantity (total protein), morphology (cryogenic electron microscopy), size distribution (dynamic light scattering), purity and EV-protein markers (flow cytometry; albumin, CD81, HSP90ß). The characterization confirmed the existence of two subsets of high purity (low albumin content) sEVs that differed in size (S- and L-sEVs). In vitro fertilization was performed with in vitro matured oocytes and frozen-thawed spermatozoa and the IVF medium was supplemented during gamete coincubation (1 h at 38.5 °C, 5 % CO2 in a humidified atmosphere) with three different concentrations of each sEV subset: 0 (control, without sEVs), 0.1, and 0.2 mg/mL. The first experiment showed that sEVs, regardless of subset and concentration, decreased penetration rates and total IVF efficiency (P < 0.0001). In a subsequent experiment, it was shown that sEVs, regardless of subset and concentration, impaired the ability of spermatozoa to bind to the zona pellucida of oocytes (P < 0.0001). The following experiment showed that sEVs, regardless of the subset, bound to frozen-thawed sperm but not to in vitro matured oocytes, indicating that sEVs would affect sperm functionality but not oocyte functionality. The lack of effect on oocytes was confirmed by incubating sEVs with oocytes prior to IVF, achieving sperm-zona pellucida binding results similar to those of control. In the last experiment, conducted under IVF conditions, sperm functionality was analyzed in terms of tyrosine phosphorylation, acrosome integrity and metabolism. The sEVs, regardless of the subset, did not affect sperm tyrosine phosphorylation or acrosome integrity, but did influence sperm metabolism by decreasing sperm ATP production under capacitating conditions. In conclusion, this study demonstrated that the presence of sEVs on IVF medium impairs IVF outcomes, most likely by altering sperm metabolism.

Immunophenotype profile by flow cytometry reveals different subtypes of extracellular vesicles in porcine seminal plasma.

BACKGROUND: Porcine seminal plasma (SP) is endowed with a heterogeneous population of extracellular vesicles (sEVs). This study evaluated the immunophenotypic profile by high-sensitivity flow cytometry of eight sEV subpopulations isolated according to their size (small [S-sEVs] and large [L-sEVs]) from four different SP sources, namely three ejaculate fractions (the first 10 mL of the sperm rich fraction [SRF-P1], the remaining SRF [SRF-P2], and the post-SRF [PSRF]) and entire ejaculate (EE). METHODS: Seminal EVs were isolated using a size exclusion chromatography-based protocol from six SP pools (five ejaculates/pool) of each SP source and characterized using complementary approaches including total protein (BCA™assay), particle size distribution (dynamic light scattering), morphology (transmission electron microscopy), and purity (albumin by Western blot). Expression of CD9, CD63, CD81, CD44 and HSP90ß was analyzed in all sEV subpopulations by high-sensitivity flow cytometry according to MIFlowCyt-EV guidelines, including an accurate calibration, controls, and discrimination by CFSE-labelling. RESULTS: Each sEV subpopulation exhibited a specific immunophenotypic profile. The percentage of sEVs positive for CD9, CD63, CD81 and HSP90ß differed between S- and L-sEVs (P < 0.0001). Specifically, the percentage of sEVs positive for CD9 and CD63 was higher and that for CD81 was lower in S- than L-sEVs in the four SP sources. However, the percentage of HSP90ß-positive sEVs was lower in S-sEVs than L-sEVs in the SRF-P1 and EE samples. The percentage of sEVs positive for CD9, CD63, and CD44 also differed among the four SP sources (P < 0.0001), being highest in PSRF samples. Notably, virtually all sEV subpopulations expressed CD44 (range: 88.04-98.50%). CONCLUSIONS: This study demonstrated the utility of high-sensitivity flow cytometry for sEV immunophenotyping, allowing the identification of distinct sEV subpopulations that may have different cellular origin, cargo, functions, and target cells.

Chromatin condensation but not DNA integrity of pig sperm is greater in the sperm-rich fraction.

BACKGROUND: Protamination and condensation of sperm chromatin as well as DNA integrity play an essential role during fertilization and embryo development. In some mammals, like pigs, ejaculates are emitted in three separate fractions: pre-sperm, sperm-rich (SRF) and post sperm-rich (PSRF). These fractions are known to vary in volume, sperm concentration and quality, as well as in the origin and composition of seminal plasma (SP), with differences being also observed within the SRF one. Yet, whether disparities in the DNA integrity and chromatin condensation and protamination of their sperm exist has not been interrogated. RESULTS: This study determined chromatin protamination (Chromomycin A3 test, CMA3), condensation (Dibromobimane test, DBB), and DNA integrity (Comet assay) in the pig sperm contained in the first 10 mL of the SRF (SRF-P1), the remaining portion of the sperm-rich fraction (SRF-P2), and the post sperm-rich fraction (PSRF). While chromatin protamination was found to be similar between the different ejaculate fractions (P > 0.05), chromatin condensation was seen to be greater in SRF-P1 and SRF-P2 than in the PSRF (P = 0.018 and P = 0.004, respectively). Regarding DNA integrity, no differences between fractions were observed (P > 0.05). As the SRF-P1 has the highest sperm concentration and ejaculate fractions are known to differ in antioxidant composition, the oxidative stress index (OSi) in SP, calculated as total oxidant activity divided by total antioxidant capacity, was tested and confirmed to be higher in the SRF-P1 than in SRF-P2 and PSRF (0.42 ± 0.06 vs. 0.23 ± 0.09 and 0.08 ± 0.00, respectively; P < 0.01); this index, in addition, was observed to be correlated to the sperm concentration of each fraction (Rs = 0.973; P < 0.001). CONCLUSION: While sperm DNA integrity was not found to differ between ejaculate fractions, SRF-P1 and SRF-P2 were observed to exhibit greater chromatin condensation than the PSRF. This could be related to the OSi of each fraction.

Mating modifies the expression of crucial oxidative-reductive transcripts in the pig oviductal sperm reservoir: is the female ensuring sperm survival?

Background: Mating induces large changes in the female genital tract, warranting female homeostasis and immune preparation for pregnancy, including the preservation of crucial oxidative status among its pathways. Being highly susceptible to oxidative stress, sperm survival and preserved function depend on the seminal plasma, a protection that is removed during sperm handling but also after mating when spermatozoa enter the oviduct. Therefore, it is pertinent to consider that the female sperm reservoir takes up this protection, providing a suitable environment for sperm viability. These aspects have not been explored despite the increasing strategies in modulating the female status through diet control and nutritional supplementation. Aims: To test the hypothesis that mating modifies the expression of crucial oxidative-reductive transcripts across the entire pig female genital tract (cervix to infundibulum) and, particularly in the sperm reservoir at the utero-tubal junction, before ovulation, a period dominated by estrogen stimulation of ovarian as well as of seminal origin. Methods: The differential expression of estrogen (ER) and progesterone (PR) receptors and of 59 oxidative-reductive transcripts were studied using a species-specific microarray platform, in specific segments of the peri-ovulatory sow reproductive tract in response to mating. Results: Mating induced changes along the entire tract, with a conspicuous downregulation of both ER and PR and an upregulation of superoxide dismutase 1 (SOD1), glutaredoxin (GLRX3), and peroxiredoxin 1 and 3 (PRDX1, PRDX3), among other NADH Dehydrogenase Ubiquinone Flavoproteins, in the distal uterus segment. These changes perhaps helped prevent oxidative stress in the area adjacent to the sperm reservoir at the utero-tubal junction. Concomitantly, there were a downregulation of catalase (CAT) and NADH dehydrogenase (ubiquinone) oxidoreductases 1 beta subcomplex, subunit 1 (NDUFB1) in the utero-tubal junction alongside an overall downregulation of CAT, SOD1, and PRDX3 in the ampullar and infundibulum segments. Conclusions: Natural mating is an inducer of changes in the expression of female genes commanding antioxidant enzymes relevant for sperm survival during sperm transport, under predominant estrogen influence through the bloodstream and semen. The findings could contribute to the design of new therapeutics for the female to improve oxidative-reductive balance.

Seminal Extracellular Vesicles and Their Involvement in Male (In)Fertility: A Systematic Review.

Seminal plasma contains numerous extracellular vesicles (sEVs). Since sEVs are apparently involved in male (in)fertility, this systematic review focused on studies specifically investigating such relationship. Embase, PubMed, and Scopus databases were searched up to 31 December 2022, primarily identifying a total of 1440 articles. After processing for screening and eligibility, 305 studies were selected as they focused on sEVs, and 42 of them were considered eligible because they included the word fertility or a related word such as infertility, subfertility, fertilization, and recurrent pregnancy loss in the title, objective(s), and/or keywords. Only nine of them met the inclusion criteria, namely (a) conducting experiments aimed at associating sEVs with fertility concerns and (b) isolating and adequately characterizing sEVs. Six studies were conducted on humans, two on laboratory animals, and one on livestock. The studies highlighted some sEV molecules, specifically proteins and small non-coding RNAs, that showed differences between fertile and subfertile or infertile males. The content of sEVs was also related to sperm fertilizing capacity, embryo development, and implantation. Bioinformatic analysis revealed that several of the highlighted sEV fertility-related proteins would be cross-linked to each other and involved in biological pathways related to (i) EV release and loading and (ii) plasma membrane organization.

Extracellular vesicles would be involved in the release and delivery of seminal TGF-ß isoforms in pigs.

Introduction: Pig seminal plasma (SP) is rich in active forms of all three isoforms (1-3) of transforming growth factor ß (TGF-ß), a chemokine modulatory of the immune environment in the female genital tract once semen is delivered during mating or artificial insemination (AI). The present study aimed to examine how TGF-ßs are secreted by the epithelium of the male reproductive tract and how they are transported in semen, emphasizing the interplay with seminal extracellular vesicles (sEVs). Methods: Source of TGF-ßs was examined by immunohistochemistry in testis, epididymis, and accessory sex glands, by immunocytochemistry in ejaculated spermatozoa, and by Luminex xMAP® technology in SP and sEVs retrieved from healthy, fertile male pigs used as breeders in AI programs. Results: All three TGF-ß isoforms were expressed in all reproductive tissues explored and would be released into ductal lumen either in soluble form or associated with sEVs. Ejaculated spermatozoa expressed all three TGF-ß isoforms, both inside and outside, probably the outer one associated with membrane-bound sEVs. The results confirmed that pig SP contains all three TGF-ß isoforms and demonstrated that a substantial portion of them is associated with sEVs. Discussion: Seminal EVs would be involved in the cellular secretion of the active forms of seminal TGF-ß isoforms and in their safe transport from the male to the female reproductive tract.

The Proteome of Large or Small Extracellular Vesicles in Pig Seminal Plasma Differs, Defining Sources and Biological Functions.

Seminal plasma contains many morphologically heterogeneous extracellular vesicles (sEVs). These are sequentially released by cells of the testis, epididymis, and accessory sex glands and involved in male and female reproductive processes. This study aimed to define in depth sEV subsets isolated by ultrafiltration and size exclusion chromatography, decode their proteomic profiles using liquid chromatography-tandem mass spectrometry, and quantify identified proteins using sequential window acquisition of all theoretical mass spectra. The sEV subsets were defined as large (L-EVs) or small (S-EVs) by their protein concentration, morphology, size distribution, and EV-specific protein markers and purity. Liquid chromatography-tandem mass spectrometry identified a total of 1034 proteins, 737 of them quantified by SWATH in S-EVs, L-EVs, and non-EVs-enriched samples (18-20 size exclusion chromatography-eluted fractions). The differential expression analysis revealed 197 differentially abundant proteins between both EV subsets, S-EVs and L-EVs, and 37 and 199 between S-EVs and L-EVs versus non-EVs-enriched samples, respectively. The gene ontology enrichment analysis of differentially abundant proteins suggested, based on the type of protein detected, that S-EVs could be mainly released through an apocrine blebbing pathway and be involved in modulating the immune environment of the female reproductive tract as well as during sperm-oocyte interaction. In contrast, L-EVs could be released by fusion of multivesicular bodies with the plasma membrane becoming involved in sperm physiological processes, such as capacitation and avoidance of oxidative stress. In conclusion, this study provides a procedure capable of isolating subsets of EVs from pig seminal plasma with a high degree of purity and shows differences in the proteomic profile between EV subsets, indicating different sources and biological functions for the sEVs.

Seminal extracellular vesicles subsets modulate gene expression in cumulus cells of porcine in vitro matured oocytes.

Seminal plasma (SP), a fluid composed mainly by secretions from accessory sex glands, contains a heterogenous population of extracellular vesicles (EVs), involved in several reproductive physiological processes. Seminal plasma has been found to modulate ovary function, in terms of hormone secretion and immune regulation. This study evaluated the potential effect of SP-EV-subsets on the modulation of cumulus-oocyte-complex (COCs) physiology during in vitro maturation (IVM). Two SP-EV-subsets, small-EVs (S-EVs) and large-EVs (L-EVs), were isolated from pig SP by size-exclusion-chromatography. Next, COCs were IVM in the absence (control) or presence of each SP-EV-subset to evaluate their uptake by COCs (PKH67-EVs labelling) and their effect on oocyte and cumulus cells (CCs) (gene expression, and progesterone and estradiol-17ß levels). S-EVs and L-EVs were able to bind CCs but not oocytes. Supplementation with L-EVs induced changes (P ≤ 0.05) in the transcript levels of oocyte maturation- (HAS2) and steroidogenesis-related genes (CYP11A1 and HSD3B1) in CCs. No effect on nuclear oocyte maturation and progesterone and estradiol-17ß levels was observed when COCs were IVM with any of the two SP-EV-subsets. In conclusion, while SP-EV-subsets can be integrated by CCs during IVM, they do not affect oocyte maturation and only L-EVs are able to modulate CCs function, mainly modifying the expression of steroidogenesis-related genes.

Extracellular vesicles in seminal plasma: A safe and relevant tool to improve fertility in livestock?

Seminal plasma (SP) is not a pre-requisite for pregnancy. Yet, this heterogeneous, composite SP has proven relevant for fertility, as mediator for cell-to-cell communication between producing cells, spermatozoa and the female internal genital tract, regulating complex reproductive processes. Bearing hormones, proteins, cytokines as well as nuclei acids in nano-sized lipid bilayer seminal extracellular vesicles (sEVs), the SP concerts signaling to the female. Signals influence timing of ovulation, sperm transport and, particularly, enable the female immune system to balance her cryptic choice to engage into pregnancy or reject an eventual fertilization. This essay, focusing on livestock in general and pigs in particular, discusses the intrinsic roles of sEVs with regards to reproductive homeostasis, while binding and internalizing their cargo in spermatozoa and female tract epithelia to regulate their functional activity. Since prior studies had inconclusive results using bulk SP or single SP-contained free molecules, argumentation is hereby provided to increase the current incipient research on livestock sEVs, where fragile molecules relevant for fertility are shielded from degradation during handling. Seminal EVs isolated from SP can be used for andrological diagnosis and perhaps to select breeders with optimal fertility. Moreover, sEVs can be laboratory-uploaded with specific molecules or even engineered as lipid nanodroplets used as additives for extenders to improve fertility after artificial insemination (AI) or reproductive biotechnologies.

miRNA-Profiling in Ejaculated and Epididymal Pig Spermatozoa and Their Relation to Fertility after Artificial Insemination.

MicroRNAs (miRNAs) are short non-coding RNAs (20-25 nucleotides in length) capable of regulating gene expression by binding -fully or partially- to the 3'-UTR of target messenger RNA (mRNA). To date, several studies have investigated the role of sperm miRNAs in spermatogenesis and their remaining presence toward fertilization and early embryo development. However, little is known about the miRNA cargo in the different sperm sources and their possible implications in boar fertility. Here, we characterized the differential abundance of miRNAs in spermatozoa from the terminal segment of the epididymis and three different fractions of the pig ejaculate (sperm-peak, sperm-rich, and post-sperm rich) comparing breeding boars with higher (HF) and lower (LF) fertility after artificial insemination (AI) using high-output small RNA sequencing. We identified five sperm miRNAs that, to our knowledge, have not been previously reported in pigs (mir-10386, mir-10390, mir-6516, mir-9788-1, and mir-9788-2). Additionally, four miRNAs (mir-1285, mir-92a, mir-34c, mir-30), were differentially expressed among spermatozoa sourced from ejaculate fractions and the cauda epididymis, and also different abundance was found between HF and LF groups in mir-182, mir-1285, mir-191, and mir-96. These miRNAs target genes with key roles in fertility, sperm survival, immune tolerance, or cell cycle regulation, among others. Linking the current findings with the expression of specific sperm proteins would help predict fertility in future AI-sires.

Extracellular vesicles in seminal fluid and effects on male reproduction. An overview in farm animals and pets.

Extracellular vesicles (EVs) are lipid bilayer nanovesicles released by most functional cells to body fluids, containing bioactive molecules, mainly proteins, lipids, and nucleic acids having actions at target cells. The EVs have essential functions in cell-to-cell communication by regulating different biological processes in target cells. Fluids from the male reproductive tract, including seminal plasma, contain many extracellular vesicles (sEVs), which have been evaluated to a lesser extent than those of other body fluids, particularly in farm animals and pets. Results from the few studies that have been conducted indicated epithelial cells of the testis, epididymis, ampulla of ductus deferens and many accessory sex glands release sEVs mainly via apocrine mechanisms. The sEVs are morphologically heterogeneous and bind to functional cells of the male reproductive tract, spermatozoa, and cells of the functional tissues of the female reproductive tract after mating or insemination. The sEVs encapsulate proteins and miRNAs that modulate sperm functions and male fertility. The sEVs, therefore, could be important as reproductive biomarkers in breeding sires. Many of the current findings regarding sEV functions, however, need experimental confirmation. Further studies are particularly needed to characterize both membranes and contents of sEVs, as well as the interaction between sEVs and target cells (spermatozoa and functional cells of the internal female reproductive tract). A priority for conducting these studies is development of methods that can be standardized and that are scalable, cost-effective and time-saving for isolation of different subtypes of EVs present in the entire population of sEVs.

How does the boar epididymis regulate the emission of fertile spermatozoa?

The epididymis is responsible for peripheral immune tolerance of maturing spermatozoa even though these have xeno-antigens foreign to the male and female immune system. The epididymis also produces factors required for fertilization and serves as a sperm repository until the time of ejaculation. These reproduction-relevant epididymal functions occur in the mesonephros-derived duct-system that is composed of absorptive and secretory epithelial cells with the capacity for merocrine and apocrine secretion of proteins, antioxidative- and electrolyte/pH-regulating enzymes and small, non-coding RNAs (sncRNAs), many stored in epididymosomes for sperm adhesion and long-lasting modifications of sperm functions. This paper provides a review summary of current and new knowledge of how the boar epididymis affects the quality of spermatozoa in the ejaculate of breeding boars. There is a particular focus on sperm maturation, survival, function and the role of signaling to the female immune system in fertility modulation. Furthermore, aspects related to the ductus epithelial contributions regarding electrolyte control, protein production, release of epididymosomes that contain sncRNAs are emphasized as are novel associations with fertility of the male, sperm quiescence during storage in the cauda epididymis, and on changes occurring in sperm subsequent to ejaculation.

mRNA expression of oxidative-reductive proteins in boars with documented different fertility can identify relevant prognostic biomarkers.

Oxidative stress unbalance is a major factor causing impairment of sperm function and, ultimately, sperm death. In this study, we identified transcriptomic and proteomic markers for oxidative-related protectors from the generation of reactive oxygen species (ROS) in spermatozoa from breeding boars with documented high- or low-fertility. Particular attention was paid to glutathione peroxidases, and to transcripts related to DNA stabilization and compaction, as protamine and transition proteins. mRNA cargo analysis was performed using porcine-specific micro-arrays (GeneChip® miRNA 4.0 and GeneChip® Porcine Gene 1.0 ST) and qPCR validation. Differences between fertility-classed boars were ample among biomarkers; some upregulated only at protein level (catalase (CAT), superoxide dismutase 1 (SOD1) and glutathione proteins), or only at the mRNA level (ATOX1, Antioxidant Protein 1). In addition, protamines 2 and 3, essential for sperm DNA condensation and also transition proteins 1 and 2 (TNP1 and TNP2), required during histone-to-protamine replacement, were overexpressed in spermatozoa from high-fertile boars. This up-regulation seems concerted to reduce DNA accessibility to ROS attack, protecting the DNA. The upregulated intracellular phospholipid hydroperoxide glutathione peroxidase (GPx4), in high-fertile boars at mRNA level, can be considered a most relevant biomarker for fertility disclosure during sperm evaluation.

Metabolomic fingerprinting of pig seminal plasma identifies in vivo fertility biomarkers.

BACKGROUND: Metabolomic approaches, which include the study of low molecular weight molecules, are an emerging -omics technology useful for identification of biomarkers. In this field, nuclear magnetic resonance (NMR) spectroscopy has already been used to uncover (in) fertility biomarkers in the seminal plasma (SP) of several mammalian species. However, NMR studies profiling the porcine SP metabolome to uncover in vivo fertility biomarkers are yet to be carried out. Thus, this study aimed to evaluate the putative relationship between SP-metabolites and in vivo fertility outcomes. To this end, 24 entire ejaculates (three ejaculates per boar) were collected from artificial insemination (AI)-boars throughout a year (one ejaculate every 4 months). Immediately after collection, ejaculates were centrifuged to obtain SP-samples, which were stored for subsequent metabolomic analysis by NMR spectroscopy. Fertility outcomes from 1525 inseminations were recorded over a year, including farrowing rate, litter size, stillbirths per litter and the duration of pregnancy. RESULTS: A total of 24 metabolites were identified and quantified in all SP-samples. Receiver operating characteristic (ROC) curve analysis showed that lactate levels in SP had discriminative capacity for farrowing rate (area under the curve [AUC] = 0.764) while carnitine (AUC = 0.847), hypotaurine (AUC = 0.819), sn-glycero-3-phosphocholine (AUC = 0.833), glutamate (AUC = 0.799) and glucose (AUC = 0.750) showed it for litter size. Similarly, citrate (AUC = 0.743), creatine (AUC = 0.812), phenylalanine (AUC = 0.750), tyrosine (AUC = 0.753) and malonate (AUC = 0.868) levels had discriminative capacity for stillbirths per litter; and malonate (AUC = 0.767) and fumarate (AUC = 0.868) levels for gestation length. CONCLUSIONS: The assessment of selected SP-metabolites in ejaculates through NMR spectroscopy could be considered as a promising non-invasive tool to predict in vivo fertility outcomes in pigs. Moreover, supplementing AI-doses with specific metabolites should also be envisaged as a way to improve their fertility potential.

Oxytocin in pig seminal plasma is positively related with in vivo fertility of inseminated sows.

BACKGROUND: Identification of relevant in vivo biomarkers for fertility remains a challenge for the livestock industry. Concentrations of the small peptide hormone oxytocin (OXT), involved in male reproductive function and present in the seminal plasma (SP) of several species could be a robust one. This study characterized concentrations of SP-OXT in ejaculates from boars used in artificial insemination (AI) programs aiming to evaluate its relationship with sperm quality variables and in vivo fertility of their liquid-stored AI-semen. Seminal OXT concentrations (ng/mL) were measured in 169 ejaculates from 61 boars of the Duroc, Pietrain, Landrace and Large White breeds using a direct competitive immunoassay test based on AlphaLISA® technology. Ejaculate (ejaculate volume, sperm concentration, total sperm count) and sperm parameters (motility, viability, intracellular generation of reactive oxygen species, plasma membrane fluidity) were assessed at 0 h and 72 h in AI-semen samples stored at 17 °C. In vivo fertility included only 18 Large White and Landrace boars whose AI-semen was used to inseminated > 100 sows and evaluated both farrowing rate and litter size of 3,167 sows. RESULTS: The results showed that SP-OXT differed between boars and between ejaculates within boar (P < 0.05) but not between breeds (Duroc, Pietrain, Landrace and Large White). Ejaculates with higher SP-OXT concentration/mL (hierarchically grouped; P < 0.001) had larger volume and came from younger boars (P < 0.05). Ejaculates of boars showing positive farrowing rate deviation exhibited higher (P < 0.05) SP-OXT concentration/mL than those with negative farrowing rate deviation. CONCLUSION: The SP concentrations of OXT are boar, ejaculate and age dependent, and positively related with ejaculate volume and farrowing rates of liquid-stored semen AI-doses.

Measurement of Oxidative Stress Index in Seminal Plasma Can Predict In Vivo Fertility of Liquid-Stored Porcine Artificial Insemination Semen Doses.

The study evaluated the relation between the oxidative stress index (OSI) in porcine seminal plasma (n = 76) with sperm resilience and in vivo fertility (farrowing rate and litter size of 3137 inseminated sows) of liquid-stored artificial insemination (AI) semen doses. The OSI was assessed as the ratio of advanced oxidation protein products to Trolox-equivalent antioxidant capacity, both measured using an automated analyzer. Sperm motility (computer-assisted sperm analyzer) and viability (flow cytometry) were evaluated in semen AI-doses at 0 and 72 h of storage at 17 °C. Sperm resilience was defined as the difference between storage intervals. Semen AI-doses were hierarchically clustered as having high, medium and low seminal OSI (p < 0.001) with those of low displaying higher resilience (p < 0.01). Boars were hierarchically clustered into two groups (p < 0.001) as having either positive or negative farrowing rate and litter size deviation; the negative one showing higher seminal OSI (p < 0.05). In sum, seminal OSI was negatively related to sperm motility and the in vivo fertility of liquid-stored boar semen AI-doses, with the receiver operating characteristic curve presenting seminal OSI as a good predictive biomarker of in vivo fertility of AI-boars (area under the curve: 0.815, p < 0.05).

Metabolite Profiling of Pig Seminal Plasma Identifies Potential Biomarkers for Sperm Resilience to Liquid Preservation.

Metabolomic approaches allow the study of downstream gene expression events since metabolites are considered as the products of cell signaling pathways. For this reason, many studies in humans have already been conducted to determine the influence of the metabolites present in seminal plasma (SP) on sperm physiology, and to identify putative biomarkers. However, in livestock species, these relationships are yet to be uncovered. Thus, the present study aimed to explore: (i) if concentrations of metabolites in pig SP are related to sperm quality and functionality, and (ii) if they could predict the sperm resilience to liquid storage at 17°C. To this end, 28 ejaculates were individually collected and split into three aliquots: one was used for SP analysis through nuclear magnetic resonance (NMR) spectroscopy; another served for the evaluation of sperm concentration and morphology; and the last one was utilized to determine sperm functionality parameters using computer-assisted sperm analysis (CASA) and flow cytometry after 0 h and 72 h of liquid-storage at 17°C. NMR analysis allowed the identification and quantification of 23 metabolites present in pig SP which, except for fumarate, were not observed to follow a breed-dependent behavior. Moreover, specific relationships between metabolites and sperm variables were identified: (i) glutamate, methanol, trimethylamine N-oxide, carnitine, and isoleucine were seen to be related to some sperm quality and functionality parameters evaluated immediately after semen collection; (ii) leucine, hypotaurine, carnitine and isoleucine were found to be associated to the sperm ability to withstand liquid storage; and (iii) Bayesian multiple regression models allowed the identification of metabolite patterns for specific sperm parameters at both 0 h and 72 h. The identification of these relationships opens up the possibility of further investigating these metabolites as potential sperm functional biomarkers.

Aldose Reductase B1 in Pig Seminal Plasma: Identification, Localization in Reproductive Tissues, and Relationship With Quality and Sperm Preservation.

Aldose reductase B1 (AKR1B1), a NADPH-dependent enzyme that belongs to the aldo-keto reductase protein superfamily, has been reported to be involved in both male and female reproductive physiology. The objectives of this study were: (1) to evaluate the concentration of SP-AKR1B1 in pig ejaculate fractions; (2) to describe the immunohistochemical localization of AKR1B1 alongside the boar genital tract; (3) to evaluate the relationship between SP-AKR1B1 and sperm quality/functionality parameters. Ejaculates from seven boars (one ejaculate per boar) were collected in separate portions [the first 10 mL of the sperm rich fraction (SRF-P1), the rest of the SRF (SRF-P2), and the post-SRF (PSRF)], and the concentration of SP-AKR1B1 was assessed using an enzyme-linked immunosorbent assay (ELISA). Immunohistochemistry and immunoblotting targeting was conducted in the reproductive tissues of these boars. Additionally, the entire ejaculates of 14 boars (one ejaculate per boar) were collected and split into three separate aliquots for: (i) SP-AKR1B1 quantification; (ii) assessment of sperm concentration and morphology; and (iii) evaluation of sperm quality and functionality parameters upon ejaculate collection (0 h) and after 72 h of liquid storage at 17°C. Concentration of AKR1B1 in the SP of SRF-P1 (458.2 ± 116.33 ng/mL) was lower (P < 0.05) than that of SRF-P2 (1105.0 ± 229.80 ng/mL) and PSRF (1342.4 ± 260.18 ng/mL). Monomeric and dimeric AKR1B1 forms were expressed alongside the reproductive tissues, except in the bulbourethral glands. No relationship between SP-AKR1B1 and sperm quality/functionality parameters was observed either at 0 h or after 72 h of storage at 17°C. In conclusion, AKR1B1 is expressed in the reproductive organs of boars (except bulbourethral glands) and a higher concentration is found in the PSRF suggesting that seminal vesicles would be the main secretory source. However, this enzyme does not appear to be related to sperm quality/functionality or to the sperm ability to withstand liquid storage at 17°C.