Inflammatory responses to induced infectious endometritis in mares resistant or susceptible to persistent endometritis

BACKGROUND: The objective of the study was to evaluate the gene expression of inflammatory cytokines (interleukin [IL]-1β, IL-6, IL-8, IL-10, tumor necrosis factor [TNF]-α, IL-1 receptor antagonist [ra] and serum amyloid A (SAA) in endometrial tissue and circulating leukocytes in response to uterine inoculation of 105 colony forming units (CFU) Escherichia coli in mares. Before inoculation, mares were classified as resistant or susceptible to persistent endometritis based on their uterine inflammatory response to infusion of 109 killed spermatozoa and histological assessment of the endometrial quality. Endometrial biopsies were obtained 3, 12, 24 and 72 hours (h) after bacterial inoculation and blood samples were obtained during the 7 day period post bacterial inoculation. Expression levels of cytokines and SAA were determined by quantitative real-time reverse transcriptase PCR (qRT-PCR). RESULTS: Compared to levels in a control biopsy (obtained in the subsequent estrous), resistant mares showed an up-regulation of IL-1β, IL-6, IL-8 and TNF-α at 3 h after E. coli inoculation, while susceptible mares showed increased gene expression of IL-6 and IL-1ra. Susceptible mares had a significant lower gene expression of TNF-α,IL-6 and increased expression of IL-1ra 3 h after E. coli inoculation compared to resistant mares. Susceptible mares showed a sustained and prolonged inflammatory response with increased gene expression levels of IL-1β, IL-8, IL-1ra and IL-1β:IL-1ra ratio throughout the entire study period (72 h), whereas levels in resistant mares returned to estrous control levels by 12 hours. Endometrial mRNA transcripts of IL-1β and IL-1ra were significantly higher in mares with heavy uterine bacterial growth compared to mares with no/mild growth.All blood parameters were unaffected by intrauterine E. coli infusion, except for a lower gene expression of IL-10 at 168 h and an increased expression of IL-1ra at 48 h observed in susceptible mares compared to resistant mares. CONCLUSIONS: The current investigation suggests that endometrial mRNA transcripts of pro-inflammatory cytokines in response to endometritis are finely regulated in resistant mares, with initial high expression levels followed by normalization within a short period of time. Susceptible mares had a prolonged expression of pro-inflammatory cytokines, supporting the hypothesis that an unbalanced endometrial gene expression of inflammatory cytokines might play an important role in the pathogenesis of persistent endometritis

Estrogens Regulate Placental Angiogenesis in Horses

A sufficient vascular network within the feto-maternal interface is necessary for placental function. Several pregnancy abnormalities have been associated with abnormal vascular formations in the placenta. We hypothesized that growth and expansion of the placental vascular network in the equine (Equus caballus) placenta is regulated by estrogens (estrogen family hormones), a hormone with a high circulating concentration during equine gestation. Administration of letrozole, a potent and specific inhibitor of aromatase, during the first trimester (D30 to D118), decreased circulatory estrone sulfate concentrations, increased circulatory testosterone and androstenedione concentrations, and tended to reduce the weight of the fetus (p \u3c 0.1). Moreover, the gene expression of CYP17A1 was increased, and the expression of androgen receptor was decreased in the D120 chorioallantois (CA) of letrozole-treated mares in comparison to that of the control mares. We also found that at D120, the number of vessels tended to decrease in the CAs with letrozole treatment (p = 0.07). In addition, expression of a subset of angiogenic genes, such as ANGPT1, VEGF, and NOS2, were altered in the CAs of letrozole-treated mares. We further demonstrated that 17β-estradiol increases the expression of ANGPT1 and VEGF and increases the angiogenic activity of equine endothelial cells in vitro. Our results from the estrogen-suppressed group demonstrated an impaired placental vascular network, suggesting an estrogen-dependent vasculogenesis in the equine CA during the first trimester

Equine Arteritis Virus Has Specific Tropism for Stromal Cells and CD8\u3csup\u3e+\u3c/sup\u3e T and CD21\u3csup\u3e+\u3c/sup\u3e B Lymphocytes but Not for Glandular Epithelium at the Primary Site of Persistent Infection in the Stallion Reproductive Tract

Equine arteritis virus (EAV) has a global impact on the equine industry as the causative agent of equine viral arteritis (EVA), a respiratory, systemic, and reproductive disease of equids. A distinctive feature of EAV infection is that it establishes long-term persistent infection in 10 to 70% of infected stallions (carriers). In these stallions, EAV is detectable only in the reproductive tract, and viral persistence occurs despite the presence of high serum neutralizing antibody titers. Carrier stallions constitute the natural reservoir of the virus as they continuously shed EAV in their semen. Although the accessory sex glands have been implicated as the primary sites of EAV persistence, the viral host cell tropism and whether viral replication in carrier stallions occurs in the presence or absence of host inflammatory responses remain unknown. In this study, dual immunohistochemical and immunofluorescence techniques were employed to unequivocally demonstrate that the ampulla is the main EAV tissue reservoir rather than immunologically privileged tissues (i.e., testes). Furthermore, we demonstrate that EAV has specific tropism for stromal cells (fibrocytes and possibly tissue macrophages) and CD8+ T and CD21+ B lymphocytes but not glandular epithelium. Persistent EAV infection is associated with moderate, multifocal lymphoplasmacytic ampullitis comprising clusters of B (CD21+) lymphocytes and significant infiltration of T (CD3+, CD4+, CD8+, and CD25+) lymphocytes, tissue macrophages, and dendritic cells (Iba-1+ and CD83+), with a small number of tissue macrophages expressing CD163 and CD204 scavenger receptors. This study suggests that EAV employs complex immune evasion mechanisms that warrant further investigation

Genome Diversity and the Origin of the Arabian Horse

The Arabian horse, one of the world\u27s oldest breeds of any domesticated animal, is characterized by natural beauty, graceful movement, athletic endurance, and, as a result of its development in the arid Middle East, the ability to thrive in a hot, dry environment. Here we studied 378 Arabian horses from 12 countries using equine single nucleotide polymorphism (SNP) arrays and whole-genome re-sequencing to examine hypotheses about genomic diversity, population structure, and the relationship of the Arabian to other horse breeds. We identified a high degree of genetic variation and complex ancestry in Arabian horses from the Middle East region. Also, contrary to popular belief, we could detect no significant genomic contribution of the Arabian breed to the Thoroughbred racehorse, including Y chromosome ancestry. However, we found strong evidence for recent interbreeding of Thoroughbreds with Arabians used for flat-racing competitions. Genetic signatures suggestive of selective sweeps across the Arabian breed contain candidate genes for combating oxidative damage during exercise, and within the Straight Egyptian subgroup, for facial morphology. Overall, our data support an origin of the Arabian horse in the Middle East, no evidence for reduced global genetic diversity across the breed, and unique genetic adaptations for both physiology and conformation

Galectinology of Equine Pregnancy

Galectins are a family of proteins that bind to glycans, acting in a cytokine-like manner throughout the body. In the majority of mammalians, galectins have been found to be involved in pregnancy maintenance, but few studies have evaluated this in the horse. Therefore, the objective of this study was to examine the expression of various galectins in pregnant and nonpregnant mares. Next-generation RNA sequencing was performed on the chorioallantois and endometrium of healthy pregnant mares at 120, 180, 300, and 330 days of gestation (n = 4/stage), as well as 45-day chorioallantois (n = 4), postpartum chorioallantois (n = 3), and diestrus endometrium (n = 3). In the endometrium, galectin-1 and galectin-13 were found in the highest expression in the nonpregnant mare, with decreasing levels of expression noted throughout gestation. In contrast, galectin-8 and galectin-12 were found to be the lowest in the nonpregnant mare and reached the highest expression levels in mid-gestation before declining as parturition neared. In the chorioallantois, galectin-1, galectin-3, and galectin-3BP were found to have heightened expression levels at 45 d of gestation, with lesser expression levels noted throughout gestation. In contrast, galectin-9, galectin-12, and galectin-13 experienced the highest expression levels in the late-term chorioallantois (300 d/330 d), with lesser expression noted in early- to mid-gestation. Of note, galectin-1, galectin-3BP, galectin-9, galectin-12, and galectin-13 all experienced the lowest expression levels in the postpartum placenta, with heightened expression noted during gestation. In conclusion, galectins appear to be involved in equine pregnancy, and this is dependent on both the tissue within the feto-maternal interface and the specific galectin involved

Expression of receptors for ovarian steroids and prostaglandin E2 in the endometrium and myometrium of mares during estrus, diestrus and early pregnancy

The objective of this study was to compare expression of estrogen receptor alpha (ER-alpha), beta (ER-beta), progesterone receptor (PR), as well as prostaglandin E2 type 2 (EP2) and 4 (EP4) receptors in the equine myometrium and endometrium during estrus, diestrus and early pregnancy. Tissues were collected during estrus, diestrus, and early pregnancy. Transcripts for ER-alpha (ESR1), ER-beta (ESR2), PR (PGR), EP2 (PTGER2) and EP4 (PTGER4) were quantified by qPCR. Immunohistochemistry was used to localize ER-alpha, ER-beta, PR, EP2 and EP4. Differences in transcript in endometrium and myometrium were compared by the Delta Delta CT method. Expression for ESR1 (P < 0.05) tended to be higher during estrus than diestrus in the endometrium (P=0.1) and myometrium (P=0.06). In addition, ESR1 expression was greater during estrus than pregnancy (P < 0.05) in the endometrium and tended to be higher in estrus compared to pregnancy in the myometrium (P = 0.1). Expression for PGR was greater (P < 0.05) in the endometrium during estrus and diestrus than during pregnancy. In the myometrium, PGR expression was greater in estrus than pregnancy (P = 0.05) and tended to be higher during diestrus in relation to pregnancy (P = 0.07). There were no differences among reproductive stages in ESR2, PTGER2 and PTGER4 mRNA expression (P > 0.05). Immunolabeling in the endometrium appeared to be more intense for ER-a during estrus than diestrus and pregnancy. In addition, immunostaining for PR during pregnancy appeared to be more intense in the stroma and less intense in glands and epithelium compared to estrus and diestrus. EP2 immunoreactivity appeared to be more intense during early pregnancy in both endometrium and myometrium, whereas weak immunolabeling for EP4 was noted across reproductive stages. This study demonstrates differential regulation of estrogen receptor (ER) and PR in the myometrium and endometrium during the reproductive cycle and pregnancy as well as abundant protein expression of EP2 in the endometrium and myometrium during early pregnancy in mares. (C) 2014 Elsevier B.V. All rights reserved.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP

The influence of age, antral follicle count and diestrous ovulations on estrous cycle characteristics of mares

Reproductive aging must be well understood to optimize the reproductive management of older mares and to predict their reproductive life-span. The objectives of this study were to: 1) examine age-related differences in follicular dynamics, endocrine profiles, and primordial follicle counts, 2) evaluate the influence of antral follicle count (AFC) on age-related changes in follicular parameters, and 3) determine the influence of diestrous ovulations on the estrous cycle. Young (3-8yr; n = 10), middle-aged (9-18 yr; n = 16), and old (>18 yr; n = 19) light horse mares were examined with transrectal ultrasonography to monitor follicular growth over two consecutive estrous cycles. Jugular blood samples were taken and plasma progesterone and FSH concentrations were determined by an enzyme immunoassay and radioimmunoassay, respectively. Both interovulatory intervals and follicular phases were longer and the day of follicle deviation occurred later in aged mares. Furthermore, older mares had a tendency to ovulate smaller follicles. Neither follicular growth rate, the number of ovulations nor the length of luteal phase was influenced by mare age. Interestingly, as mare age increased, mares with low AFC had longer interovulatory intervals and follicular phases than mares with medium or high AFC. In addition, the number of primordial follicles declined with an increase in mare age but varied considerably between mares of the same age. Progesterone concentrations were positively influenced by age, whereas FSH concentrations were not, despite that FSH concentrations appeared higher in aged mares during the follicular phase. Estrous cycles with a diestrous ovulation had a longer interovulatory interval as well as a longer follicular and luteal phase while day of deviation occurred later. Progesterone concentrations were significantly higher on day 14 and 16 in estrous cycles with a diestrous ovulation than without a diestrous ovulation. In conclusion, aging in mares is associated with changes in follicular parameters which in turn are closely linked to differences in antral follicle count suggesting a relationship with ovarian reserve. Therefore, determination of antral follicle counts in aged mares can provide valuable information about the reproductive aging process. Finally, diestrous ovulations have a significant influence on different estrous cycle parameters

Binding of Equine Seminal Lactoferrin/Superoxide Dismutase (SOD-3) Complex Is Biased towards Dead Spermatozoa

Sperm-neutrophil binding is an important facet of breeding and significantly impacts fertility. While a specific seminal plasma protein has been found to reduce this binding and improve fertility (CRISP-3), additional molecule(s) appear to promote binding between defective sperm and neutrophils. Recent work has suggested one of these proteins is lactoferrin (LF), an 80 kDa iron-binding protein found throughout the body, but the purity of the protein was not confirmed. It is unknown if LF binds to sperm selectively based on viability, and if receptors for LF are located on equine sperm. To evaluate this, we attempted to purify equine seminal LF from five stallions (n = 5), biotinylate LF, and evaluate potential binding site(s) on spermatozoa. LF was consistently associated with superoxide dismutase (SOD-3), and all attempts to separate the two proteins were unsuccessful. Flow cytometric and microscopic analyses were used to compare LF/SOD-3 binding to viable and nonviable spermatozoa. Additionally, various methods of biotinylation were assessed to optimize this methodology. Biotinylation of seminal plasma protein was an effective and efficient method to study seminal plasma protein properties, and the binding site for LF/SOD-3 was found to be broadly localized to the entire sperm cell surface as well as selective towards nonviable/defective sperm. Although we were not able to determine if the binding to equine spermatozoa was through LF or SOD-3, we can conclude that equine seminal LF is tightly bound to SOD-3 and this protein complex binds selectively to nonviable spermatozoa, possibly to mark them for elimination by neutrophil phagocytosis