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Abstract The establishment of the fetomaternal interface depends on precisely regulated communication between the conceptus and the uterine environment. Recent evidence suggests that microRNAs (miRNAs) may play an important role in embryo-maternal dialogue. This study aimed to determine the expression profile of endometrial miRNAs during days 26–28 of equine pregnancy. Additionally, the study aimed to predict target genes for differentially expressed miRNAs (DEmiRs) and their potential role in embryo attachment, adhesion, and implantation. Using next-generation sequencing, we identified 81 DEmiRs between equine endometrium during the pre-attachment period of pregnancy (day 26–28) and endometrium during the mid-luteal phase of the estrous cycle (day 10–12). The identified DEmiRs appear to have a significant role in regulating the expression of genes that influence cell fate and properties, as well as endometrial receptivity formation. These miRNAs include eca-miR-21, eca-miR-126-3p, eca-miR-145, eca-miR-451, eca-miR-491-5p, members of the miR-200 family, and the miRNA-17-92 cluster. The target genes predicted for the identified DEmiRs are associated with ion channel activity and sphingolipid metabolism. Furthermore, it was noted that the expression of mucin 1 and leukemia inhibitory factor, genes potentially regulated by the identified DEmiRs, was up-regulated at day 26–28 of pregnancy. This suggests that miRNAs may play a role in regulating specific genes to create a favorable uterine environment that is necessary for proper attachment, adhesion, and implantation of the embryo in mares.

Abstract Background Intrauterine devices (IUD) are used in the veterinary practice as the non-pharmacological method of oestrus suppression in mares. When placed in the uterus, IUD create a physical contact with the endometrium that mimics the presence of an equine embryo. However, the mechanism of their action has not been fully elucidated. The objective of the present study was to examine the effect of mechanical stimulation of IUD on mare`s endometrium in both in vitro and in vivo study. For this purpose, we demonstrated the effect of IUD on prostaglandin (PG) F2α and PGE2 secretion, and mRNA transcription of genes involved in PG synthesis pathway in equine endometrial cells in vitro. In the in vivo study, we aimed to compare short-term effect of IUD inserted on day 0 (oestrus) with day 5–6 post-ovulation (the specific time when embryo reaches uterus after fertilization) on PG secretion from equine endometrium. To determine the long-term effect on PG synthase mRNA transcription, a single endometrial biopsy was taken only once within each group of mares at certain time points of the estrous cycle from mares placement with IUD on days 0 or 5–6 post-ovualtion. Results We showed for the first time that the incubation of the endometrial cells with the presence of IUD altered the pattern of PG synthase mRNA transcription in equine epithelial and stromal endometrial cells. In vivo, in mares placement with IUD on day 0, PGE2 concentrations in blood plasma were upregulated between 1 and 6, and at 10 h after the IUD insertion, compared with the control mares (P

Abstract Mare endometrial fibrosis (endometrosis), is one of the main causes of equine infertility. Despite the high prevalence, both ethology, pathogenesis and the nature of its progression remain poorly understood. Recent studies have shown that microRNAs (miRNAs) are important regulators in multiple cellular processes and functions under physiological and pathological circumstances. In this article, we reported changes in miRNA expression at different stages of endometrosis and the effect of transforming growth factor (TGF)-β1 on the expression of the most dysregulated miRNAs. We identified 1, 26, and 5 differentially expressed miRNAs (DEmiRs), in categories IIA (mild fibrosis), IIB (moderate fibrosis), and III (severe fibrosis) groups compared to category I (no fibrosis) endometria group, respectively (Padjusted

We examined the effect of prostaglandin (PG) E2 on the secretory function of equine corpus luteum (CL), according to the application site: intra-CL injection vs. an intrauterine (intra-U) administration. Moreover, the effect of intra-CL injection vs. intra-U administration of both luteotropic factors: PGE2 and human chorionic gonadotropin (hCG) as a positive control, on CL function was additionally compared. Mares were assigned to the groups (n = 6 per group): (1) an intra-CL saline injection (control); (2) an intra-CL injection of PGE2 (5 mg/ml); (3) an intra-CL injection of hCG (1,500 IU/ml); (4) an intra-U saline administration (control); (5) an intra-U administration of PGE2 (5 mg/5 ml); (6) an intra-U administration of hCG (1,500 IU/5 ml). Progesterone (P4) and PGE2 concentrations were measured in blood plasma samples collected at −2, −1, and 0 (pre-treatment), and at 1, 2, 3, 4, 6, 8, 10, 12, and 24 h after treatments. Moreover, effects of different doses of PGE2 application on the concentration of total PGF2α (PGF2α and its main metabolite 13,14-dihydro-15-keto-prostaglandin F2α– PGFM) was determined. The time point of PGE2, hCG, or saline administration was defined as hour “0” of the experiment. An intra-CL injection of PGE2 increased P4 and PGE2 concentrations between 3 and 4 h or at 3 and 12 h, respectively (p < 0.05). While intra-U administration of PGE2 elevated P4 concentrations between 8 and 24 h, PGE2 was upregulated at 1 h and between 3 and 4 h (p < 0.05). An intra-CL injection of hCG increased P4 concentrations at 1, 6, and 12 h (p < 0.05), while its intra-U administration enhanced P4 and PGE2 concentrations between 1 and 12 h or at 3 h and between 6 and 10 h, respectively (p < 0.05). An application of PGE2, dependently on the dose, supports equine CL function, regardless of the application site, consequently leading to differences in both P4 and PGE2 concentrations in blood plasma.

Cryopreservation deteriorates boar sperm quality and lifespan, which restricts the use of artificial insemination with frozen-thawed boar semen in field conditions. The objective of this study was to test the effects of post-thaw storage time and temperature on boar sperm survival. Semen ejaculates from five Landrace boars (one ejaculate per boar) were collected and frozen following a 0.5 mL-straw protocol. Straws from the five boars were thawed and diluted 1:1 (v:v) in BTS. The frozen-thawed semen samples were aliquoted into three parts and respectively stored at 5 °C, 17 °C, and 37 °C for up to 6 h. At 0.5, 2, and 6 h of storage, sperm motility, viability, mitochondrial membrane potential, and intracellular reactive oxygen species (ROS) levels and apoptotic changes were measured. Antioxidant and oxidant levels were tested in boar sperm (SPZ) and their surrounding environment (SN) at each timepoint. The results showed significant effects of post-thaw storage time and temperature and an impact on boar sperm quality (total and progressive motility, VCL, viability, acrosome integrity), early and late sperm apoptotic changes, and changes in MDA levels in SPZ and SN. Compared to storage at 5 °C and 37 °C, frozen-thawed semen samples stored at 17 °C displayed better sperm quality, less apoptotic levels, and lower levels of SPZ MDA and SN MDA. Notably, post-thaw storage at 17 °C extended boar sperm lifespan up to 6 h without obvious reduction in sperm quality. In conclusion, storage of frozen-thawed boar semen at 17 °C preserves sperm quality for up to 6 h, which facilitates the use of cryopreserved boar semen for field artificial insemination.

Semen delivery practice is crucial to the efficiency of artificial insemination using high-quality boar sperm. The present study aimed to evaluate the effect of a common semen delivery method, a Styrofoam box, under elevated temperatures on boar sperm quality and functionality and to investigate the underlying molecular responses of sperm to the temperature rise. Three pooled semen samples from 10 Duroc boars (3 ejaculates per boar) were used in this study. Each pooled semen sample was divided into two aliquots. One aliquot was stored at a constant 17 °C as the control group. Another one was packaged in a well-sealed Styrofoam box and placed in an incubator at 37 °C for 24 h to simulate semen delivery on hot summer days and subsequently transferred to a refrigerator at 17 °C for 3 days. The semen temperature was continuously monitored. The semen temperature was 17 °C at 0 h of storage and reached 20 °C at 5 h, 30 °C at 14 h, and 37 °C at 24 h. For each time point, sperm quality and functionality, apoptotic changes, expression levels of phosphorylated AMPK, and heat shock proteins HSP70 and HSP90 were determined by CASA, flow cytometry, and Western blotting. The results showed that elevated temperature during delivery significantly deteriorated boar sperm quality and functionality after 14 h of delivery. Storage back to 17 °C did not recover sperm motility. An increased temperature during delivery apparently promoted the conversion of sperm early apoptosis to late apoptosis, showing a significant increase in the expression levels of Bax and Caspase 3. The levels of phosphorylated AMPK were greatly induced by the temperature rise to 20 °C during delivery but reduced thereafter. With the temperature elevation, expression levels of HSP70 and HSP90 were notably increased. Our results indicate that a temperature increase during semen delivery greatly damages sperm quality and functionality by promoting sperm apoptosis. HSP70 and HSP90 could participate in boar sperm resistance to temperature changes by being associated with AMPK activation and anti-apoptotic processes.

Endometrium type I (COL1) and III (COL3) collagen accumulation, periglandular fibrosis and mare infertility characterize endometrosis. Metalloproteinase-2 (MMP-2), MMP-9 and tissue inhibitors of metalloproteinases (TIMP-1 and TIMP-2) are involved in collagen turnover. Since epigenetic changes may control fibroproliferative diseases, we hypothesized that epigenetic mechanisms could modulate equine endometrosis. Epigenetic changes can be reversed and therefore extremely promising for therapeutic use. Methylation pattern analysis of a particular gene zone is used to detect epigenetic changes. DNA methylation commonly mediates gene repression. Thus, this study aimed to evaluate if the transcription of some genes involved in equine endometrosis was altered with endometrial fibrosis, and if the observed changes were epigenetically modulated, through DNA methylation analysis. Endometrial biopsies collected from cyclic mares were histologically classified (Kenney and Doig category I, n = 6; category IIA, n = 6; category IIB, n = 6 and category III, n = 6). Transcription of COL1A1, COL1A2, COL3A1, MMP2, MMP9, TIMP1, and TIMP2 genes and DNA methylation pattern by pyrosequencing of COL1A1, MMP2, MMP9, TIMP1 genes were evaluated. Both MMP2 and MMP9 transcripts decreased with fibrosis, when compared with healthy endometrium (category I) (P < 0.05). TIMP1 transcripts were higher in category III, when compared to category I endometrium (P < 0.05). No differences were found for COL1A1, COL1A2, COL3A1 and TIMP2 transcripts between endometrial categories. There were higher methylation levels of (i) COL1A1 in category IIB (P < 0.05) and III (P < 0.01), when compared to category I; (ii) MMP2 in category III, when compared to category I (P < 0.001) and IIA (P < 0.05); and (iii) MMP9 in category III, when compared to category I and IIA (P < 0.05). No differences in TIMP1 methylation levels were observed between endometrial categories. The hypermethylation of MMP2 and MMP9, but not of COL1A1 genes, occurred simultaneously with a decrease in their mRNA levels, with endometrial fibrosis, suggesting that this hypermethylation is responsible for repressing their transcription. Our results show that endometrosis is epigenetically modulated by anti-fibrotic genes (MMP2 and MMP9) inhibition, rather than fibrotic genes activation and therefore, might be promising targets for therapeutic use.

Endometrosis negatively affects endometrial function and fertility in mares, due to excessive deposition of type I (COL1) and type III (COL3) collagens. The pro-fibrotic transforming growth factor (TGF-β1) induces myofibroblast differentiation, characterized by α-smooth muscle actin (α-SMA) expression, and collagen synthesis. In humans, fibrosis has been linked to epigenetic mechanisms. To the best of our knowledge, this has not been described in mare endometrium. Therefore, this study aimed to investigate the in vitro epigenetic regulation in TGF-β1-treated mare endometrial fibroblasts and the use of 5-aza-2′-deoxycytidine (5-aza-dC), an epigenetic modifier, as a putative treatment option for endometrial fibrosis. Methods and Results: The in vitro effects of TGF-β1 and of 5-aza-dC on DNA methyltransferases (DNMT1, DNMT3A, and DNMT3B), COL1A1, COL3A1, and α-SMA transcripts were analyzed in endometrial fibroblasts, and COL1 and COL3 secretion in a co-culture medium. TGF-β1 upregulated DNMT3A transcripts and collagen secretion. In TGF-β1-treated endometrial fibroblasts, DNA methylation inhibitor 5-aza-dC decreased collagen transcripts and secretion, but not α-SMA transcripts. Conclusion: These findings suggest a possible role of epigenetic mechanisms during equine endometrial fibrogenesis. The in vitro effect of 5-aza-dC on collagen reduction in TGF-β1-treated fibroblasts highlights this epigenetic involvement. This may pave the way to different therapeutic approaches for endometrosis.

Activation of the AMP-activated protein kinase (AMPK) has been demonstrated to be beneficial for boar sperm quality and functionality, while the underlying mechanism of AMPK activation of boar spermatozoa remains obscure. This study aimed to explore the effect of antioxidants and oxidants in boar spermatozoa and their surrounding fluid (SF) on the activation of AMPK during the liquid storage. Ejaculates from Duroc boars, routinely used for semen production, were collected and diluted to a final concentration of 25 × 106/mL. In experiment 1, twenty-five semen samples from eighteen boars were stored at 17 °C for 7 days. In experiment 2, three pooled semen samples created from nine ejaculates of nine boars were used, and each sample was treated with 0, 0.1, 0.2, and 0.4 μM/L H2O2 and stored at 17 °C for 3 h. Sperm quality and functionality, antioxidants and oxidants in boar spermatozoa and SF, the intracellular AMP/ATP ratio, and the expression levels of the phosphorylated AMPK (Thr172) were determined. Sperm quality significantly decreased with storage time in terms of viability (p < 0.05). Antioxidant and oxidant levels were markedly affected with storage time, with a decline in the SF total antioxidant capacity (TAC) (p < 0.05), SF malondialdehyde (MDA) (p < 0.05), and the sperm’s total oxidant status (TOS), as well as a fluctuation in sperm superoxidase dismutase-like (SOD-like) activity (p < 0.05). The intracellular AMP/ATP ratio increased (p < 0.05) on day 4 and subsequently decreased to its lowest value on days 6 and 7 (p < 0.05). The phosphorylated AMPK levels increased from day 2 to day 7 (p < 0.05). Correlation analyses indicate that sperm quality during liquid storage was correlated to antioxidants and oxidants in spermatozoa and SF (p < 0.05), which were correlated to the phosphorylation of sperm AMPK (p < 0.05). Treatment with H2O2 induced damages in sperm quality (p < 0.05), a decline in antioxidant levels (SF TAC, p < 0.05; sperm SOD-like activity, p < 0.01), an increase in oxidant levels (SF MDA, p < 0.05; intracellular ROS production, p < 0.05), a higher AMP/ATP ratio (p < 0.05), and phosphorylated AMPK levels (p < 0.05) in comparison with the control. The results suggest that antioxidants and oxidants in boar spermatozoa and SF are involved in AMPK activation during liquid storage.

Abstract Background Equine endometrosis is a chronic degenerative condition, described as endometrial fibrosis that forms in the stroma, under the basement membrane and around the endometrial glands. The role of lysophosphatidic acid (LPA) in the development of tissue fibrosis varies depending on the organ, and its profibrotic role in mare endometrosis remains unclear. The study aimed to establish the endometrial presence of LPA and its receptors (LPAR1–4), together with its effects on connective tissue growth factor (CTGF) and prostaglandins (PG) secretion from equine endometrium under physiological (estrous cycle), or pathological conditions (endometrosis). Mare endometria in the mid-luteal phase (n = 5 for each category I, IIA, IIB, III of Kenney and Doig) and in the follicular phase (n = 5 for each category I, IIA, III and n = 4 for IIB) were used. In experiment 1, the levels of LPA, LPAR1–4 mRNA level and protein abundance were investigated in endometria at different stages of endometrosis. In experiment 2, the in vitro effect of LPA (10− 9 M) on the secretion of CTGF and PGs from endometrial tissue explants at different stages of endometrosis were determined. Results Endometrial LPA concentration was higher in the mid-luteal phase compared to the follicular phase in category I endometrium (P

In older mares, increasing collagen fibers (fibrosis) in the endometrium and oviduct predisposes to sub-fertility and infertility. In this study, (i) gene transcription of collagen (qPCR: COL1A1, COL1A2, COL3A1, COL5A1); (ii) total collagen protein (hydroxyproline); (iii) collagen distribution (Picrosirius red staining; polarized light microscopy); and (iv) microvascular density (Periodic acid-Schiff staining), were evaluated in mares' placenta, and related to mares age, and placenta and neonate weights. Samples were collected from the gravid horn, non-gravid horn, and body of the placenta from younger (n = 7), and older mares (n = 9) of different breeds. Transcripts of COL1A1, COL3A1 and COL5A1, total collagen protein, chorionic plate connective tissue thickness, and microvascularization increased in the gravid horn of older mares' placentas, compared to the youngest (P < 0.05). Although in other species placenta fibrosis may indicate placental insufficiency and reduced neonate weight, this was not observed here. It appears that older fertile mares, with more parities, may develop a heavier, more vascularized functional placenta with more collagen, throughout a longer gestation, which enables the delivery of heavier foals. Thus, these features might represent morphological and physiological adaptations of older fertile mares' placentas to provide the appropriate nutrition to the equine fetus.

Myeloperoxidase is an enzyme released by neutrophils when neutrophil extracellular traps (NETs) are formed. Besides myeloperoxidase activity against pathogens, it was also linked to many diseases, including inflammatory and fibrotic ones. Endometrosis is a fibrotic disease of the mare endometrium, with a large impact on their fertility, where myeloperoxidase was shown to induce fibrosis. Noscapine is an alkaloid with a low toxicity, that has been studied as an anti-cancer drug, and more recently as an anti-fibrotic molecule. This work aims to evaluate noscapine inhibition of collagen type 1 (COL1) induced by myeloperoxidase in equine endometrial explants from follicular and mid-luteal phases, at 24 and 48 h of treatment. The transcription of collagen type 1 alpha 2 chain (COL1A2), and COL1 protein relative abundance were evaluated by qPCR and Western blot, respectively. The treatment with myeloperoxidase increased COL1A2 mRNA transcription and COL1 protein, whereas noscapine was able to reduce this effect with respect to COL1A2 mRNA transcription, in a time/estrous cycle phase-dependent manner (in explants from the follicular phase, at 24 h of treatment). Our study indicates that noscapine is a promising drug to be considered as an anti-fibrotic molecule to prevent endometrosis development, making noscapine a strong candidate to be applied in future endometrosis therapies.

The expression of genes of various proinflammatory chemokines and cytokines is controlled, among others, by the signaling pathway of the nuclear factor kappaB (NF-κB) superfamily of proteins, providing an impact on immune system functioning. The present review addresses the influence and role of the NF-κB pathway in the development and progression of most vital endometrial diseases in human and animal species. Immune modulation by NF-κB in endometritis, endometrosis, endometriosis, and carcinoma results in changes in cell migration, proliferation, and inflammation intensity in both the stroma and epithelium. In endometrial cells, the NF-κB signaling pathway may be activated by multiple stimuli, such as bacterial parts, cytokines, or hormones binding to specific receptors. The dysregulation of the immune system in response to NF-κB involves aberrant production of chemokines and cytokines, which plays a role in endometritis, endometriosis, endometrosis, and endometrial carcinoma. However, estrogen and progesterone influence on the reproductive tract always plays a major role in its regulation. Thus, sex hormones cannot be overlooked in endometrial disease physiopathology. While immune system dysregulation seems to be NF-κB-dependent, the hormone-independent and hormone-dependent regulation of NF-κB signaling in the endometrium should be considered in future studies. Future goals in this research should be a step up into clinical trials with compounds affecting NF-κB as treatment for endometrial diseases.

Collagen pathological deposition in equine endometrium (endometrosis) is responsible for infertility. Kenney and Doig’s endometrial biopsy histopathological classification is the gold standard method for endometrosis evaluation, whereby blood biomarkers identification would be less invasive and could provide additional information regarding endometrosis diagnosis and fertility prognosis. This study aimed to identify blood biomarkers for endometrosis diagnosis (42 mares were used in experiment 1), and fertility assessment (50 mares were used in experiment 2). Reproductive examination, endometrial biopsy histopathological classification (Kenney and Doig) and blood collection were performed. Endometrium and serum collagen type I (COL1) and type III (COL3), and hydroxyproline concentrations were measured (ELISA). Serum COL3 cut-off value of 60.9 ng/mL allowed healthy endometria (category I) differentiation from endometria with degenerative/fibrotic lesions (categories IIA, IIB or III) with 100% specificity and 75.9% sensitivity. This cut-off value enabled category I + IIA differentiation from IIB + III (76% specificity, 81% sensitivity), and category III differentiation from others (65% specificity, 92.3% sensitivity). COL1 and hydroxyproline were not valid as blood biomarkers. Serum COL3 cut-off value of 146 ng/mL differentiated fertile from infertile mares (82.4% specificity, 55.6% sensitivity), and was not correlated with mares’ age. Only COL3 may prove useful as a diagnostic aid in mares with endometrial fibrosis and as a fertility indicator.

Endometrosis is a frequently occurring disease decreasing mares’ fertility. Thus, it is an important disease of the endometrium associated with epithelial and stromal cell alterations, endometrium gland degeneration and periglandular fibrosis. Multiple degenerative changes are found in uterine mucosa, the endometrium. However, their pathogenesis is not well known. It is thought that nuclear factor-κB (NF-κB), a cell metabolism regulator, and its activation pathways take part in it. The transcription of the profibrotic pathway genes of the NF-κB in fibrotic endometria differed between the follicular (FLP) and mid-luteal (MLP) phases of the estrous cycle, as well as with fibrosis progression. This study aimed to investigate the transcription of genes of estrogen (ESR1, ESR2) and progesterone receptors (PGR) in equine endometria to find relationships between the endocrine environment, NF-κB-pathway, and fibrosis. Endometrial samples (n = 100), collected in FLP or MLP, were classified histologically, and examined using quantitative PCR. The phase of the cycle was determined through the evaluation of ovarian structures and hormone levels (estradiol, progesterone) in serum. The transcription of ESR1, ESR2, and PGR decreased with the severity of endometrial fibrosis and degeneration of the endometrium. Moreover, differences in the transcription of ESR1, ESR2, and PGR were noted between FLP and MLP in the specific categories and histopathological type of equine endometrosis. In FLP and MLP, specific moderate and strong correlations between ESR1, ESR2, PGR and genes of the NF-κB pathway were evidenced. The transcription of endometrial steroid receptors can be subjected to dysregulation with the degree of equine endometrosis, especially in both destructive types of endometrosis, and mediated by the canonical NF-κB pathway depending on the estrous cycle phase.

In this paper, the evolution of our understanding about post breeding endometritis (PBE), the susceptibility of mares, and events leading to endometrosis are reviewed. When sperm arrive in the uterus, pro-inflammatory cytokines and chemokines are released. They attract neutrophils and induce modulatory cytokines which control inflammation. In susceptible mares, this physiological defense can be prolonged since the pattern of cytokine release differs from that of resistant mares being delayed and weaker for anti-inflammatory cytokines. Delayed uterine clearance due to conformational defects, deficient myometrial contractions, and failure of the cervix to relax is detected by intrauterine fluid accumulation and is an important reason for susceptibility to endometritis. Multiparous aged mares are more likely to be susceptible. Untreated prolonged PBE can lead to bacterial or fungal endometritis called persistent or chronic endometritis. Exuberant or prolonged neutrophilia and cytokine release can have deleterious and permanent effects in inducing endometrosis. Interactions of neutrophils, cytokines, and prostaglandins in the formation of collagen and extracellular matrix in the pathogenesis of fibrosis are discussed. Endometritis and endometrosis are interconnected, influencing each other. It is suggested that they represent epigenetic changes induced by age and hostile uterine environment.

Although proteases found in neutrophil extracellular traps (NETs) have antimicrobial properties, they also stimulate collagen type 1 (COL1) production by the mare endometrium, contributing for the development of endometrosis. Cathepsin G (CAT), a protease present in NETs, is inhibited by specific inhibitors, such as cathepsin G inhibitor I (INH; β-keto-phosphonic acid). Matrix metallopeptidases (MMPs) are proteases involved in the equilibrium of the extracellular matrix. The objective of this study was to investigate the effect of CAT and INH (a selective CAT inhibitor) on the expression of MMP-2 and MMP-9 and on gelatinolytic activity. In addition, the putative inhibitory effect of INH on CAT-induced COL1 production in mare endometrium was assessed. Endometrial explants retrieved from mares in follicular phase or midluteal phase were treated for 24 or 48 h with CAT, inhibitor alone, or both treatments. In explants, transcripts (quantitative polymerase chain reaction) of COL1A2, MMP2, and MMP9, as well as the relative abundance of COL1 protein (Western blot), and activity of MMP-2 and MMP-9 (zymography) were evaluated. The protease CAT induced COL1 expression in explants, at both estrous cycle phases and treatment times. The inhibitory effect of INH was observed on COL1A2 transcripts in follicular phase at 24-h treatment, and in midluteal phase at 48 h (P < 0.05), and on the relative abundance of COL protein in follicular phase and midluteal phase explants, at 48 h (P < 0.001). Our study suggests that MMP-2 might also be involved in an earlier response to CAT, and MMP-9 in a later response, mainly in the follicular phase. While the use of INH reduced CAT-induced COL1 endometrial expression, MMPs might be involved in the fibrogenic response to CAT. Therefore, in mare endometrium, the use of INH may be a future potential therapeutic means to reduce CAT-induced COL1 formation and to hamper endometrosis establishment.

Collagen fibers and inflammatory cells are the basis for jenny endometrium Kenney and Doig's classification developed for the mare. The infiltration of a large number of eosinophils in the jenny endometrium is intriguing. Eosinophil and fibroblast produced IL33, which has been related to fibrosis development and chronicity. This work on the endometrium consisted of (i) quantification of collagen type I (COL1A2), type III (COL3A1), and IL33 transcripts; (ii) histological localization and quantification of COL1 and COL3 proteins; and (iii) eosinophil and neutrophil count and correlation with collagen area and IL33 transcripts. Localization of COL protein in the jenny endometrium was also compared to the mare endometrium. As fibrosis increased, eosinophil and neutrophil count decreased (P < 0.05). A 5-fold increase in IL33 transcripts was noted from categories IIA to III. There was a tendency toward a positive correlation between eosinophil count and IL33 transcripts in category IIA endometrium (P = 0.055). Neither transcripts of COL1A2 nor COL3A1 nor the areas of COL1 or COL3 differed with endometrial categories. Unlike for the mare, and regardless of the jenny endometrium classification, COL3 was always found to different extents in the stratum compactum, while COL1 was mainly present in deep stroma. As fibrosis progressed in the mare, an extensive increase in COL1 fibers was notorious under the surface epithelium. Correlations between neutrophil count and COL1 and COL3 areas were observed in the jenny endometrium, although no correlation was found for eosinophil count. Neutrophil count positive correlation with the COL1 area and negative correlation with the COL3 area in endometria with mild lesions suggest that neutrophils in the jenny endometrium may be involved in fibrogenesis. In addition, when eosinophilia subsides, the endometrium reacts with fibrosis establishment, which could be stimulated by the pro-fibrotic cytokine IL33, whose release might then be ascribed to fibroblasts. Further studies are needed to analyze the effect of the presence of COL3 next to the surface epithelium in the stratum compactum, or around the endometrial glands on jenny's endometrial function and fertility.

Background: Seminal plasma (SP) promotes sperm survival and fertilizing capacity, and potentially affects embryo development, presumably via specific signaling pathways to the internal female genital tract.Objectives: This study evaluated how heterologous SP, infused immediately before postcervical artificial insemination (AI) affected embryo development and the transcriptional pattern of the pig endometria containing embryos.Materials and Methods: Postweaning estrus sows (n = 34) received 40-mL intrauterine infusions of either heterologous pooled SP or Beltsville Thawing Solution (BTS; control) 30 min before AI of semen extended to 10% of homologous SP. Embryos (all sows) and endometrium samples (3 sows/group) were removed during laparotomy 6 days after the infusion of SP or BTS to morphologically evaluate the embryos to determine their developmental stage and to analyze the endometrial transcriptome using microarrays (PORGENE 1.0 ST GeneChip array, Affymetrix) followed by qPCR for further validation.Results: Embryo viability was equal between the groups (~93%), but embryo development was significantly (P < 0.05) more advanced in the SP-treated group compared to control. A total of 1,604 endometrium transcripts were differentially expressed in the SP group compared to the control group. An enrichment analysis showed an overrepresentation of genes and pathways associated with the immune response, cytokine signaling, cell cycle, cell adhesion, and hormone response, among others.Conclusions: SP infusions prior to AI positively impacted the preimplantation embryo development and altered the expression of the endometrial genes and pathways potentially involved in embryo development.

Endometrosis is an important mares’ disease which considerably decreases their fertility. As classic endometrial classification methods might be insufficient for tissue pathological evaluation, further categorization into active/inactive and destructive/non-destructive types was developed by Hoffmann and others. This study aimed to compare NF-κB pathway genes transcription among histopathological types of endometrosis, following Hoffmann and co-authors’ classification. Endometrial samples, collected postmortem from cyclic mares (n = 100) in estrus or diestrus, were classified histologically and used for gene transcription assessment. Gene transcription of NF-κB subunits (RelA, NF-κB1, NF-κB2), pro-inflammatory molecules (MCP-1, IL-6), and hyaluronan synthases (HAS 1, HAS 2, HAS 3) was compared among endometrosis types (active, non-active, destructive, non-destructive). Most individual mRNA samples showed high expression of RelA, NF-κB1, and MCP-1 gene transcripts and the destructive type of endometrosis, simultaneously. The expression of RelA and NF-κB1 genes was higher in active destructive group than in the other groups only in the follicular phase, as well as being higher in the inactive destructive group than in the others, only in the mid-luteal phase. The increase in gene transcription of the NF-κB canonical activation pathway in destructive endometrosis may suggest the highest changes in extracellular matrix deposition. Moreover, the estrous cycle phase might influence fibrosis pathogenesis.

Cathepsin G (CAT) is a protease released by neutrophils when forming neutrophil extracellular traps that was already associated with inducing type I collagen (COL1) in equine endometrium in vitro. Endometrosis is a fibrotic condition mainly characterized by COL1 deposition in the equine endometrium. The objective was to evaluate if noscapine (an alkaloid for cough treatment with anti-neoplastic and anti-fibrotic properties) would reduce COL1A2 transcription (evaluated by qPCR) and COL1 protein relative abundance (evaluated by western blot) induced by CAT in equine endometrial explants from follicular and mid-luteal phases treated for 24 or 48 h. The explants treated with CAT increased COL1 expression. Noscapine decreased COL1A2 transcription at both estrous cycle phases, but COL1 relative protein only at the follicular phase, both induced by CAT. Additionally, the noscapine anti-fibrotic action was found to be more effective in the follicular phase. The CAT treatment caused more fibrosis at the longest period of treatment, while noscapine acted better at the shortest time of treatment. Our results showed that noscapine could act as an anti-fibrotic drug in equine endometrosis by inhibiting CAT in vitro. Noscapine offers a new promising therapeutic tool for treating fibrosis as a single non-selective agent to be considered in the future.

Endometrosis, a fibrotic disease of mare endometrium, impairs uterine function. Prostaglandins (PG), despite modulating reproductive physiological functions, may also cause local pathological collagen deposition (fibrogenesis). We have previously shown that neutrophil extracellular traps (NETs) may also favor mare endometrosis. The aim of this study was to investigate the effect of enzymes present in NETs on PGF2α-pathway activation. Kenney and Doig’s type I/IIA and IIB/III mare endometria, from follicular phase (FLP) and mid-luteal (MLP) phase, were cultured in vitro in the presence of NETs enzymes (elastase, cathepsin-G or myeloperoxidase). Production of PGF2α (EIA) and transcription (qPCR) of its synthases (PTGS2, AKR1C3) and receptor (PTGFR) genes were evaluated. PGF2α and PTGFR were influenced by endometrial category and estrous cycle phase. In FLP endometrium, NETs enzymes induced both high PGF2α production and/or PTGFR transcription. In MLP type I/IIA tissues, down-regulation of PTGFR transcripts occurred. However, in MLP type IIB/III endometrium, high levels of PTGFR transcripts were induced by NETs enzymes. As PGF2α-pathway activation facilitates fibrogenesis in other tissues, PGF2α may be involved in endometrosis pathogenesis. In the mare, the endocrine microenvironment of healthy and pathological endometrium might modulate the PGF2α pathway, as well as fibrosis outcome on endometrium challenged by NETs enzymes.

The regulation of corpus luteus (CL) luteolysis is a complex process involving a myriad of factors. Previously, we have shown the involvement of Nodal in functional luteolysis in mares. Presently, we ask the extent of which Nodal mediation of luteolysis is done through regulation of angioregression. We demonstrated the interaction between Nodal and hypoxia-inducible factor 1 α (HIF1α) and thrombospondin 1/thrombospondin receptor (TSP1/CD36) systems, could mediate angioregression during luteolysis. First, we demonstrated the inhibitory effect of Nodal on the vascular marker platelet/endothelial cell adhesion molecule 1 (CD31). Also, treatment of mid CL explants with vascular endothelial growth factor A (VEGFA) showed a trend on activin-like kinase 7 (Alk7) protein inhibition. Next, Nodal was also shown to activate HIF1α and in vitro culture of mid CL explants under decreased oxygen level promoted Nodal expression and SMAD family member 3 (Smad3) phosphorylation. In another experiment, the crosstalk between Nodal and TSP1/CD36 was investigated. Indeed, Nodal increased the expression of the anti-angiogenic TSP1 and its receptor CD36 in mid CL explants. Finally, the supportive effect of prostaglandin F2α (PGF2α) on TSP1/CD36 was blocked by SB431542 (SB), a pharmacological inhibitor of Nodal signaling. Thus, we evidenced for the first time the in vitro interaction between Nodal and both HIF1α and TSP1 systems, two conserved pathways previously shown to be involved in vascular regression during luteolysis. Considering the given increased expression of Nodal in mid CL and its role on functional luteolysis, the current results suggest the additional involvement of Nodal in angioregression during luteolysis in the mare, particularly in the activation of HIF1α and TSP1/CD36.

Development of a healthy musculoskeletal system is of high concern for horse breeders and users. A longitudinal field study was performed in order to: (i) evaluate growth patterns and long-term changes on bone quality, bone metabolism, growth factors and metabolic variables in the Lusitano horse; and (ii) retrospectively assess whether these changes were related with radiographic findings regarding osteochondrosis-like lesions (OC) at the onset of training. Thirty-four Lusitano foals born and raised at four stud-farms, were periodically weighed (BW), and measured (withers height-WH) from birth to 36 months of age. On the same days, blood samples were collected for determination of osteocalcin, bone alkaline phosphatase, insulin-like growth factor I (IGF-I), leptin, insulin, glucose, parathyroid hormone (PTH), calcium, phosphorus and magnesium plasma concentrations, and quantitative ultrasound measurements were performed on the right third metacarpal bone (McIII). At the end of the study horses underwent radiographic examination of the four fetlocks, hocks and stifles. According to their radiographic status (OC negative vs. OC positive), Richards growth function was adjusted to BW and WH data. Instantaneous BW and WH growth rates (BW IADG and WH IADG) were calculated for each foal, from the resolution of the first derivative of growth models for seven age-classes. The presence of radiographic findings compatible with OC at the onset of training was associated with changes in BW and WH growth rates. Positive horses presented higher BW IADG from six to 18 months of age and lower WH IADG before 45 days of age (P

Endometrosis is a reproductive pathology that is responsible for mare infertility. Our recent studies have focused on the involvement of neutrophil extracellular traps enzymes, such as elastase (ELA), in the development of equine endometrosis. Noscapine (NOSC) is an alkaloid derived from poppy opium with anticough, antistroke, anticancer, and antifibrotic properties. The present work investigates the putative inhibitory in vitro effect of NOSC on collagen type I alpha 2 chain (COL1A2) mRNA and COL1 protein relative abundance induced by ELA in endometrial explants of mares in the follicular or mid-luteal phases at 24 or 48 h of treatment. The COL1A2 mRNA was evaluated by qPCR and COL1 protein relative abundance by Western blot. In equine endometrial explants, ELA increased COL 1 expression, while NOSC inhibited it at both estrous cycle phases and treatment times. These findings contribute to the future development of new endometrosis treatment approaches. Noscapine could be a drug capable of preventing collagen synthesis in mare’s endometrium and facilitate the therapeutic approach.

The oviduct presents the ideal conditions for fertilization and early embryonic development. In this study, (i) vascularization pattern; (ii) microvascular density; (iii) transcripts of angiogenic factors (FGF1, FGF2, VEGF) and their receptors—FGFR1, FGFR2, KDR, respectively, and (iv) the relative protein abundance of those receptors were assessed in cyclic mares’ oviducts. The oviductal artery, arterioles and their ramifications, viewed by means of vascular injection-corrosion, differed in the infundibulum, ampulla and isthmus. The isthmus, immunostained with CD31, presented the largest vascular area and the highest number of vascular structures in the follicular phase. Transcripts (qPCR) and relative protein abundance (Western blot) of angiogenic factors fibroblast growth factor 1 (FGF1) and 2 (FGF2) and vascular endothelial growth factor (VEGF), and their respective receptors (FGFR1, FGFR2, VEGFR2 = KDR), were present in all oviduct portions throughout the estrous cycle. Upregulation of the transcripts of angiogenic receptors FGF1 and FGFR1 in the ampulla and isthmus and of FGF2 and KDR in the isthmus were noted. Furthermore, in the isthmus, the relative protein abundance of FGFR1 and KDR was the highest. This study shows that the equine oviduct presents differences in microvascular density in its three portions. The angiogenic factors VEGF, FGF1, FGF2 and their respective receptors are expressed in all studied regions of the mare oviduct, in agreement with microvascular patterns.

Seminal plasma (SP) in the female genital tract induces changes that affect multiple reproductive processes. One of the active components in SP is the transforming growth factor β1 (TGF-β1), which has major roles in embryo development and pregnancy. Embryo transfer (ET) technology is welcomed by the pig industry provided that embryo quality at embryo collection as well as the fertility and prolificacy of the recipients after the ET is increased. This study evaluated different intrauterine infusion treatments at estrus (40 mL of SP, TGF-β1 cytokine in the extender, or the extender alone (control)) by mimicking an ET scenario in so-called “donor” (inseminated) and “recipient” (uninseminated) sows. On day 6 (day 0—onset of estrus), all “donors” were laparotomized to determine their pregnancy status (presence and developmental stage of the embryos). In addition, endometrial explants were collected from pregnant “donors” and cyclic “recipients,” incubated for 24 h, and analyzed for cytokine production. SP infusions (unlike TGF-β1 infusions) positively influenced the developmental stage of day 6 embryos. Infusion treatments differentially influenced the endometrial cytokine production, mainly in donors. We concluded that SP infusions prior to AI not only impacted the porcine preimplantation embryo development but also influenced the endometrial cytokine production six days after treatment, both in donors and recipients.

Neutrophils can originate neutrophil extracellular traps (NETs). Myeloperoxidase (MPO) is a peroxidase found in NETs associated to equine endometrosis and can be inhibited by 4-aminobenzoic acid hydrazide (ABAH). Metallopeptidases (MMPs) participate in extracellular matrix stability and fibrosis development. The objectives of this in vitro work were to investigate, in explants of mare’s endometrium, (i) the ABAH capacity to inhibit MPO-induced collagen type I (COL1) expression; and (ii) the action of MPO and ABAH on the expression and gelatinolytic activity of MMP-2/-9. Explants retrieved from the endometrium of mares in follicular or mid-luteal phases were treated with MPO, ABAH, or their combination, for 24 or 48 h. The qPCR analysis measured the transcription of COL1A2, MMP2, and MMP9. Western blot and zymography were performed to evaluate COL1 protein relative abundance and gelatinolytic activity of MMP-2/-9, respectively. Myeloperoxidase elevated COL1 relative protein abundance at both treatment times in follicular phase (p < 0.05). The capacity of ABAH to inhibit MPO-induced COL1 was detected in follicular phase at 48 h (p < 0.05). The gelatinolytic activity of activated MMP-2 augmented in mid-luteal phase at 24 h after MPO treatment, but it was reduced with MPO+ABAH treatment. The activity of MMP-9 active form augmented in MPO-treated explants. However, this effect was inhibited by ABAH in the follicular phase at 48 h (p < 0.05). By inhibiting the pro-fibrotic effects of MPO, it might be possible to reduce the development of endometrosis. Metallopeptidase-2 might be involved in an acute response to MPO in the mid-luteal phase, while MMP-9 might be implicated in a prolonged exposition to MPO in the follicular phase.

In the context of porcine embryo transfer (ET) technology, understanding the tightly regulated local uterine immune environment is crucial to achieve an adequate interaction between the transferred embryos and the receiving endometrium. However, information is limited on the uterine immune status of cyclic-recipient sows when receiving embryos during ET. The present study postulated that the anti- and proinflammatory cytokine profile 6 days after the onset of estrus differs between endometria from uninseminated cyclic sows and blastocyst-bearing sows. On Day 6 of the cycle, endometrial explants were collected from sows inseminated or not inseminated during the postweaning estrus and cultured for 22 h. The culture medium was then analyzed for the contents of a total of 16 cytokines using Luminex MAP® technology. The results showed important differences in the endometrial production of most cytokines between the sow categories, with a predominant anti-inflammatory environment displayed by the blastocyst-bearing endometria. These findings suggest that sperm, seminal plasma (SP) and/or early embryos modify the uterine environment by inducing an immune-tolerant cytokine profile already visible at Day 6. Whether the SP or some of its active components may help to develop strategies to maximize the reproductive performance of recipients after ET needs further investigation.

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

Neutrophil extracellular traps (NETs) fight endometritis, and elastase (ELA), a protease found in NETs, might induce collagen type I (COL1) accumulation in equine endometrium. Metallopeptidases (MMPs) are involved in extracellular matrix balance. The aim was to evaluate the effects of ELA and sivelestat (selective elastase inhibitor) on MMP-2 and MMP-9 expression and gelatinolytic activity, as well as the potential inhibitory effect of sivelestat on ELA-induced COL1 in equine endometrium. Endometrial explants from follicular (FP) and mid-luteal (MLP) phases were treated for 24 or 48 h with ELA, sivelestat, and their combination. Transcripts of COL1A2, MMP2, and MMP9 were evaluated by qPCR; COL1 protein relative abundance by Western blot, and MMP-2 and MMP-9 gelatinolytic activity by zymography. In response to ELA treatment, there was an increase in MMP2 mRNA transcription (24 h) in active MMP-2 (48 h), both in FP, and in MMP9 transcripts in FP (48 h) and MLP (24 h) (p < 0.05). Sivelestat inhibited ELA-induced COL1A2 transcripts in FP (24 h) and MLP (24 h, 48 h) (p < 0.05). The sivelestat inhibitory effect was detected in MMP9 transcripts in FP at 48 h (p < 0.05), but proteases activity was unchanged. Thus, MMP-2 and MMP-9 might be implicated in endometrium fibrotic response to ELA. In mare endometrium, sivelestat may decrease ELA-induced COL1 deposition and hinder endometrosis development.

Early life stress is a major factor underlying the vulnerability to respond to stressful events later in life. The present study attempted to evaluate the role of prenatal stress affecting the development of stress-related disorders and their reversion by postnatal exposure to Sertraline (SERT), a front-line medication for medication for posttraumatic stress disorder (PTSD) in humans. To achieve this, adult male and female prenatally stressed (PS) or unstressed (Controls) offspring rats, following oral chronic treatment with SERT (5 mg/kg/day; from 1 month to 4 months old), or not, were studied prior to and after a traumatic event. First, anxiety-like behavior during the prepulse inhibition (PPI) test, a modulation of the startle reflex, was examined in all animals. Subsequently, the animals were subjected to a session of mild inescapable footshocks (IS; 0.35 mA, 5 s) in a shuttle box that was followed by 4 days of situational reminders in the aversive context. Prior to the footshocks no effects of PS or SERT were shown, and no changes in PPI and the habituation to the shuttle box were found. After them, PS led animals to exhibit behavioral alterations. When compared to the Controls, PS animals of both sexes displayed less rearing activity in the aversive environment. PS males responded less to footshock delivery and, in most of the animals, fear extinction was impaired. Moreover, the early postnatal exposure to SERT lessened the behavioral impact of PS in females, while in males it had no effect. Current results extend previous data from our laboratory, showing that PS heightened vulnerability to stress later on, and that SERT acts differently in males and females.

Post-parturient behavior of mammalian females is essential for early parent–offspring contact. After delivery, lambs need to ingest colostrum for obtaining the related immunological protection, and early interactions between the mother and the lamb are crucial. Despite visual and auditory cues, olfactory cues are decisive in lamb orientation to the mammary gland. In sheep, the inguinal sinus is located bilaterally near the mammary gland as a skin pouch (IGS) that presents a gland that secretes a strong-smelling wax. Sheep IGS gland functions have many aspects under evaluation. The objective of the present study was to evaluate sheep IGS gland functional aspects and mRNA transcription and the protein expression of several hormone receptors, such as progesterone receptor (PGR), estrogen receptor 1 (ESR1), and 2 (ESR2) and prolactin receptor (PRLR) present. In addition, another aim was to achieve information about IGS ultrastructure and chemical compounds produced in this gland. All hormone receptors evaluated show expression in IGS during the estrous cycle (follicular/luteal phases), pregnancy, and the post-partum period. IGS secretion is rich in triterpenoids that totally differ from the surrounding skin. They might be essential substances for the development of an olfactory preference of newborns to their mothers.

Metabolic hormones have been associated with reproductive function modulation. Thus, the aim of this study was: (i) to characterize the immunolocalization, mRNA and protein levels of leptin (LEP), Ghrelin (GHR) and respective receptors LEPR and Ghr-R1A, throughout luteal phase; and (ii) to evaluate the role of LEP and GHR on progesterone (P4), prostaglandin (PG) E2 and PGF2α, nitric oxide (nitrite), tumor necrosis factor-α (TNF); macrophage migration inhibitory factor (MIF) secretion, and on angiogenic activity (BAEC proliferation), in equine corpus luteum (CL) from early and mid-luteal stages. LEPR expression was decreased in late CL, while GHR/Ghr-R1A system was increased in the same stage. Regarding secretory activity, GHR decreased P4 in early CL, but increased PGF2α, nitrite and TNF in mid CL. Conversely, LEP increased P4, PGE2, angiogenic activity, MIF, TNF and nitrite during early CL, in a dose-dependent manner. The in vitro effect of LEP on secretory activity was reverted by GHR, when both factors acted together. The present results evidence the presence of LEP and GHR systems in the equine CL. Moreover, we suggest that LEP and GHR play opposing roles in equine CL regulation, with LEP supporting luteal establishment and GHR promoting luteal regression. Finally, a dose-dependent luteotrophic effect of LEP was demonstrated.

The present research aimed to evaluate the effects of foaling season and feeding management in extensive systems on the nutritional status of Lusitano broodmares throughout the gestation/lactation cycle, by assessment of body condition (BC), body weight (BW), and some blood metabolic indicators. Four groups of Lusitano broodmares (A, B, C and D) were monitored during four years, in a total of 119 gestation/lactation cycles. All mares were kept on pasture. A and B mares were daily supplemented. Monthly, mares were weighed and BC evaluated. Suckling foals from these mares were also monitored for BW and withers height (WH). Glucose, NEFA, urea and albumin concentrations were determined in blood. BW changes were influenced by reproductive stage and foaling season (P

In adults, physiological angiogenesis is a rare event, with few exceptions as the vasculogenesis needed for tissue growth and function in female reproductive organs. Particularly in the corpus luteum (CL), regulation of angiogenic process seems to be tightly controlled by opposite actions resultant from the balance between pro- and antiangiogenic factors. It is the extremely rapid sequence of events that determines the dramatic changes on vascular and nonvascular structures, qualifying the CL as a great model for angiogenesis studies. Using the mare CL as a model, reports on locally produced cytokines, such as tumor necrosis factor α (TNF), interferon gamma (IFNG), or Fas ligand (FASL), pointed out their role on angiogenic activity modulation throughout the luteal phase. Thus, the main purpose of this review is to highlight the interaction between immune, endothelial, and luteal steroidogenic cells, regarding vascular dynamics/changes during establishment and regression of the equine CL.