MicroRNA expression in specific segments of the pig periovulatory internal genital tract is differentially regulated by semen or by seminal plasma.

microRNAs play pivotal roles during mammalian reproduction, including the cross-talk between gametes, embryos and the maternal genital tract. Mating induces changes in the expression of mRNA transcripts in the female, but whether miRNAs are involved remains to be elucidated. In the current study, we mapped 181 miRNAs in the porcine peri-ovulatory female reproductive tract: Cervix (Cvx), distal and proximal uterus (Dist-Ut, Prox-Ut), Utero-tubal-junction (UTJ), isthmus (Isth), ampulla (Amp), and infundibulum (Inf) when exposed to semen (natural mating (NM) or artificial insemination (AI-P1)) or to infusions of sperm-free seminal plasma (SP): the first 10 mL of the sperm rich fraction (SP-P1) or the entire ejaculate (SP-E). Among the most interesting findings, NM decreased mir-671, implicated in uterine development and pregnancy loss prior to embryo implantation, in Cvx, Dist-UT, Prox-UT, Isth, and Inf, while it increased in Amp. NM and SP-E induced the downregulation of miR-let7A-1 (Dist-UT, Prox-UT), a regulator of immunity during pregnancy. miR-34C-1, a regulator of endometrial receptivity gene expression, was increased in Dist-UT, UTJ and Amp (NM), in Prox-UT (AI-P1), and in Amp (SP-P1). miR-296, a modulator of the inflammatory response and apoptosis, was upregulated in the UTJ (all treatments). NM elicited the highest miRNA activity in the sperm reservoir (UTJ), suggesting that key-regulators such as miR-34c or miR-296 may modulate the metabolic processes linked to the adequate preparation for gamete encounter in the oviduct. Our results suggest that SP should be maintained in AI to warrant miRNA regulation within the female genital tract for reproductive success.

The landscape of long noncoding RNA expression in the goat brain.

The brain regulates multiple metabolic processes, such as food intake, energy expenditure, insulin secretion, hepatic glucose production, and glucose and fatty acid metabolism in adipose tissue, which are fundamental for the maintenance of energy and glucose homeostasis during lactation and pregnancy. In addition, brain expression has a fundamental impact on the development of maternal behavior. Although brain functions are partly regulated by long noncoding RNAs (lncRNAs), their expression profiles have not been characterized in depth in any ruminant species. We have sequenced the transcriptome of 12 brain tissues from 3 goats that were 1 mo pregnant and 4 nonpregnant goats to investigate their lncRNA expression patterns. Between 4,363 (adenohypophysis) and 4,604 (olfactory bulb) lncRNAs were expressed in brain tissues, leading us to establish a set of 794 already annotated lncRNAs and 5,098 novel lncRNA candidates. The detected lncRNAs shared features with those of other mammals, and tissue-specific lncRNAs were enriched in brain development-related terms. Differential expression analyses between goats that were 1 mo pregnant and nonpregnant goats showed that the lncRNA expression profiles of certain brain regions experience substantial changes associated with early pregnancy (238 lncRNAs are differentially expressed in the olfactory bulb), but others do not. Enrichment analysis showed that differentially expressed lncRNAs from the olfactory bulb are co-expressed with genes previously linked to behavioral changes related to pregnancy. These findings provide a first characterization of the landscape of lncRNA expression in the goat brain and provides valuable clues to understand the molecular events triggered by early pregnancy in the central nervous system.

Effect of Honey, Coenzyme Q10, and ß-Carotene/α-Tocopherol as Novel Additives in Rabbit-Sperm Cryopreservation Extender.

The effectiveness of rabbit-sperm cryopreservation is still below average compared to other domestic species. After the sperm cryopreservation process, post-thawing parameters like motility and membrane integrity are significantly compromised. The use of new extender constituents is an approach that can be used to improve the effectiveness of cryopreservation. Accordingly, we used honey (1.25, 2.5, 5, and 10%), coenzyme Q10 (100 and 200 µM), and ß-carotene/α-tocopherol (500 µM/620 µM and 250 µM/310 µM) as candidate components for rabbit-sperm extenders during cryopreservation. Ejaculates from commercial adult rabbit bucks (n = 5) were cryopreserved using conventional freezing. Several post-thawing sperm parameters were assessed, including total motility, membrane integrity, viability, nuclear membrane integrity, acrosome reaction, and mitochondrial membrane potential and activation. Additionally, we performed hormonal analyses of the seminal plasma. Moreover, we analyzed the post-thawing levels of a molecular marker of sperm quality, proAKAP4, which was used in rabbits for the first time. Our findings showed that the 2.5% honey supplementation increased the post-thawing sperm motility (13.75 ± 3.75%) compared to the greater concentrations employed. However, the post-thawing motility was negatively affected by the coenzyme Q10 (0%, in both groups) but was not affected by the ß-carotene/α-tocopherol supplementation (22 ± 18.15%, and 11.67 ± 10.17%). In conclusion, the cryopreservation protocols of this study did not help to maintain the sperm parameters after thawing. Further studies are required to identify novel protocols to mitigate the damage caused to rabbit sperm during cryopreservation.

A protein-coding gene expression atlas from the brain of pregnant and non-pregnant goats.

Background: The brain is an extraordinarily complex organ with multiple anatomical structures involved in highly specialized functions related with behavior and physiological homeostasis. Our goal was to build an atlas of protein-coding gene expression in the goat brain by sequencing the transcriptomes of 12 brain regions in seven female Murciano-Granadina goats, from which three of them were 1-month pregnant. Results: Between 14,889 (cerebellar hemisphere) and 15,592 (pineal gland) protein-coding genes were expressed in goat brain regions, and most of them displayed ubiquitous or broad patterns of expression across tissues. Principal component analysis and hierarchical clustering based on the patterns of mRNA expression revealed that samples from certain brain regions tend to group according to their position in the anterior-posterior axis of the neural tube, i.e., hindbrain (pons and medulla oblongata), midbrain (rostral colliculus) and forebrain (frontal neocortex, olfactory bulb, hypothalamus, and hippocampus). Exceptions to this observation were cerebellum and glandular tissues (pineal gland and hypophysis), which showed highly divergent mRNA expression profiles. Differential expression analysis between pregnant and non-pregnant goats revealed moderate changes of mRNA expression in the frontal neocortex, hippocampus, adenohypophysis and pons, and very dramatic changes in the olfactory bulb. Many genes showing differential expression in this organ are related to olfactory function and behavior in humans. Conclusion: With the exception of cerebellum and glandular tissues, there is a relationship between the cellular origin of sampled regions along the anterior-posterior axis of the neural tube and their mRNA expression patterns in the goat adult brain. Gestation induces substantial changes in the mRNA expression of the olfactory bulb, a finding consistent with the key role of this anatomical structure on the development of maternal behavior.

Anthrax bio-surveillance of livestock in Arua District, Uganda, 2017-2018.

Anthrax, caused by Bacillus anthracis, is a widespread zoonotic disease with many human cases, especially in developing countries. Even with its global distribution, anthrax is a neglected disease with scarce information about its actual impact on the community level. Due to the ecological dynamics of anthrax transmission at the wildlife-livestock interface, the Sub-Saharan Africa region becomes a high-risk zone for maintaining and acquiring the disease. In this regard, some subregions of Uganda are endemic to anthrax with regular seasonal trends. However, there is scarce data about anthrax outbreaks in Uganda. Here, we confirmed the presence of B. anthracis in several livestock samples after a suspected anthrax outbreak among livestock and humans in Arua District. Additionally, we explored the potential risk factors of anthrax through a survey within the community kraals. We provide evidence that the most affected livestock species during the Arua outbreak were cattle (86%) compared to the rest of the livestock species present in the area. Moreover, the farmers' education level and the presence of people's anthrax cases were the most critical factors determining the disease's knowledge and awareness. Consequently, the lack of understanding of the ecology of anthrax may contribute to the spread of the infection between livestock and humans, and it is critical to reducing the presence and persistence of the B. anthracis spores in the environment. Finally, we discuss the increasingly recognized necessity to strengthen global capacity using a One Health approach to prevent, detect, control, and respond to public threats in Uganda.

NR3C1 and glucocorticoid-regulatory genes mRNA and protein expression in the endometrium and ampulla during the bovine estrous cycle.

The bovine reproductive tract exhibits changes during the estrous cycle modulated by the interplay of steroid hormones. Glucocorticoids can be detrimental when stress-induced but are relevant at baseline levels for appropriate reproductive function. Here, an analysis of quantitative real-time PCR was performed to study the bovine glucocorticoid-related baseline gene transcription in endometrial and ampullar tissue samples derived from three time points of the estrous cycle, stage I (Days 1-4), stage III (Days 11-17) and stage IV (Days 18-20). Our results revealed expression differences during stages, as expression observed in the ampulla was higher during the post-ovulatory phase (stage I), including the glucocorticoid receptor NR3C1, and some of its regulators, involved in glucocorticoid availability (HSD11B1 and HSD11B2) and transcriptional actions (FKBP4 and FKBP5). In contrast, in the endometrium, higher expression of the steroid receptors was observed during the late luteal phase (stage III), including ESR1, ESR2, PGRMC1 and PGRMC2, and HSD11B1 expression decreased, while HSD11B2 increased. Moreover, at protein level, FKBP4 was higher expressed during the late luteal phase, and NR3C1 during the pre-ovulatory phase (stage IV). These results suggest that tight regulation of the glucocorticoid activity is promoted in the ampulla, when reproductive events are taking place, including oocyte maturation. Moreover, most expression changes in the endometrium were observed during the late luteal phase, and may be related to the embryonic maternal recognition. In conclusion, the glucocorticoid regulation changes across the estrous cycle and may be playing a role on the reproductive events occurring in the bovine ampulla and endometrium.

Apoptosis and glucocorticoid-related genes mRNA expression is modulated by coenzyme Q10 supplementation during in vitro maturation and vitrification of bovine oocytes and cumulus cells.

Oocyte in vitro maturation (IVM) and vitrification procedures lead to detrimental effects on the overall oocyte quality. The addition of antioxidants during IVM, such as the coenzyme Q10 (Q10), has been demonstrated to positively impact on the cumulus-oocyte complexes due to its role in protection from oxidative damage and modulating gene transcription. Furthermore, glucocorticoids (GC) regulate gene transcription, energy metabolism and apoptosis during the early steps of reproduction. In this sense, most GC actions are mediated by the glucocorticoid receptor (NR3C1), a transcription factor. However, the specific roles of GC in ovarian physiology and oocyte maturation are still unknown. In this regard, a better knowledge on the expression of GC-related and apoptosis-related genes during IVM and cryopreservation procedures could potentially benefit the refinement of assisted reproductive techniques in the bovine species. The present study aims to explore the expression of NR3C1 mRNA in fresh and vitrified bovine oocytes and cumulus cells in response to Q10 (50 µM), and the effect of cortisol addition (0.25 µM, 0.5 µM) on the expression of NR3C1. We also studied the mRNA expression of NR3C1-related genes belonging to the GC regulation pathway, such as hydroxysteroid dehydrogenases (HSD11B1; HSD11B2), immunophilins (FKBP4; FKBP5), signal transducers and activators of transcription (STAT3; STAT5A), the mineralocorticoid receptor (NR3C2), and to the apoptosis pathway, such as the anti- (BCL2) and pro-apoptotic (BAX) mRNA transcripts in oocytes and cumulus cells 1) after IVM, and 2) after vitrification, both in presence or absence of Q10 supplementation during IVM. Our results show that there is an increase in the NR3C1 receptor expression after vitrification of oocytes, but not after exogenous cortisol supplementation during IVM. In addition, Q10 reduces the mRNA expression of HSD11B1 and FKBP5 in oocytes at levels of immature oocytes (HSD11B1 mRNA expression also in cumulus cells), and the BAX:BCL2 ratio mRNA expression. After vitrification in the presence of Q10, HSD11B2 mRNA expression increases in cumulus cells, while HSD11B1 and BAX:BCL2 mRNA expression decreases significantly both in oocytes and cumulus cells. In conclusion, our results show for the first time the effect of IVM, vitrification and Q10 supplementation on the mRNA relative expression of GC-related and apoptosis genes, and the effect of vitrification in the protein expression of NR3C1.

The mRNA expression of the three major described cold-inducible proteins, including CIRBP, differs in the bovine endometrium and ampulla during the estrous cycle.

The cold-inducible proteins (CIPs) are essential for post-transcriptional gene regulation playing diverse tissue-specific roles in maintaining normal cellular function and morphogenesis. The potential implications of CIPs in reproductive events raise questions about their role in the physiology of the bovine reproductive tract. However, the expression changes of CIPs during the bovine estrous cycle have not been studied so far. Here, we hypothesized that the bovine estrous cycle could affect the mRNA expression of the CIPs and other candidate transcripts in the reproductive tract. This study aimed to examine estrous cycle-dependent mRNA expression patterns in the bovine endometrium and ampulla of three of the major described CIPs (CIRBP, RBM3, SRSF5), a set of inflammatory cytokines (IL-10, IL-18, IL-1ß), and other candidate genes (IL-10RA, IL-10RB, BCL2, NLRP3, STAT1, STAT3, STAT5A, STAT6). Endometrial and ampullar tissues were assessed by RT-qPCR. Additionally, the mRNA expression levels were correlated among them and with follicular progesterone and estradiol concentrations. The transcript levels of CIPs increased in the endometrium during stage III (Days 11-17) compared to stage I (Days 1-4) and IV (Days 18-20). In the ampulla, the mRNA expression of CIRBP increased during the late luteal phase (stage III), but no differences in the expression of other CIPs were observed. This study expands the current knowledge regarding mRNA expression in the endometrium and oviductal ampulla of cycling heifers, focusing mainly on the CIPs. A better understanding of the mechanisms within the uterus and oviduct during the estrous cycle is crucial to improving the fertility rate.

Changes in aquaporins mRNA expression and liquid storage at 17°C: A potential biomarker of boar sperm quality?

Artificial insemination (AI) for pigs relies on liquid storage of extended semen at 17°C, which preserves sperm quality and ensures its fertilizing capacity. Routine quality controls include the evaluation of sperm motility, viability and capacitation status. The physiological functions of all these features depend on transmembrane aquaporins (AQPs), proteins playing key roles in osmoadaptation. In this study, we made a relative quantification, using RT-qPCR, of the mRNA of several sperm AQPs in AI-liquid semen doses before and after a 48-hr incubation period, aiming to determine possible quantitative compromising expression changes during the process that could serve as a diagnostic tool. Our results showed a decrease in classical sperm motility variables (total and progressive motility and velocity) and sperm viability after 48-hr storage, whereas capacitation status increased overtime. mRNA expression increased in the orthodox AQP4 and AQP6 after 48-hr incubation, relative to control (0 hr) and 24-hr time-points. Moreover, mRNA expression of aquaglyceroporins AQP3, AQP7 and AQP10 was higher after 48-hr incubation, confirmed by AQP7-protein validation using Western blot. Our results indicate that expression levels of AQPs-mRNA can change in ejaculated pig spermatozoa under conditions of ex-vivo incubation that could modify sperm homeostasis, suggesting it could eventually become a relevant molecular biomarker to assess the efficiency of liquid storage of pig semen.

Semen Modulates Cell Proliferation and Differentiation-Related Transcripts in the Pig Peri-Ovulatory Endometrium.

Uterine homeostasis is maintained after mating by eliminating pathogens, foreign cells, and proteins by a transient inflammation of the uterus. Such inflammation does not occur in the oviductal sperm reservoir (utero-tubal junction, UTJ), colonized by a population of potentially fertile spermatozoa before the inflammatory changes are triggered. Semen entry (spermatozoa and/or seminal plasma) modifies the expression of regulatory genes, including cell proliferation and differentiation-related transcripts. Considering pigs display a fractionated ejaculation, this study aims to determine whether different ejaculate fractions differentially modulate cell proliferation and differentiation-related transcripts in the sow reproductive tract during the peri-ovulatory stage. Using species-specific microarray analyses, the differential expression of 144 cell proliferation and differentiation-related transcripts was studied in specific segments: cervix (Cvx), distal and proximal uterus (DistUt, ProxUt), UTJ, isthmus (Isth), ampulla (Amp), and infundibulum (Inf) of the peri-ovulatory sow reproductive tract in response to semen and/or seminal plasma cervical deposition. Most mRNA expression changes were induced by mating. In addition, while mating upregulates the fibroblast growth factor 1 (FGF1, p-value DistUt = 0.0007; ProxUt = 0.0253) transcript in the endometrium, both its receptor, the fibroblast growth factor receptor 1 (FGFR1, p-value DistUt = 2.14 e-06; ProxUt = 0.0027; UTJ = 0.0458) transcript, and a potentiator of its biological effect, the fibroblast growth factor binding protein 1 (FGFBP1), were downregulated in the endometrium (p-value DistUt = 0.0068; ProxUt = 0.0011) and the UTJ (p-value UTJ = 0.0191). The FGFBP1 was downregulated in the whole oviduct after seminal depositions (p-value Isth = 0.0007; Amp = 0.0007; Inf = 6.87 e-05) and, interestingly, FGFR1 was downregulated in the endometrium in the absence of semen (p-value DistUt = 0.0097; ProxUt = 0.0456). In conclusion, the findings suggest that spermatozoa, seminal components, and the act of mating trigger, besides inflammation, differential mechanisms in the peri-ovulatory female reproductive tract, relevant for tissue repair.

Mild hypothermia and vitrification increase the mRNA expression of cold-inducible proteins in bovine oocytes and cumulus cells.

The cold-inducible RNA-binding protein (CIRBP) assists cells in adapting to new environmental conditions stabilizing specific mRNAs and promoting their translation. CIRBP participates in anti-apoptotic and anti-senescence processes, and its expression is induced by mild hypothermia, which may be advantageous to oocytes during vitrification. Several newly discovered small molecules, like zr17-2, mimic the effects of cold temperatures by increasing the expression of CIRBP at normothermia. This study aimed to evaluate the mRNA changes of a group of cold-inducible protein-encoding and apoptotic genes in response to exogenous supplementation of zr17-2 (experiment 1) or CIRBP (experiment 2) in vitro matured bovine oocytes and their cumulus cells. In experiment 1, cumulus-oocyte complexes (COCs) were randomly exposed to three concentrations of zr17-2 (1, 10, and 100 µM) during 24 h of in vitro maturation (IVM) under normothermia (38.5 °C) or mild hypothermia (34 °C) culture conditions. In experiment 2, COCs were randomly exposed to three concentrations of CIRBP (2, 4, and 6 µg/mL) or subjected to mild hypothermia (34 °C), followed by oocyte vitrification/warming after 20 h of IVM. The quantification of the selected gene transcript expression was performed separately in oocytes and cumulus cells by quantitative real-time PCR. We show that oocytes and cumulus cells exhibited similar mRNA expression responses to mild hypothermia and vitrification. However, minor differences were observed when COCs were exposed to exogenous supplementation with zr17-2 and CIRBP. In conclusion, the alterations observed in the mRNA expression in these experimental conditions may impact the quality of the cumulus-oocyte complexes in terms of vitrification and sublethal hypothermia challenges. In this sense, our results should complement other oocyte quality assessments for its application in future assisted reproductive techniques in the bovine species, including improving oocyte cryotolerance to vitrification.

The Role of Aquaporin 7 in the Movement of Water and Cryoprotectants in Bovine In Vitro Matured Oocytes.

Aquaglyceroporins are known as channel proteins, and are able to transport water and small neutral solutes. In this study, we evaluate the effect of exposure of in vitro matured bovine oocytes to hyperosmotic solutions containing ethylene glycol (EG), dimethyl sulfoxide (Me2SO) or sucrose on the expression levels of AQP3, AQP7 and AQP9. Moreover, we studied whether artificial protein expression of AQP7 in bovine oocytes increases their permeability to water and cryoprotectants. Exposure to hyperosmotic solutions stimulated AQP3 and AQP7 but not AQP9 expression. Oocytes exposed to hyperosmotic Me2SO solution exhibited upregulated AQP3 expression, while AQP7 expression was upregulated by EG hyperosmotic exposure. Microinjection of oocytes at the germinal vesicle stage with enhanced green fluorescent protein (EGFP) or EGFP+AQP7 cRNAs resulted in the expression of the corresponding proteins in ≈86% of the metaphase-II stage oocytes. AQP7 facilitated water diffusion when bovine MII oocytes were in presence of Me2SO solution but not EG or sucrose solution. However, the overexpression of this aquaporin did not increase membrane permeability to Me2SO or EG. In summary, cryoprotectant-induced increase of AQP3 and AQP7 expression could be one of the mechanisms underlying oocyte tolerance to hyperosmotic stress. Water diffusion appears to be improved when AQP7 overexpressed oocytes are exposed to Me2SO, shortening the time required for oocytes to achieve osmotic balance with cryoprotectant solutions.

Hair Cortisol, Testosterone, Dehydroepiandrosterone Sulfate and Their Ratios in Stallions as a Retrospective Measure of Hypothalamic-Pituitary-Adrenal and Hypothalamic-Pituitary-Gonadal Axes Activity: Exploring the Influence of Seasonality.

The monitoring of stress physiology includes studying a wide range of endocrinological mechanisms, which can be assessed using multiple tissue samples. This study aimed to evaluate the seasonal variations of hair C, T and DHEA-S in horses for a whole year, as well as to assess the variations between seasons of C/DHEA-S and T/C ratios as a retrospective measure of the hypothalamic-pituitary-adrenal and hypothalamic-pituitary-gonadal axis activity. Ten pure-breed Menorca stallions were included in the study. The hair samples were collected approximately every two months following the shave-reshave method caudally to the sternum. After a methanol-based extraction, samples were analyzed by enzyme immunoassay for cortisol, testosterone, and dehydroepiandrosterone sulphate. Following our findings, we detected that cortisol, testosterone and dehydroepiandrosterone sulphate were significantly affected by seasonality, with the highest values of cortisol during summer and the lowest values of testosterone during spring. Dehydroepiandrosterone sulphate concentrations were increased in autumn compared to the other studied periods. Additionally, the studied hormone ratios showed variations between seasons. To conclude, season should, therefore, be considered when assessing sexual and stress hormones in stallion hair, since this variable can be a potential influencing factor and led to misinterpretations.

Semen Modulates Inflammation and Angiogenesis in the Reproductive Tract of Female Rabbits.

The maternal environment modulates immune responses to facilitate embryo development and ensure pregnancy. Unraveling this modulation could improve the livestock breeding systems. Here it is hypothesized that the exposure of the female rabbit reproductive tract to semen, as well as to early embryos, modulates inflammation and angiogenesis among different tissue segments. qPCR analysis of the gene expression changes of the anti-inflammatory interleukin-10 (IL10) and transforming growth factor beta family (TGFß1-3) and the angiogenesis mediator vascular endothelial growth factor (VEGF-A) were examined in response to mating or insemination with sperm-free seminal plasma (SP). Reproductive tract segment (cervix to infundibulum) samples were obtained in Experiment 1, 20 h after gonadotropin-releasing hormone (GnRH) stimulation (control), natural mating (NM) or vaginal infusion with sperm-free SP (SP-AI). Additionally, segmented samples were also obtained at 10, 24, 36, 68 or 72 h after GnRH-stimulation and natural mating (Experiment 2). The results of gene expression, analyzed by quantitative PCR, showed that NM effects were mainly localized in the uterine tissues, depicting clear temporal variation, while SP-AI effects were restricted to the oviduct. Changes in anti-inflammatory and angiogenesis mediators indicate an early response in the uterus and a late modulation in the oviduct either induced by semen or preimplantation embryos. This knowledge could be used in the implementation of physiological strategies in breeding systems to face the new challenges on rabbit productivity and sustainability.

Seminal Plasma Triggers the Differential Expression of the Glucocorticoid Receptor (NR3C1/GR) in the Rabbit Reproductive Tract.

Rabbits are interesting as research animal models for reproduction, due to their condition of species of induced ovulation, with the release of endogenous gonadotropin-releasing hormone (GnRH) due to coitus. Glucocorticoid (GC) signaling, crucial for physiological homeostasis, is mediated through a yet unclear mechanism, by the GC receptor (NR3C1/GR). After mating, the female reproductive tract undergoes dynamic modifications, triggered by gene transcription, a pre-amble for fertilization and pregnancy. This study tested the hypothesis that when ovulation is induced, the expression of NR3C1 is influenced by sperm-free seminal plasma (SP), similarly to after mating (whole semen), along the different segments of the internal reproductive tract of female rabbits. Semen (mating) was compared to vaginal infusion of sperm-free SP (Experiment 1), and changes over time were also evaluated, i.e., 10, 24, 36, 68, and 72 h post-mating, corresponding to specific stages, i.e., ovulation, fertilization, and the interval of early embryo development up to the morula stage (Experiment 2). All does were treated with GnRH to induce ovulation. Samples were retrieved from seven segments of the reproductive tract (from the cervix to infundibulum), at 20 h post-mating or sperm-free SP infusion (Experiment 1) or at 10, 24, 36, 68, and 72 h post-mating (Experiment 2). Gene expression of NR3C1 was analyzed by qPCR. Results showed an increase in NR3C1 expression in the infundibulum compared to the other anatomical regions in the absence of spermatozoa when sperm-free SP infusion was performed (Experiment 1). Moreover, during the embryo transport through the oviduct, the distal isthmus was time-course upregulated, especially at 72 h, when morulae are retained in this anatomical region, while it was downregulated in the distal uterus at 68 h (Experiment 2). The overall results suggest that NR3C1, the GC receptor gene, assessed in the reproductive tract of does for the first time, shows differential expression changes during the interval of oviductal and uterine embryo transport that may imply a relevant role of the GC action, not only close to the site of ovulation and fertilization, but also in the endometrium.

The Expression of Cold-Inducible RNA-Binding Protein mRNA in Sow Genital Tract Is Modulated by Natural Mating, But Not by Seminal Plasma.

The RNA-binding proteins (RBPs), some of them induced by transient receptor potential (TRP) ion channels, are crucial regulators of RNA function that can contribute to reproductive pathogenesis, including inflammation and immune dysfunction. This study aimed to reveal the influence of spermatozoa, seminal plasma, or natural mating on mRNA expression of RBPs and TRP ion channels in different segments of the internal genital tract of oestrous, preovulatory sows. Particularly, we focused on mRNA expression changes of the cold-inducible proteins (CIPs) and related TRP channels. Pre-ovulatory sows were naturally mated (NM) or cervically infused with semen (Semen-AI) or sperm-free seminal plasma either from the entire ejaculate (SP-TOTAL) or the sperm-rich fraction (SP-AI). Samples (cervix to infundibulum) were collected by laparotomy under general anaesthesia for transcriptomic analysis (GeneChip® Porcine Gene 1.0 ST Array) 24 h after treatments. The NM treatment induced most of the mRNA expression changes, compared to Semen-AI, SP-AI, and SP-TOTAL treatments including unique significative changes in CIRBP, RBM11, RBM15B, RBMS1, TRPC1, TRPC4, TRPC7, and TRPM8. The findings on the differential mRNA expression on RBPs and TRP ion channels, especially to CIPs and related TRP ion channels, suggest that spermatozoa and seminal plasma differentially modulated both protein families during the preovulatory phase, probably related to a still unknown early signalling mechanism in the sow reproductive tract.

Semen Modulates the Expression of NGF, ABHD2, VCAN, and CTEN in the Reproductive Tract of Female Rabbits.

Semen changes the gene expression in endometrial and oviductal tissues modulating important processes for reproduction. We tested the hypothesis that mating and/or sperm-free seminal plasma deposition in the reproductive tract affect the expression of genes associated with sperm-lining epithelium interactions, ovulation, and pre-implantation effects (nerve growth factor, NGF; α/ß hydrolase domain-containing protein 2, ABHD2; C-terminal tensin-like protein, CTEN or TNS4; and versican, VCAN) in the period 10-72 h post-mating. In Experiment 1, does (n = 9) were treated with gonadotropin-releasing hormone (GnRH) (control), GnRH-stimulated, and vaginally infused with sperm-free seminal plasma (SP-AI), or GnRH-stimulated and naturally mated (NM). In Experiment 2, does (n = 15) were GnRH-stimulated and naturally mated. Samples were retrieved from the internal reproductive tracts (cervix-to-infundibulum) 20 h post-treatment (Experiment 1) or sequentially collected at 10, 24, 36, 68, or 72 h post-mating (Experiment 2, 3 does/period). All samples were processed for gene expression analysis by quantitative PCR. Data showed an upregulation of endometrial CTEN and NGF by NM, but not by SP-AI. The findings suggest that the NGF gene affects the reproductive tract of the doe during ovulation and beyond, influencing the maternal environment during early embryonic development.

Natural Mating Differentially Triggers Expression of Glucocorticoid Receptor (NR3C1)-Related Genes in the Preovulatory Porcine Female Reproductive Tract.

Mating initiates dynamic modifications of gene transcription in the female reproductive tract, preparing the female for fertilization and pregnancy. Glucocorticoid signaling is essential for the homeostasis of mammalian physiological functions. This complex glucocorticoid regulation is mediated through the glucocorticoid receptor, also known as nuclear receptor subfamily 3 group C member 1 (NR3C1/GR) and related genes, like 11ß-hydroxysteroid dehydrogenases (HSD11Bs) and the FK506-binding immunophilins, FKBP5 and FKBP4. This study tested the transcriptome changes in NR3C1/GR regulation in response to natural mating and/or cervical deposition of the sperm-peak ejaculate fraction collected using the gloved-hand method (semen or only its seminal plasma), in the preovulatory pig reproductive tract (cervix to infundibulum, 24 h after mating/insemination/infusion treatments). Porcine cDNA microarrays revealed 22 NR3C1-related transcripts, and changes in gene expression were triggered by all treatments, with natural mating showing the largest differences, including NR3C1, FKBP5, FKBP4, hydroxysteroid 11-beta dehydrogenase 1 and 2 (HSD11B1, HSD11B2), and the signal transducer and activator of transcription 5A (STAT5A). Our data suggest that natural mating induces expression changes that might promote a reduction of the cortisol action in the oviductal sperm reservoir. Together with the STAT-mediated downregulation of cytokine immune actions, this reduction may prevent harmful effects by promoting tolerance towards the spermatozoa stored in the oviduct and perhaps elicit spermatozoa activation and detachment after ovulation.

Temporary Relocation during Rest Periods: Relocation Stress and Other Factors Influence Hair Cortisol Concentrations in Horses.

Horse transportation for temporary relocation during rest periods is a common and widespread practice among horse owners, either from sport competition or working tasks. This study aimed to determine the effect of a relocation period and the multiple factors associated with a rest period on hair cortisol concentrations (HCCs) in horses. Additionally, this study reports the seasonal effect on HCCs and hair growth over a year. Thirteen police horses, Pure Spanish stallions of various ages (5-13 y), were selected to participate in this study. Hair sample collection was carried out approximately every 30 d for seven months (Study 1) and a year (Study 2). Cortisol determinations were performed by enzyme immunoassay. Interestingly, Study 1 revealed that relocated horses (n = 4) exhibited elevated HCCs compared with control horses (n = 4) after the relocation period (p < 0.05). Study 2 (n = 5) showed higher HCCs during summer compared with autumn and winter, and higher hair growth rates in winter compared with the other seasons (p < 0.05). Relocated horses had higher HCCs, suggesting a change in their welfare status, probably related to the sudden change in their surrounding conditions. However, these results should be interpreted cautiously due to the low sample size used. The nature of the relationship between HCCs and horse welfare needs to be further examined.

Induction of CIRBP expression by cold shock on bovine cumulus-oocyte complexes.

The aim of this study was to induce the cold-inducible RNA-binding protein (CIRBP) expression on cumulus-oocyte complexes (COCs) through exposure to a sub-lethal cold shock and determine the effects of hypothermic temperatures during the in vitro maturation of bovine oocytes. Nuclear maturation, cortical granule redistribution and identification of cold-inducible RNA-binding protein (CIRBP) were assessed after 24 hr of in vitro maturation of control (38.5°C) and cold-stressed oocytes (33.5°C). The presence of CIRBP was assessed by Western blot in COCs or denuded oocytes and their respective cumulus cells. Based on the odds ratio, cold-stressed oocytes presented higher abnormal cytoplasmic distribution of cortical granules and nuclear maturation than the control group. Although CIRBP was detected in both control and cold-stressed groups, cold-stressed COCs had 2.17 times more expression of CIRBP than control COCs. However, when denuded oocytes and cumulus cells were assessed separately, CIRBP only was detected in cumulus cells in both groups. In conclusion, cold shock induced CIRBP expression, but it negatively affected nuclear maturation and cortical granule distribution of bovine oocytes. Moreover, the expression of CIRBP was only identified in cumulus cells but not in oocytes.