Norandrogens are significant anabolic-androgenic steroids used initially for the treatment of debilitating conditions and later as performance-enhancing drugs. Regulatory challenges of their abuse in human, equine, and canine sports stem from the question of endogenous norandrogens. Evidence from mass spectrometry for 19-norandrostenedione (19-norA4) in the sulfate fraction of conjugated steroids in equine yolk-sac fluid suggested a need for reexamination. In vitro biosynthesis of 19-norandrogens was examined in highly purified preparations of porcine Leydig cells from mature pigs by incubation in -medium containing [3H]-androstenedione (A4) or nonradioactive A4. Steroids were recovered from media by solid-phase extraction, separately as unconjugated and conjugated fractions. High performance liquid chromatographic (HPLC) profiles were obtained from liquid-scintillaion counting of radioctivity. Several peaks in the conjugated fractions were investigated further by HPLC. Two peaks widely separated on HPLC as conjugated steroids yielded evidence of norandrostenedione (19-norA4) after solvolysis. Other peaks identified by HPLC retention times matching standards were 19-hydroxy-A4, A4 (substrate), and epiandrosterone. Incubations with non-radioactive A4 provided further evidence from radioimmunoassay of sulfated steroids as precursors of 19-norA4. With known high sulfotransferase activity in porcine Leydig cells, it is proposed that two 3-enol sulfates were formed as stable "precursors" of 19-norA4 by the distinctive action of the third aromatase isoform of the boar testes. Although their identities remain unknown, it was shown that 19-norA4 itself was not present as a 3-enol sulfate. These findings have implications regarding endogenous anabolic agents in normal growth and relevance to illegal use of anabolic androgens in sports and animal production.

BackgroundHeat stress (HS) can impair boar testicular function, leading to reproductive issues. However, chlorogenic acid (CGA) has been shown to mitigate HS-induced damage in various livestock and poultry species. Prepuberty is an important stage of testicular development in boars after birth. However, the protective effect of CGA on testicular HS injury during prepuberty boars and the underlying mechanisms are still not fully understood.ResultsIn vivo, a total of 30 healthy boars with similar body weights and ages were obtained and randomly divided into 3 groups, which were fed a basal diet supplemented with CGA 0 (the ND_TN group), 0 (the ND_HS group) or 1,000 (the CGA_HS group) mg/kg. After being fed for 28 d, all the groups, except the ND_TN group, were treated with high temperature for 7 d, after which samples were collected from the boars and analysed. The results showed that CGA significantly mitigated the HS-induced reduction in T-AOC content in testicular tissue and sperm density. Mechanistically, multiomics analysis revealed that the genes differentially expressed by CGA and HS were predominantly associated with the glutathione metabolism pathway. The combined analysis of transcriptomics and proteomics revealed that only BLVRA was affected by both HS and CGA when the mRNA and protein levels of a gene showed differential expression with the same trend. In vitro studies confirmed that CGA modulated GPX3 expression via BLVRA, affected GPx activity, and attenuated HS-induced ROS accumulation.ConclusionsIn conclusion, prepubertal HS impairs the spermatogenic capacity of boars. BLVRA may mediate the testicular protective effect of CGA, although in vivo validation of this pathway is needed. This study contributes to elucidating the mechanisms underlying the effects of HS on prepubertal boar testicular development using multiomics approaches, laying a foundation for the potential utilization of CGA in swine production.Supplementary InformationThe online version contains supplementary material available at 10.1186/s40104-025-01336-0.

BackgroundPostnatal testicular development involves the proliferation and differentiation of somatic and spermatogenic cells. The entire process is governed by spatial and temporal cues that coordinate spermatogenesis. Comparative analyses of gene expression and transcriptional regulation across multiple stages can provide insights into the complexity of developmental dynamics. Therefore, we systematically analyzed the cellular composition and molecular features of boar testes at different time points using single-cell RNA sequencing (scRNA-seq) to reveal their roles in testicular development.ResultsWe performed scRNA-seq on 115,248 testicular cells from eight pigs at 0, 2, 5, 24, and 48 months of age, identifying 10 distinct cell types, including three rare populations: pericytes, smooth muscle cells, and neutrophils. Trajectory analysis and RNA velocity modeling uncovered dynamic cell fate transitions during spermatogenesis. Furthermore, we identified testis cell-specific transcription factors and regulators. Notably, PRDX5 and LUZP2 exhibited developmental stage-specific expression after M5 and varied with the proportion of germ cells. Cross-species comparative analysis revealed the evolutionary conservation among testicular cell populations, especially endothelial and germ cells, and identified several conserved transcription factors such as SOX9, IRF8, and KDM5B, highlighting conserved regulatory mechanisms during testicular development.ConclusionsOur findings provide novel insights into the cellular dynamics of spermatogenesis and offer a valuable foundation for future research on the molecular mechanisms underlying testicular development in pigs and other mammals.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12864-025-12280-8.

Abstract Boar fertility is critical for swine reproductive efficiency. Sperm production capacity is driven by Sertoli cell numbers within the testis. Sertoli cell proliferation is malleable in early life but ceases post-puberty. Extensive Sertoli cell proliferation occurs in neonates, a period termed mini-puberty that overlaps temporally with nursing. Previous data demonstrates that colostrum consumption promotes neonatal Sertoli cell proliferation and subsequent sperm production in the boar, a phenomenon termed lactocrine programming. For example, boars who consumed more colostrum as neonates produced 17 billion more sperm per ejaculate (15% increase) compared to boars with lower colostrum intake. However, the biological mechanisms underlying this effect are unclear. The objective of this study was to determine how consumption of sow milk affects testicular development and reproductive endocrinology in neonatal pigs. After birth, white crossbred neonatal boars nursed colostrum ad libitum for 2 days prior to treatment. Boars were randomly assigned to consume either sow milk (n = 4) or milk replacer (n = 4; NutraStart Liqui-Wean) three times daily (90 ml/feeding) for eight days. At 10 days of age, boars were weighed and euthanized prior to testis and blood collection. Testes were weighed and snap frozen for subsequent proteomics. Serum concentrations of thyroxine (T4), gonadal steroids (Leydig cell products), and anti-müllerian hormone (AMH; Sertoli cell product) were quantified. Milk-fed boars had 35% heavier testes (P = 0.031) compared with replacer-fed pigs, despite no difference in body weight (P > 0.05). Serum concentrations of T4 were 30% greater in milk-fed boars compared with replacer-fed animals (P = 0.0339). Circulating concentrations of AMH and gonadal steroids (e.g., testosterone, 17β-estradiol) did not differ between treatments (P > 0.05). Regarding testicular proteomics, a total of 1,517 proteins were detected. Of these, 49 proteins were upregulated and 721 proteins were downregulated within the testes of milk-fed boars compared to replacer-fed (P < 0.05). Downregulated proteins within testes of milk-fed boars included those important for germ cell differentiation (e.g., DDX4), steroidogenesis (e.g., HSD3B), and apoptosis (e.g., PDCD4). In addition, AMH was also downregulated within testes of milk-fed boars, indicating an effect on Sertoli cells. Upregulated testicular proteins included mediators of cellular metabolism (e.g., IDH1), steroidogenesis (e.g., StAR), cell proliferation (e.g., CHD5), and antioxidant defenses (e.g., GSTO1). Notably, INSL3 – a biomarker of Leydig cell functionality – was also upregulated within milk-fed versus replacer-fed testes. Interestingly, transthyretin (thyroid hormone transporter) was also elevated, consistent with increased circulating T4 concentrations in milk-fed boars. To conclude, these data elucidate the biological mechanisms underlying lactocrine programming of the porcine testis. Neonatal intake of sow milk increased testicular weight, indicative of a proliferative effect on Sertoli cells. Therefore, manipulation of neonatal diet may be a novel intervention to develop boars with greater reproductive potential.

Abstract Boar fertility is critical for swine reproductive efficiency. Recent data demonstrates that neonatal colostrum intake is associated with enhanced sperm counts in adult boars. These data support the “Lactocrine Hypothesis,” which states that bioactive factors from maternal milk directly promote neonatal tissue development, including reproductive organs. Notably, maternal milk consumption increased the number of Sertoli cells within the testes of nursed versus formula-fed boars. However, the biological mechanisms mediating this effect are unknown. We hypothesized that milk extracellular vesicles (EVs) may contribute because EVs are abundant in milk, carry bioactive cargo (e.g., miRNAs), and protect these cargo from digestion before uptake by the intestinal mucosa and entry into the neonatal circulatory system. Our group previously demonstrated that maternal milk EVs bioaccumulate in distant neonatal tissues (e.g., brain), but testes were not evaluated. Recently, we reported that acute consumption of milk EVs alters the testicular proteome in neonatal boars, including key Sertoli cell markers (e.g., AMH, GATA-4, SOX-9). Together, these data suggest that milk EVs directly alter testis biology. The objective of this study was to determine the bioavailability of milk EVs to the neonatal testis using a transgenic swine model. Hemizygous transgenic sows ubiquitously expressing the fluorescent protein, ZsGreen1, and their male offspring, were utilized for this study. Milk was collected and pooled from 3 lactating (20–22 days post-partum) transgenic sows. Milk EVs were isolated via ultracentrifugation and filtration (220 nm). Milk EVs were validated via nano-flow cytometry (nFCM; size and concentration) and transmission electron microscopy (TEM; size and structure). The presence of ZsGreen1 (488 nm) in milk EVs was evaluated via nFCM and confocal microscopy. At necropsy, blood and testes were collected from wild type (WT) boars nursed (8–14 days) by transgenic (TSG) sows (n=4), WT boars nursed by WT sows (negative control; n=2) and a TSG boar nursed by a TSG sow (positive control; n=1). Serum was isolated for subsequent EV collection, validation, and fluorescent evaluation as described above for milk EVs. Testis samples were snap frozen and protein was extracted for HPLC/MS-MS. Results indicate that transgenic sows produce milk EVs endogenously labeled with ZsGreen1 compared with WT sow milk EVs. In addition, WT boars nursed by transgenic sows exhibited ZsGreen1-labeled milk EVs in their serum. Finally, ZsGreen1 was detectable in testes of WT boars nursed by TSG sows. The present study is the first to evaluate if maternal milk EVs are bioavailable to the neonatal testis. Using a novel swine model, these data suggest the transfer of maternal milk EVs to offspring blood and testes after a natural ingestion route (ad libitum suckling). Hence, milk EVs are bioavailable to the testis and may help mediate lactocrine programming.

The study investigated the sperm motility and morphometry of pre-freeze and post-thaw boar epididymal semen cooled at increasing holding times at 18 °C. A total of 50 testes of heterogeneous boars were collected (5 testes/day) from the local abattoir and transported to the laboratory at 5 °C within 30 min after slaughter. Semen was retrieved from the caudal part of the epididymis using the slicing float-up method, diluted with Beltsville Thawing Solution extender, pooled in a 50 mL centrifuge tube/5 testes/day, and cooled at 18 °C. Following each holding time (0, 3, 6, 9, 12, 24, and 48 h), the cooled semen sample was re-suspended with Fraction A extender and stored at 5 °C for an additional 45 min. A cooled resuspended semen sample was then diluted with Fraction B extender, loaded into 0.25 mL straws, and frozen using liquid nitrogen vapour. Thawing was accomplished by immersing the semen straws in warm (37 °C) water for 1 min and the samples were evaluated for sperm motility and morphometry traits using the computer-assisted sperm analyzer system. The data were analyzed using variance analysis. Descriptive statistics were used to assess sperm morphometry, establishing the minimum and maximum values. Boar epididymal sperm survived for up to 48 h when held at 18 °C. Furthermore, the highest post-thawed sperm motility rates were observed in semen frozen after 3 h of holding time, with a sperm total motility of 85.9%, a progressive motility of 60.3%, and a rapid motility of 33.2%, as compared to other holding times (p < 0.05). The acceptable ranges for pre-freeze and post-thawed sperm morphology were head length (8.4-9.1 µm), width (4.4-4.8 µm), area (29.9-38.2 µm2), perimeter (20.1-23.7 µm), midpiece width (1.1-2.8 µm), and sperm shape, were consistent regardless of the holding time. A holding time of 3 h enhances the cryoresistance of sperm cooled at 18 °C. Therefore, these findings suggest that boar epididymal sperm can be effectively conserved and can maintain fertilization capability when cooled for 3 h at 18 °C before freezing.

Abstract The boar has the greatest cumulative impact on swine reproductive performance and drives genetic progress in the herd. However, developing boars may be exposed to agrichemicals directly and/or indirectly via drinking water. Atrazine is an herbicide heavily used in U.S. corn production and an endocrine-disrupting chemical. Atrazine concentrations over 20 µg/L have been detected in groundwater of agricultural states, exceeding the Environmental Protection Agency limit (3 µg/L). This study aims to determine if maternal consumption of environmentally relevant atrazine levels in drinking water affects testicular development in exposed offspring. Pregnant littermate gilts drank water containing either atrazine (20 µg/L; n = 4) or vehicle control [0.002% (v/v) ethanol; n = 3)] ad libitum from 28 days of gestation through parturition (~127 days total). Atrazine-exposed (ATZ; n = 24) and control (n = 12) boars were necropsied on post-natal day 10. Body weight was recorded, and blood was collected for quantification of thyroxine and anti-müllerian hormone (AMH; product of Sertoli cells). Testes were trimmed, weighed, and fixed for histological analysis. Testicular samples stained with hematoxylin and eosin were utilized to quantify interstitial and tubular area, average area of individual seminiferous tubules and the number of seminiferous tubules per field. Data were analyzed via the MIXED procedure of the Statistical Analysis System with a model that included treatment as the fixed effect, boar as the experimental unit, and body weight as a covariate for testis weight. Results indicated an effect of treatment on testis development. For example, the testes of ATZ males were 29% smaller than control boars (2.7±0.2 versus 3.8±0.3 g; P = 0.0021), indicating a reduction in Sertoli cell number. Histological analysis revealed alterations in testis composition. ATZ exposed boars had larger seminiferous tubules than control boars (P = 0.0246), leading to a greater total tubular area (P = 0.0101). Consequently, total interstitial area – a region containing steroidogenic Leydig cells – was reduced in ATZ exposed males (P = 0.0116). Results also indicated an effect of atrazine on endocrine function. For example, serum concentrations of thyroxine were reduced by 9.5% in ATZ boars compared to controls (P = 0.0124). Thyroxine is a known modulator of neonatal Sertoli cell proliferation. In addition, serum AMH concentrations were 52.5% greater in ATZ boars compared with control animals (P = 0.0011). Previous work in other species has linked elevated serum AMH concentrations with poor spermatogenesis and testicular degeneration. These data collectively suggest that maternal ATZ exposure impairs testicular development and Sertoli cell function in offspring. Given that Sertoli cells determine sperm production capacity, these alterations may predispose a lifetime of reproductive failure. Therefore, maternal consumption of environmentally relevant ATZ levels may hinder the development of fertile offspring destined for the boar stud.

A distinctive feature of chlamydia is the often-asymptomatic course of the disease, and without timely, high-quality diagnosis, the pathogen can persist in the body for many months and even years, causing serious diseases and spreading unhindered among animals. The purpose of research is to study the morphological changes in the parenchyma of the testes of wild boar with chlamydia. The objects of the study are the testes of wild boars aged 10–11 months. Histological sections were cut on an MZP-01 technomy microtome with a thickness of 5 microns. The finished histological preparations were stained with hematoxylin-eosin, according to Van Gieson and Pavlovsky. Using microscopy of histological preparations, the structure of the testes and changes in them were studied. Microscopic examinations were carried out with a Micros microscope at a magnification of 200–400 times in 10 fields of view of correctly oriented sections. At least 100 cells were studied. The established numerical data were subjected to variable statistical processing according to Student using MS Excel 2010. When analyzing morphometric studies of the spermatogenic epithelium of the convoluted seminiferous tubules of the testes, it was noted that in the convoluted tubules that retained their structure, the thickness of the spermatogenic epithelium was 14.19 ± 0.23 μm, the number of cells Sertoli are smaller than spermatogonium and spermatocytes. As a result of the studies, we can conclude that in sick animals, already at a young age, the parenchyma is affected with the development of alterative processes, in which there is a decrease in spermatogenesis, which negatively affects the function of the testes, and mechanical transmission of infectious agents during mating is possible.

Bacteria present in fresh extended boar semen may impair the fertility of artificial insemination doses in swine. To date, information regarding the presence or composition of bacterial communities within the boar’s urogenital tract is lacking. These unexplored communities may contribute to the bacterial composition of semen and thereby influence boar fertility. Moreover, hormonal and anatomical changes that occur during puberty could also alter the urogenital tract bacterial communities. Therefore, the objective of this study was to evaluate pre- and post-pubertal shifts in bacterial communities and diversity in boar urogenital tissues (i.e., testis, epididymis, seminal vesicle, prostate, bulbourethral gland, bladder, and preputial diverticulum) using 16S rRNA gene amplicon community sequencing. Crossbred boars were euthanized at 74 ± 2 days of age (pre-pubertal; n = 4) or 276 ± 3 days of age (post-pubertal; n = 6), and intact reproductive tracts were harvested. Sterile swab samples were collected from each tissue of interest for microbiota analysis, and plasma was collected to analyze circulating hormone concentrations of testosterone and dihydrotestosterone. Circulating testosterone was greater (P < 0.01) in post-pubertal boars compared to pre-pubertal boars (3.01 ± 0.26 vs. 0.96 ± 0.36 ng/mL) yet no differences were observed in dihydrotestosterone concentrations. The relative abundance of the phylum Firmicutes was elevated (P < 0.05) in the testis, epididymis, seminal vesicle, bulbourethral gland, and preputial diverticulum of pre-pubertal boars and negatively correlated (P < 0.05) with testosterone. Alternatively, the relative abundance of the phylum Proteobacteria was greater in those same tissues from post-pubertal boars (P < 0.05) and was positively correlated (P < 0.05) with testosterone. Alpha-diversity was reduced in the urogenital tracts of post-pubertal boars compared to pre-pubertal boars (P < 0.01). The bladder had greater alpha-diversity compared to other tissues (P < 0.05). Pre- and post-pubertal boar urogenital tissues have distinct bacterial communities and shifts in these communities following puberty attainment may be associated with elevated testosterone. Future research is warranted to compare bacterial compositions of the boar urogenital tissues to the animal’s ejaculate, which would provide greater insight into the origin of bacteria within boar semen.

BackgroundThe Hezuo (HZ) pig, a famous indigenous breed in China, is characterized by precocious puberty compared with foreign-introduced pig breeds. Sexual maturation is a complex physiological process, and in recent years, circular RNAs (circRNAs), a new class of noncoding RNAs with endogenous regulatory functions, have been shown to play important roles in regulating sexual maturation. However, the dynamic expression and regulatory mechanism of circRNAs during sexual maturation in HZ pigs remain unclear. In this study, we performed RNA sequencing and bioinformatics analysis to reveal circRNA expression patterns in the testes of HZ boars at 30 days (sexual immaturity; Ha) and 120 days (sexual maturity; Hb), with Landrace (LC) boars of the same age (La and Lb) as controls. Subsequently, an abundant circ_005678 (circPAN3) transcribed from the PAN3 gene, was functionally investigated by RT-qPCR, Western Blot, CCK-8, and flow cytometry.ResultsWe identified 31,134 circRNAs in 12 samples, and 2,562, 2,401, 749, and 831 differentially expressed (DE) circRNAs were identified in the Ha-vs-Hb, La-vs-Lb, Ha-vs-La, and Hb-vs-Lb groups, respectively. The results of functional enrichment analyses indicated that these source genes of the DE circRNAs were involved mainly in testicular development and spermatogenesis. Furthermore, we constructed a circRNA-miRNA-mRNA interaction network and functionally analyzed the target genes. GO functional annotation of the target genes suggested that they were mainly involved in biological processes such as gland development, cell proliferation, and reproduction. KEGG pathway analysis further revealed that these genes were enriched mainly in signaling pathways involved in testicular development and spermatogenesis, including the PI3K-Akt and MAPK signaling pathways. Cellular assays revealed that circPAN3 promoted proliferation and inhibited apoptosis in immature Sertoli cells, whereas opposite changes were observed by circPAN3 knockdown.ConclusionsThis study revealed the dynamic expression profiles and regulatory mechanisms of circRNAs during sexual maturation in HZ pigs. Further functional studies demonstrated that circPAN3 promoted the proliferation and inhibited the apoptosis of immature Sertoli cells, suggesting that circPAN3 may be closely related to the characteristics of precocious puberty in HZ boars. These findings provide a new perspective for exploring the regulatory mechanism of circRNAs in precocious puberty in HZ pigs.

Environmental pollution results in serious health hazards to animals, reflected in biogenic and risk element concentrations in animal tissues. The objective of this study was to examinate concentration of selected elements in testes, and epididymal spermatozoa motility of wild boars. Wild boars were hunted in region Žuhračka - Levice, Slovak Republic. Testes were removed postmortem, spermatozoa were collected from cauda epididymis and assessed by CASA system. Elements concentrations were measured by ICP and by CV-AAS. Spermatozoa motility was 44.29% and progressive motility 18.47%. Concentration of elements in testes was in following order: K > Na > Mg > Ca > Fe > Zn > Al > Cu > Se > Mn > As > Cr > Pb > Mo > Sr > Ni > Ba > Cd > Li > Hg. The most notable correlations indicate association between Se and total spermatozoa motility, as well as with progressive motility, furthermore between As and velocity curved line and beat cross frequency. A high positive significant correlation was found between mercury and beat cross frequency. The data may serve as a fine control indicator to detect potentially toxic elements accumulated from polluted environment that can affect reproduction of wild animals.

BackgroundThe health and size of the testes are crucial for boar fertility. Testicular development is tightly regulated by epigenetics. N6-methyladenosine (m6A) modification is a prevalent internal modification on mRNA and plays an important role in development. The mRNA m6A methylation in boar testicular development still needs to be investigated.ResultsUsing the MeRIP-seq technique, we identify and profile m6A modification in boar testes between piglets and adults. The results showed 7783 distinct m6A peaks in piglets and 6590 distinct m6A peaks in adults, with 2,471 peaks shared between the two groups. Enrichment of GO and KEGG analysis reveal dynamic m6A methylation in various biological processes and signalling pathways. Meanwhile, we conjointly analyzed differentially methylated and expressed genes in boar testes before and after sexual maturity, and reproductive related genes (TLE4, TSSK3, TSSK6, C11ORF94, PATZ1, PHLPP1 and PAQR7) were identified. Functional enrichment analysis showed that differential genes are associated with important biological functions, including regulation of growth and development, regulation of metabolic processes and protein catabolic processes.ConclusionThe results demonstrate that m6A methylation, differential expression and the related signalling pathways are crucial for boar testicular development. These results suggest a role for m6A modification in boar testicular development and provided a resource for future studies on m6A function in boar testicular development.

Selenium is counted among the trace elements necessary to maintain metabolic processes occurring in the animal body, including reproductive processes. The aim of the study was to assess selenium content in testes and ovaries of wild boars (Sus scrofa) in an attempt to establish a range of reference values for individuals of this species. Selenium concentrations in tissues tested were determined using spectrofluorometric method after wet mineralization in HNO3 and HClO4 mixture. Based on the results, it was found that male wild boars accumulated significantly higher concentrations of Se than females. The average content of this element in the testes of wild boars was 0.32 mg · kg−1 while in the ovaries it was at the level of 0.14 mg · kg−1 wet weight. In addition, the gonads of males were characterized by a higher variability of the analyzed parameter in relation to samples obtained from female wild boars. The coefficient of variation in their case was almost 40.9, while the value of the coefficient of variation for selenium content in the parenchymal layer of the ovaries was 24.9. On the basis of statistical analysis, highly significant differences were found at p ≤ 0.01 according to the sex of the individuals studied. Despite the fact that samples were taken from individuals living in areas with selenium deficiencies due to the biogeochemical background, it should be considered that the supply of Se is optimal, which is evident in the population growth dynamics of individuals of this species in the study area.

Efficient isolation and purification of spermatogonia, spermatocytes, and spermatids from mice, piglets, and adult boars using an optimized STA-PUT method

In a previous study, our group detected the cholecystokinin (CCK) protein in the porcine oviduct. This fact, together with the involvement of CCK in the regulation of sperm protein tyrosine phosphorylation by the modulation of HCO3 - uptake (in mice and humans) suggests a role for CCK during sperm capacitation. Therefore, on the one hand, the expression of CCK receptors (CCK1R and CCK2R) on boar testes has been investigated and probed; on the other hand, boar spermatozoa (from seminal doses of 1-day and 5-day storage) were exposed to different concentrations of CCK (0-control, 25 or 50 μM) in a medium supporting capacitation supplemented with 0, 5 or 25 mmol/L of HCO3 - for 1 h at 38.5°C. Sperm motion (total and progressive motility), kinetic parameters, viability, acrosome status, and mitochondrial activity were determined. No differences between groups (0, 25 or 50 μM of CCK) were observed when HCO3 - was absent in the media (p > .05). However, the results showed that when the media was supplemented with 5 mmol/L HCO3 - in 1-day seminal dose storage, the linearity index (LIN, %), straightness index (STR, %) and oscillation index (WOB, %) (sperm kinetics parameters) increased in the presence of CCK regardless the concentration (p < .05). Nevertheless, CCK in sperm from 5-day storage only increased the WOB parameter in comparison to the control (p < .05). Furthermore, the average amplitude of the lateral displacement of the sperm head (ALH, μm) and curvilinear velocity (VCL, μm/s) decreased when CCK was present, depending on its concentration and sperm aging (1-day vs. 5-days) (p < .05). In the case of the media supporting capacitation supplemented with 25 mmol/L HCO3 - , any differences were observed except for sperm viability in the 5-day seminal doses, which increased in the 50 μM-CCK group compared to the control (p < .05). In conclusion, these data suggest an implication of CCK protein during sperm capacitation under low bicarbonate concentration increasing the sperm linear trajectory.

Macrophage presence and location were evaluated in juvenile boar testes at the end of the first wave of Sertoli cell proliferation. Macrophage presence was compared in littermate boars treated with letrozole, a treatment which extended this first wave of proliferation beyond the sampling timepoint. Macrophages were identified as the CD68 positive cells following immunohistochemical labelling of paraffin sections and parenchymal macrophages enumerated. Macrophages present in a layer beneath the tunica albuginea received a score based on density and thickness of this layer. Density within the testicular parenchyma was highly variable in vehicle-treated boars (>100-fold) and did not differ from that observed in the letrozole-treated littermates. However, the macrophage layer beneath the tunica albuginea was denser and thicker in the letrozole-treated animals than in their vehicle-treated littermates. This suggests that macrophages might be involved in the letrozole-induced prolongation of Sertoli cell proliferation.

Precocious puberty is closely related to testicular development and spermatogenesis, and there is increasing evidence that miRNAs are involved in regulation of testicular development and spermatogenesis. However, little is known about the regulation of microRNAs (miRNAs) during precocious maturation in Hezuo (HZ) boars. In this study, serum Testosterone (T), Estradiol (E2), Follicle-stimulating hormone (FSH) and Luteinizing hormone (LH) levels were detected in HZ and Landrace (LC) boars in the postnatal period at 30, 90, 120, 180, and 240 days, and the testes of HZ and LC boars at 30 and 120 days were used for histological observation. In addition, we performed small RNA-Seq to identify miRNA at sexual immaturity (30-days-old) and maturity (120-days-old) of HZ boar testis (using LC boar as control) to reveal the key miRNA in regulation of precocious puberty. Hormone assay results showed that high levels of T, E2, FSH, and LH may be related to precocious sexual maturity of HZ boars, and that FSH may play an important function before sexual maturity. Histological observation showed that HZ boars developed earlier than LC boars and had reached sexual maturity at 120 days. Small RNA-Seq yielded a total of 359 exist miRNAs, 767 known miRNAs and 322 novel miRNAs in 12 samples; 549, 468, 133, and 247 differentially expressed (DE) miRNAs were identified between Ha vs. Hb, La vs. Lb, Ha vs. La, and Hb vs. Lb (log2 fold change >1 and p < 0.05). Enrichment analysis showed that target genes of these DE miRNAs were enriched in many gene ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) signaling pathways (such as PI3K-Akt, Hippo and Rap1 signaling pathways) were related to testicular development and spermatogenesis. Further screening, some miRNAs (such as ssc-miR-29b, ssc-miR-199b, ssc-miR-383, ssc-miR-149, ssc-miR-615, and ssc-miR-370) were possibly associated with precocious puberty. These results provide new light on miRNA regulatory mechanisms involved in precocious puberty.

The hepatitis E virus (HEV) is the main cause of viral acute hepatitis in the world, affecting more than 20 million people annually. During the acute phase of infection, HEV can be detected in various body fluids, which has a significant impact in terms of transmission, diagnosis or extrahepatic manifestations. Several studies have isolated HEV in the genitourinary tract of humans and animals, which could have important clinical and epidemiological implications. So, our main objective was to evaluate the presence of HEV in testis of naturally infected wild boars (Sus scrofa). For it, blood, liver, hepatic lymph node and testicle samples were collected from 191 male wild boars. The presence of HEV was evaluated in serum by PCR, as well as in tissues by PCR and immunohistochemistry. Four animals (2.09%; 95%CI: 0.82-5.26) showed detectable HEV RNA in serum, being confirmed the presence of HEV-3f genotype in three of them by phylogenetic analysis. HEV was also detected in liver and/or hepatic lymph nodes of the four animals by RT-PCR, as well as by immunohistochemistry analysis. Only one of these wild boars also showed detectable viral load in testis, observing HEV-specific labelling in a small number of fibroblasts and some Sertoli cells. Our results confirm the presence of HEV genotype 3 in naturally infected wild boar testis, although no associated tissue damage was evidenced. This study does not allow us to discard semen as a possible source of HEV transmission in suids. Future experimental studies are necessary to evaluate the impact of HEV genotype 3 on fertility and the possibility of transmission through sexual contact in this specie.

MeDIP-seq and RNA-seq analysis during porcine testis development reveals functional DMR at the promoter of LDHC

Testicular parenchyma is split into lobules, each lobule contains convoluted seminiferous tubules surrounded by myoid cells and the interstitial tissue contains groups of Leydig cells. The seminiferous tubules are lined by two groups of cells the first one is the spermatogenic cells and the second one is Sertoli cells.