Alterations in T cell-related transcripts at the feto-maternal interface throughout equine gestation.

INTRODUCTION: The tolerance of pregnancy by the maternal immune system is balanced between recognition and protection. In the human this is controlled by balancing helper T cell populations (Th1, Th2) in addition to immune suppression from the regulatory arm (Tregs), but this has not been evaluated in the horse. METHODS: RNA sequencing was performed on chorioallantois and endometrium of mares at 120, 180, 300 and 330 days of gestation (n = 4/stage), as well as 45-day chorioallantois (n = 4) and diestrus endometrium (n = 3). Transcripts were selected for relativity to Th1, Th2, or Treg-associated. qPCR and immunohistochemistry were used to confirm the results of select differentially expressed genes. RESULTS: In the endometrium, Th1 transcripts were highest in the diestrus mare and decreased as gestational length progressed. In contrast, Th2 transcripts were upregulated in comparison to the diestrus mare and highest in mid gestation. Treg transcripts were found increased in comparison to the diestrus mare, but decreased prepartum. In the chorioallantois no Th1 transcripts changed. The majority of Th2 transcripts increased from 45 to 300 days gestation, and then decreased prepartum. Treg-related transcripts trended down in the chorioallantois from 45 days to 120 days gestation, followed by an upregulation to 300 days and a secondary decline prepartum. DISCUSSION: The mare experiences a complex and evolving immune profile within the tissues of the feto-maternal interface. This consists of a balance between the Th1 and Th2 response, and a dynamic Treg response that is hypothesized to regulate overall events within the immune system.

The anti-inflammatory effect of exogenous lactoferrin on breeding-induced endometritis when administered post-breeding in susceptible mares.

The deposition of semen into the uterus of the horse induces a transient innate immune response that lasts 24-36 h in the normal mare. There exists a subset of mares that are unable to resolve this inflammation in a timely manner, and are classified as susceptible to the disease of persistent breeding-induced endometritis (PBIE). Lactoferrin is a protein of interest as a potential therapeutic for this persistent inflammation due to its anti-inflammatory and bactericidal properties. The addition of human recombinant lactoferrin (hrLF) to the insemination dose was previously shown to suppress mRNA expression of the pro-inflammatory cytokine tumor necrosis factor (TNF)-α at 6 h after insemination, but no studies have shown the effect of lactoferrin when infused post-breeding. Therefore, the objectives of this study were to (1) assess the safety of intra-uterine infusion of hrLF, (2) evaluate the effect of intrauterine infusion of hrLF post-breeding as a modulator of the immune response to breeding in the susceptible mare, and (3) determine the most effective concentration of hrLF. For the first experiment four normal mares received an intrauterine infusion of 500 µg/mL hrLF resuspended in 10 mL lactated Ringer's solution (LRS) and heart rate, rectal temperature, respiration, and endometrial quality were evaluated. For the second experiment, six mares classified as susceptible to PBIE were bred during estrus with 500 × 106 progressively motile sperm comprised of the ejaculates from two stallions, which were centrifuged over Androcoll-E to remove seminal plasma. Each insemination dose was resuspended in 30 mL LRS. Six hours after breeding, a 1L LRS uterine lavage was performed prior to treatments. Four treatments were administered over four consecutive estrous cycles in randomized order of: 10 mL LRS (vehicle control), 50 µg/mL hrLF resuspended in 10 mL LRS, 250 µg/mL hrLF resuspended in 10 mL LRS, and 500 µg/mL hrLF resuspended in 10 mL LRS. Twenty-four hours after breeding the mares were evaluated via transrectal ultrasonography for fluid retention. A low volume uterine lavage (250 mL LRS) was performed and the effluent was evaluated for polymorphonuclear neutrophils (PMNs). Finally, an endometrial biopsy was obtained for qPCR analysis of selected inflammatory cytokines. Lactoferrin had no significant overall effect on vital signs or endometrial quality. The addition of hrLF (50 µg/mL, 250 µg/mL, 500 µg/mL) did not significantly affect the amount of fluid detected post-breeding, but suppressed the ratio of PMNs to epithelial cells at all three concentrations compared to controls. In addition, all three concentrations of hrLF increased the mRNA expression of the anti-inflammatory cytokine interleukin-1 receptor antagonist (IL-1RN), while the 50 µg/mL dose significantly suppressed mRNA expression of the pro-inflammatory cytokine interferon gamma (IFNγ). In conclusion, the infusion of hrLF post-breeding was found to modulate the inflammatory response to breeding in the mare, and appears to be most effective at the 50 µg/mL concentration.

Characterization of the cervical mucus plug in mares.

The cervical mucus plug (CMP) is believed to play an integral role in the maintenance of pregnancy in the mare, primarily by inhibiting microbial entry. Unfortunately, very little is known about its composition or origin. To determine the proteomic composition of the CMP, we collected CMPs from mares (n = 4) at 9 months of gestation, and proteins were subsequently analyzed by nano-LC-MS/MS. Results were searched against EquCab2.0, and proteomic pathways were predicted by Ingenuity Pathway Analysis. Histologic sections of the CMP were stained with H&E and PAS. To identify the origin of highly abundant proteins in the CMP, we performed qPCR on endometrial and cervical mucosal mRNA from mares in estrus, diestrus as well as mares at 4 and 10 m gestation on transcripts for lactotransferrin, uterine serpin 14, uteroglobin, uteroferrin, deleted in malignant brain tumors 1 and mucins 4, 5b and 6. Overall, we demonstrated that the CMP is composed of a complex milieu of proteins during late gestation, many of which play an important role in immune function. Proteins traditionally considered to be endometrial proteins were found to be produced by the cervical mucosa suggesting that the primary source of the CMP is the cervical mucosa itself. In summary, composition of the equine CMP is specifically regulated not only during pregnancy but also throughout the estrous cycle. The structural and compositional changes serve to provide both a structural barrier as well as a physiological barrier during pregnancy to prevent infection of the fetus and fetal membranes.

Expression and localization of cysteine-rich secretory protein-3 (CRISP-3) in the prepubertal and postpubertal male horse.

The seminal plasma protein, cysteine-rich secretory protein-3 (CRISP-3), has been correlated with increased fertility and first-cycle conception rates, and has been suggested to be involved in the modulation of polymorphonuclear neutrophil and phagocytosis of spermatozoa during the inflammatory response to breeding in the horse. Previous research demonstrated that equine CRISP-3 is located in both the ampulla of the vas deferens and the seminal vesicles. However, this was done with nonquantitative laboratory techniques. In humans and rodents, CRISP-3 has been described as an androgen-dependent protein, but the effect of androgens on the expression of CRISP-3 has not been investigated in the horse. The objectives of this study were to (a) confirm and quantify the expression of CRISP-3 in the male equine reproductive tract, (b) describe the localization of CRISP-3 within the specific tissues which express it, and (c) determine if expression of CRISP-3 increases after puberty. We hypothesized that expression of CRISP-3 would be expressed in both the ampulla of the vas deferens and the seminal vesicles, and expression would increase after puberty. Tissues were collected postmortem from three prepubertal colts (<6 months) and six postpubertal stallions (>3 years). Tissue samples were collected from the ampulla of vas deferens, seminal vesicles, bulbourethral gland, prostate gland, testis, as well as the cauda, corpus, and caput aspects of the epididymis. Quantitative real-time polymerase chain reaction and immunohistochemistry (IHC) were performed using an equine-specific CRISP-3 designed primer and monocolonal antibody. A mixed linear additive model was used to compare mRNA expression between age groups, and significance was set to P < 0.05. There was a significant interaction between maturity and tissue type (P < 0.0001). Expression of CRISP-3 mRNA was found primarily in the ampulla of vas deferens with lesser expression in the seminal vesicles. Expression of CRISP-3 was higher in the postpubertal stallion when compared with the prepubertal colt for the ampulla (P < 0.0001) and seminal vesicles (P = 0.0013). IHC showed that equine CRISP-3 is primarily located in the glandular aspects of both the ampulla of vas deferens and the seminal vesicles, with staining concentrated in the cytoplasm of the epithelial cells that surrounded the glands of the mucosa. CRISP-3 was only observed in the postpubertal male horse suggesting that puberty plays a role in the activation of equine CRISP-3 expression.

Changes in maternal androgens and oestrogens in mares with experimentally-induced ascending placentitis.

REASONS FOR PERFORMING STUDY: While advanced stages of ascending placentitis can be diagnosed by transrectal ultrasonography and clinical signs, early stages can be missed. Thus, additional tools could enhance assessment of placental health. OBJECTIVES: To characterise peripheral dehydroepiandrosterone sulphate (DHEA-S) and testosterone concentrations in mares carrying normal pregnancies (Study 1) and compare plasma concentrations of DHEA-S, testosterone, oestradiol 17-ß (oestradiol) and oestrone sulphate (OES) in mares with or without placentitis (Study 2). STUDY DESIGN: Longitudinal cohort study of healthy mares (Study 1) and controlled experiment (Study 2). METHODS: In Study 1, mares had serum samples collected from 100 days of gestation to term. In Study 2, pregnant mares (260-280 days gestation) were assigned to a control group or a group with placentitis. Placentitis was induced via intracervical inoculation of Streptococcus equi ssp. zooepidemicus. Blood was collected at inoculation/commencement for control mares (day = 0) and daily for 12 days post inoculation (DPI) or until abortion. Steroid concentrations were determined by immunoassays. Concentrations of steroids in Study 2 were also evaluated relative to days from abortion (DFA -8 days to 0). RESULTS: In Study 1, DHEA-S peaked by 180 days gestation, while testosterone concentrations were progressively increased from Days 100 to 180 with a plateau until ~240 days and a progressive decline until 290 days of gestation. In Study 2, concentrations of DHEA-S and testosterone were not significantly different between groups. There were significant effects of time (oestradiol P = 0.0008, OES P = 0.01) and time-by-group interactions (oestradiol P<0.001, OES P<0.0001) for oestrogen concentrations. For mares with experimental placentitis, concentrations of oestradiol were significantly reduced at -6, -2, -1 and 0 DFA, while OES concentrations were significantly reduced on the day before abortion (0 DFA). CONCLUSIONS: Testosterone and DHEA-S were increased and varied through pregnancy. Oestrogens but not androgens decreased significantly in mares with experimentally-induced ascending placentitis.

Reversible downregulation of the hypothalamic-pituitary-gonadal axis in stallions with a novel GnRH antagonist.

The GnRH antagonist, acyline, has not yet been investigated in the stallion. Our study aimed to: (1) evaluate the downregulation of the stallion hypothalamic-pituitary-gonadal axis by acyline through assessment of seminal parameters, testicular volume, and sexual behavior; (2) assess hormonal response of acyline-treated stallions to GnRH stimulation; and (3) verify reversibility after treatment. Stallions were assessed pretreatment and subsequently treated (every five days) for 50 days: acyline (n = 4; 330 µg/kg acyline) or control (n = 4, vehicle). The stallions were then monitored for 62 days after the last day of treatment. Treatment-induced declines (P < 0.05) in FSH, LH, testosterone, and estrone sulfate. Gonadotropins and testosterone returned to control values within 9 days, and estrone sulfate by 14 days, after discontinuation of treatment. Acyline-treated stallions failed to respond with a rise in FSH, LH, and testosterone after exogenous GnRH stimulation (gonadorelin) at Day 46 of treatment compared to pretreatment stimulation and control stallions. Decreases (P < 0.05) were observed in total sperm numbers and motility (week 2) in acyline-treated stallions, as well as total seminal plasma protein (week 2) and testicular volume (week 5). Over the course of the study, the time to erection, time to ejaculation, and number of mounts increased (P < 0.0001) across both groups of stallions; however, there was no effect of treatment or treatment by time interactions on these parameters. Testicular volume, and most seminal parameters regained normal levels within 62 days after treatment ended; on follow-up, sperm output of acyline-treated stallions was regained within 7 months after the end of experiment. In conclusion, acyline reversibly suppresses the stallion hypothalamic-pituitary-gonadal axis.

Molecular changes in the equine follicle in relation to variations in antral follicle count and anti-Müllerian hormone concentrations.

REASONS FOR PERFORMING STUDY: The wide variation in circulating anti-Müllerian hormone (AMH) concentrations between mares is attributed to differences in antral follicle count (AFC) which may reflect follicular function. There are few data regarding variations in AFC and associated regulatory factors for AMH in the equine follicle during follicular development. OBJECTIVES: To examine molecular and hormonal differences in the equine follicle in relation to variations in AFC and circulating AMH concentrations during follicular development and to identify genes co-expressed with AMH in the equine follicle. STUDY DESIGN: Observational study. METHODS: Plasma AMH concentrations and AFC were determined in 30 cyclic mares. Granulosa cells, theca cells and follicular fluid were recovered from growing (n = 17) or dominant follicles (n = 13). The expression of several genes, known to be involved in folliculogenesis and steroidogenesis, was examined using real-time reverse transcriptase polymerase chain reaction and immunohistochemistry. Intrafollicular oestradiol and AMH concentrations were determined by immunoassay. RESULTS: Within growing follicles, the expression of AMH, AMHR2, ESR2 and INHA in granulosa cells was positively correlated with AFC and plasma AMH concentrations. In addition, the expression of ESR1 and FSHR was positively associated with plasma AMH concentrations. No significant associations were detected in dominant follicles. Furthermore, there was no association between AMH or oestradiol concentrations in follicular fluid and variations in AFC. Finally, the expression of AMH and genes co-expressed with AMH (AMHR2, ESR2 and FSHR) in granulosa cells as well as intrafollicular AMH concentrations decreased during follicular development while intrafollicular oestradiol concentrations increased and were inversely related to intrafollicular AMH concentrations. CONCLUSIONS: This study indicates that variations in AFC and circulating AMH concentrations are associated with molecular changes in the growing equine follicle.

Activation of persistent Streptococcus equi subspecies zooepidemicus in mares with subclinical endometritis.

Endometritis in horses caused by Streptococcus equi subspecies zooepidemicus (S. zooepidemicus) may be underdiagnosed due to traditional diagnostic methods lacking sensitivity and specificity. We serendipitously identified a bacterial growth medium (bActivate) that appeared capable of inducing growth of dormant S. zooepidemicus, which subsequently allowed detection by standard diagnostics. To assess the effect of bActivate we compared its ability to activate dormant S. zooepidemicus in a group of potentially infected subfertile mares with phosphate-buffered saline (PBS). All mares had to test negative for S. zooepidemicus on a low-volume uterine lavage, be negative on endometrial cytology and without clinical signs of endometritis to be included in the investigation. The mares were instilled with bActivate or PBS in the uterus. Growth of S. zooepidemicus was induced by bActivate in 64% (16/25) and PBS in 8% (1/12) of the mares, respectively (p<0.002). In vitro studies supported that some strains of S. zooepidemicus were able to form persister cells tolerating 32-times of the minimal inhibitory concentration of penicillin compared to normal growing cells. Persister cells had not acquired penicillin resistance, but seemed to tolerate the antimicrobial due to dormancy. This is, to our knowledge, the first description of controlled growth induction of dormant bacteria from a subclinical infection. Moreover we demonstrated how endometritis can origin from a reservoir of dormant bacteria residing within the endometrium, and not only as an ascending infection. Further studies should aim at determining the prevalence of dormant S. zooepidemicus, impact of activation on diagnostic and treatment efficacy, uterine health and mare fertility.

Diagnostic double-guarded low-volume uterine lavage in mares.

Endometritis constitutes a major problem in the management of broodmares; hence, diagnostic tests with a high sensitivity and specificity are highly appreciated. The aim of this study was to compare the results from endometrial, cytologic, and bacteriologic examinations obtained by a newly developed, double-guarded, flushing technique versus standard diagnostic tests, the double-guarded swab and biopsy. The described double-guarded flush technique requires the use of a disposable uterine flushing tube, a sanitary sleeve, a sterile steel speculum, and a 250 mL fluid bag. Endometrial biopsies, swabs, and low-volume lavage samples were obtained from 34 research mares at six different time points in four estrous cycles and were evaluated cytologically and bacteriologically. Endometrial biopsies from the first cycle (n = 34) were examined for the presence of polymorphonuclear neutrophils (PMNs) in the stratum compactum and stratum spongiosum and used as a gold standard for calculation of diagnostic sensitivity and specificity. In all samples, Escherichia coli was most frequently isolated (lavage, 30%; swab, 21%; and biopsy, 12%) followed by ß-hemolytic streptococci (lavage, 11%; swab, 8%; and biopsy, 7%). Positive cytology was less likely to occur when E coli was isolated from the diagnostic tests compared with the growth of ß-hemolytic streptococci. Isolation of pathogens from uterine samples was highly associated with the presence of PMNs in the stratum compactum and straum spongiosum on histology. Using the presence of PMNs in the tissue specimens as the gold standard for diagnosing endometritis, the sensitivity of low-volume lavage culture was 0.75 and the specificity was 0.72. In conclusion, the double-guarded, low-volume, lavage technique was a rapid and accurate method for diagnosing mares with endometritis, and the risk of false-positive samples is considered to be minimal compared with other flushing techniques described.

The effect of treatment with immune modulators on endometrial cytokine expression in mares susceptible to persistent breeding-induced endometritis.

REASONS FOR PERFORMING STUDY: Research has shown that 6 h after breeding is a critical time during the uterine innate immune response, and the failure to respond appropriately will result in persistent breeding-induced endometritis. This presents a potential opportunity to modulate the course of inflammation towards a timely resolution. OBJECTIVES: To evaluate the effects of immune modulation on endometrial mRNA expression of inflammatory genes in susceptible mares 6 h after breeding. The hypothesis was that immune modulation alters endometrial cytokine expression in susceptible mares. STUDY DESIGN: A randomised controlled study to evaluate the effects of mycobacterial cell wall extract and dexamethasone on endometrial gene expression after insemination in 6 mares susceptible to persistent breeding-induced endometritis. METHODS: Six susceptible mares were selected based on their uterine inflammatory response to insemination. Mares were inseminated during 3 oestrous cycles with 1 × 10(9) nonviable spermatozoa and 1) no additional treatment (control), or in combination with 2) dexamethasone (50 mg i.v.) at the time of insemination, or 3) with mycobacterial cell wall extract (1.5 ml i.v.) administered 24 h prior to insemination. Mares received one treatment per cycle in randomised order, and each mare served as her own control. Endometrial biopsies were collected 6 h after breeding, and quantitative polymerase chain reaction analysis for interleukin (IL)1ß, IL6, interferon γ, IL10 and IL1RA was performed. Relative quantification values reported fold changes in mRNA expression from the control. Data were analysed using an ANOVA and significance was set at P<0.05. RESULTS: Expression of IL1ß mRNA was lower after treatment with dexamethasone (P<0.001) and mycobacterial cell wall extract (P<0.05) when compared with control. No differences were detected in the mRNA expression of the other cytokines after any of the treatments. CONCLUSIONS: Treatment with immune modulators alters endometrial mRNA expression of IL1ß after insemination. A better understanding of the mechanisms of immune modulation in the equine uterus can help to improve treatments for persistent breeding-induced endometritis.

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-α), ß (ER-ß), 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-α (ESR1), ER-ß (ESR2), PR (PGR), EP2 (PTGER2) and EP4 (PTGER4) were quantified by qPCR. Immunohistochemistry was used to localize ER-α, ER-ß, PR, EP2 and EP4. Differences in transcript in endometrium and myometrium were compared by the ΔΔCT 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-α 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.

Acute and chronic effects of a contraceptive compound RTI-4587-073(l) on testicular histology and endocrine function in miniature horse stallions.

The objective of this study was to evaluate acute endocrine effects as well as histological changes in testicular parenchyma induced by the contraceptive compound RTI-4587-073(l). Six miniature stallions were used in this experiment. The treatment group (n = 3) received one oral dose of 12.5 mg/kg of RTI-4587-073(l), and the control group (n = 3) received placebo only. The stallions' baseline parameters (semen, testicular dimensions, endocrine values) were collected and recorded for 5 weeks before treatment and for 6 weeks after treatment. Multiple blood samples were collected for endocrine analysis. Testicular biopsies were obtained before treatment, 1 day after treatment and every other week after treatment. Ultrasound exams were performed to monitor the dimensions of the stallions' testes. All stallions were castrated 6 weeks after treatment. Sperm numbers, motility and percentage of morphologically normal sperm decreased (p < 0.05), while the number of immature germ cells increased in ejaculates from treated animals (p < 0.05). Serum concentrations of inhibin and follicle-stimulating hormone did not change. Testosterone concentrations initially transiently decreased (p < 0.05) after administration of RTI-4587-073(l), and increased several days later (p < 0.05). Testicular content of testosterone and estradiol 17-ß was lower in treated stallions than in control stallions on Day 1 after treatment (p < 0.05). Severe disorganization of the seminiferous tubules, significant loss of immature germ cells and complete depletion of elongated spermatids were observed in testicular biopsies obtained from treated stallions 1 day, 2 and 4 weeks after treatment. These changes were still present in the testicular samples taken from treated stallions after castration. The results of this study confirmed that RTI-4587-073(l) has antispermatogenic effects in stallions. Furthermore, we concluded that this compound causes acute sloughing of immature germ cells from the seminiferous tubules. RTI-4587-073(l) has significant but transient effects on Leydig cell function in stallions.

Blue light from individual light masks directed at a single eye advances the breeding season in mares.

REASONS FOR PERFORMING STUDY: Artificial lighting is commonly used to advance the breeding season in horses. Light masks have been developed that direct light at a single eye to inhibit the production of melatonin, the decoder of photoperiod for seasonally breeding animals. OBJECTIVES: To investigate whether low-intensity blue light from light masks was effective at advancing the breeding season in mares. STUDY DESIGN: Controlled experiment. METHODS: Data on reproductive activity was collected from 3 groups of mares maintained on Kentucky horse farms under various lighting conditions between 20 November 2011 and 10 February 2012: 59 nonpregnant, healthy Thoroughbred mares were used. On 1 December 2011, Group 1 (n = 16) was housed indoors under barn lighting (250 Lux) until 23.00 h daily. Group 2 (n = 25) wore light masks programmed to turn on from 16.30 h until 23.00 h daily and was maintained outdoors. Group 3 (n = 19) was maintained outdoors under the natural photoperiod as control. At 2-week intervals, rectal ultrasound examinations were performed and blood was collected for progesterone analysis. Oestrous cyclicity was defined as the presence of follicles >20 mm diameter detected in conjunction with serum progesterone >1 ng/ml and confirmation of ovulation by transrectal ultrasound examination. RESULTS: On 10 February, the number of mares exhibiting oestrous cyclicity was 14/16 (87.5%) in Group 1; 20/25 (80%) in Group 2; and 4/19 (21%), in Group 3. Pairwise comparison of groups revealed no difference in the number of cycling mares between Groups 1 and 2 (χ(2) test, P = 0.3348) whereas differences were observed between Groups 1 and 3 (χ(2) test, P<0.0001) and Groups 2 and 3 (χ(2) test, P<0.0003). CONCLUSIONS: Low-intensity blue light to a single eye from a light mask is an effective alternative to maintenance of mares indoors under lights for advancing the breeding season. Mobile light therapy for horses could have economic benefits for the breeder by reducing the costs of maintaining mares indoors, and welfare benefits for horses by permitting outdoor maintenance.

Endometrial inflammatory markers of the early immune response in mares susceptible or resistant to persistent breeding-induced endometritis.

Transient endometritis after breeding is necessary for clearance of bacteria and spermatozoa; however, in a subpopulation of mares, the inflammation fails to resolve in a timely fashion. The objective of this study was to describe the uterine inflammatory response in mares susceptible or resistant to persistent breeding-induced endometritis (PBIE) during the first 24 h after induction of uterine inflammation.Twelve mares were classified as susceptible (nZ6) or resistant (nZ6) to PBIE. Mares were inseminated over five estrous cycles and endometrial biopsies were collected at one time point per cycle before (0) and 2, 6, 12, and 24 h after insemination. qPCR analysis for IL1B, IL6, IL8, IFNG, TNF (TNFA), IL10, and IL1RN was performed, and endometrial inflammatory cells were counted for each sample. Relative quantification values reported fold changes in mRNA expression from 0 h values. A general pattern of expression post insemination was observed in both groups of mares. Cytokine mRNA increased at 2 h, peaked between 2 and 12 h, and then decreased.Differences were detected between groups of mares 6 h after challenge; resistant mares had higher mRNA expression of IL6, IL1RN,and IL10 than susceptible mares. Susceptible mares had an increased number of polymorphonuclear neutrophils in the endometrium 2 and 12 h after breeding when compared with resistant mares. These findings describe an inherent difference in the initial immune response to insemination and may help explain the transient nature of inflammation in resistant mares, whereas susceptible mares develop a persistent inflammation.

Diestrus administration of oxytocin prolongs luteal maintenance and reduces plasma PGFM concentrations and endometrial COX-2 expression in mares.

The objectives were to: (1) evaluate the efficacy of varying intervals of oxytocin administration in preventing luteolysis in mares; (2) examine PGF(2α) release in mares experiencing prolonged diestrus secondary to oxytocin treatment; and (3) evaluate the endometrial expression of oxytocin receptor, estrogen receptor α, and prostaglandin synthesis enzymes after oxytocin administration. In experiment I, mares received oxytocin (60 IU, im) daily on Days 8 to 10, 8 to 12, or 8 to 14 postovulation, and control mares received sterile saline. Prolongation of diestrus was defined by elevation of serum progesterone >1.0 ng/mL through Day 30 postovulation. The proportion of mares experiencing prolonged cycles increased (P < 0.01) as the number of days of oxytocin administration increased. Oxytocin administration on Days 8 to 10, 8 to 12, and 8 to 14 prolonged luteal maintenance in 3/7, 4/7, and 6/7 mares respectively, compared with 0/7 control mares. Treated mares with prolonged diestrus had lower (P < 0.05) plasma PGFM concentrations at Day 16 than did mares with normal diestrus periods. In experiment II, endometrial biopsies from mares treated with oxytocin from Days 8 to 14 postovulation (N = 6) had reduced cyclooxygenase-2 expression (P < 0.05) compared with control mares (N = 6) as determined by quantitative reverse transcription polymerase chain reaction and immunohistochemical staining. Oxytocin administration prolonged luteal maintenance in mares, with an increasing number of mares responding to treatment as the number of days of oxytocin administration was increased beyond Day 8 postovulation. Luteal maintenance in mares was also associated with decreased plasma PGFM concentrations and reduced endometrial cyclooxygenase-2 expression.

Macrophage migration inhibitory factor is expressed by equine conceptuses and endometrium.

Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine expressed by a wide range of tissues, which has been implicated to be involved in reproduction. Relative abundance of MIF mRNA in conceptus and endometrial tissue was assessed using real-time RT-PCR. Western blot analysis and immunohistochemistry were used to detect MIF protein expression. MIF transcript abundance was lowest in conceptuses obtained 16 days after ovulation, while the remaining stages of conceptus development that were analysed showed relatively constant expression levels. Migration inhibitory factor expression localized to trophectoderm cells, while capsular material was void of MIF immunoreactivity. Throughout the oestrous cycle, no clear statistically significant cycle-dependent expression pattern could be observed. During early pregnancy, the highest mRNA transcript levels were detected 16 days after ovulation. Pregnancy status did not affect MIF mRNA expression. Using immunohistochemistry, MIF protein expression was primarily localized in luminal and glandular epithelial cells, while stromal cells displayed weaker immunoreactivity. Taken together, we suggest that MIF is part of the molecular repertoire that contributes to normal endometrial function. The detailed functional significance of MIF expression in equine endometrium and pre-implantation stages of conceptus development remains to be determined.

Expression of aquaporin water channels in equine endometrium is differentially regulated during the oestrous cycle and early pregnancy.

The expression of 12 different aquaporin subtypes in equine endometrium was examined at the mRNA and protein level. Endometrial samples were obtained during anoestrus, oestrus, 8, and 14 days after ovulation in non-pregnant mares, and 14 days after ovulation in pregnant mares. Quantitative PCR revealed a time-dependent pattern for all aquaporin subtypes examined except for AQP10 and 12. AQP3, 5 and 7 showed highest mRNA abundance 8 days after ovulation, while AQP0 and 2 were most abundant at Day 14 of the cycle in non-pregnant mares. At 14 days of pregnancy, AQP1, 4, 8, 9 and 11 displayed highest expression levels. Western blot analysis confirmed protein expression of AQP0, 2 and 5. Immunohistochemistry localized protein expression to luminal and glandular epithelial and stromal cells. AQP0 staining intensity was highest in samples obtained on Day 14 of the oestrous cycle. AQP2 immunoreactivity seemed to be stronger in samples collected 14 days after ovulation from non-pregnant animals, in particular luminal epithelial staining. Samples collected 8 days after ovulation from cyclic animals were characterized by intense AQP5 staining of glandular epithelium, predominantly in the deeper glands. Progesterone treatment of anoestrous mares did not enhance expression of AQPs, indicating that factors other than progesterone are required for the up-regulation of certain AQP subtypes during dioestrus. In conclusion, it seems that an equine-specific collaboration of aquaporin subtypes contributes to changes in endometrial fluid content occurring throughout the oestrous cycle and contributes to endometrial receptivity during early pregnancy in the mare.

An investigation of uterine nitric oxide production in mares susceptible and resistant to persistent breeding-induced endometritis and the effects of immunomodulation.

The first objective of this study was to evaluate intrauterine nitric oxide (NO) and endometrial inducible NO synthase (iNOS) in mares susceptible or resistant to persistent breeding-induced endometritis (PBIE) within 24 h after breeding. Mares susceptible (n = 6) or resistant (n = 6) to PBIE were inseminated over five cycles, and uterine secretions and endometrial biopsies were collected before and 2, 6, 12 and 24 h after insemination. Uterine secretions were analysed for NO and biopsies were analyzed for iNOS expression. A second experiment evaluated the effect of treatment with dexamethasone or mycobacterial cell wall extract (MCWE) on uterine NO production and endometrial iNOS mRNA expression. Six susceptible mares were inseminated over three cycles with (i) killed spermatozoa without treatment (control), (ii) killed spermatozoa with 50 mg of dexamethasone IV or (iii) MCWE IV 24 h prior to insemination with killed spermatozoa. Six resistant mares were inseminated with killed spermatozoa as a control. Six hours after breeding, uterine biopsies and secretions were collected and evaluated for NO and iNOS mRNA. In Experiment 1, resistant mares had an increase in iNOS mRNA expression 2 h post-breeding compared to baseline (p = 0.045), 12 h (p = 0.014) and 24 h (p = 0.001). Susceptible mares had higher expression 2 h compared to 6 h (p = 0.046). No differences were observed in mRNA or protein expression of iNOS between resistant and susceptible mares. Resistant mares had a relatively steady amount of total intrauterine NO over 24 h, while susceptible mares had an increase over time, with a significantly higher increase in total NO than resistant mares at 6 (p = 0.04) and 12 h (p = 0.032). In Experiment 2, no differences were observed for iNOS mRNA expression. Susceptible mares had increased NO when compared to resistant mares (p = 0.008) and MCWE decreased NO (p = 0.047).

Ovarian steroid regulation of endometrial phospholipase A2 isoforms in horses.

Real-time PCR was used to investigate the role of progesterone (P4) and oestradiol (E2) in regulation of endometrial cytosolic, secretory and calcium-independent phospholipase A2 (PLA2G4A, PLA2G2A and PLA2G6, respectively) gene expression. Ovariectomized mares underwent 6 days of E2 pre-treatment followed by 14 days of P4 supplementation. At the start of P4 treatment (Day 1), mares were assigned in a 2 × 2 factorial design to receive either E2 or vehicle starting on Day 11 and endometrial biopsy collection on either Day 14 when P4 concentrations remained high (>4 ng/ml) or Day 16 when P4 concentrations had declined (0.5-2 ng/ml). Additional biopsies were collected from ovariectomized mares on Day 8, which served as control. Blood samples were collected for P4 determination. PLA2G4A expression was higher (p < 0.05) on Day 14 compared with Day 8. In contrast, PLA2G2A did not change significantly (p < 0.12). PLA2G4A and PLA2G2A gene expression increased (p < 0.05), as P4 concentration dropped, on Day 16. In contrast, PLA2G6 gene expression did not show differences between days. Treatment with oestradiol did not increase PLA2 isoforms expression when compared to treatment with the vehicle. PLA2G4A and PLA2G2A were positively correlated with each other and negatively correlated with P4 concentrations. In conclusion, P4 withdrawal upregulated PLA2G4A and PLA2G2A gene expression, and this was not affected by E2. PLA2G4A and PLA2G2A but not PLA2G6 gene expression may be involved in controlling prostaglandin F2 alpha synthesis and luteolysis.

Endometrial phospholipase A2 activity during the oestrous cycle and early pregnancy in mares.

The aim of this study was to determine phospholipase A2 (PLA2) kinetics and activity in the mare's endometrium during the oestrous cycle and early pregnancy. Phospholipase A2 is responsible for the liberation of arachidonic acid from phospholipids, which is the first limiting step in prostaglandins synthesis. Phospholipase A2 activity was measured using an assay based on the liberation of oleic acid from 1-palmitoyl-2-[(14) C] oleoyl phosphatidylcholine. The enzyme was shown to be calcium dependent, to have an optimum pH of 8 and an apparent Michaelis constant of 127 µM. Enzyme activity was low in the endometrium of early luteal phase tissue but increased significantly (p < 0.001) during the late luteal phase (5.39 ± 0.16; 3.48 ± 0.33, 6.85 ± 0.59, and 9.96 ± 1.23 nmol oleic acid released/mg protein at oestrus, and Days 3, 8 and 14 after ovulation, respectively). The mean PLA2 activity in endometrial tissue from pregnant mares (4.23 ± 0.74) was significantly lower (p < 0.01) than from cyclic animals during late dioestrus (9.96 ± 1.23). The results indicate that PLA2 activity in equine endometrium changes with the stage of the oestrous cycle and thus may be influenced by systemic hormone concentrations. The inhibitory effects of conceptus products on secretion of prostaglandin during early pregnancy were associated with a competitive inhibitor that decreased endometrial PLA2 activity.