The Effects of Treatment with N-Acetyl Cysteine on Clinical Signs in Persistent Breeding-Induced Endometritis Susceptible Mares.

Persistent breeding-induced endometritis (PBIE) is a major cause of infertility in mares. Endometrial inflammation that persists until embryonic descent ultimately results in early embryonic death. A poor endometrial biopsy grade (IIb or III) has been identified as a risk factor for PBIE. Intrauterine fluid accumulation (>2 cm in depth), pathologic endometrial edema, and elevated intrauterine neutrophil levels are all clinical features of PBIE. Commonly applied treatment options include uterine lavage and oxytocin therapy. N-acetyl cysteine (NAC), a mucolytic used to treat bacterial endometritis in mares, has anti-inflammatory properties and was investigated as a potential treatment for PBIE. A randomized, blinded, cross-over design clinical trial used NAC before breeding in PBIE-susceptible mares (n = 9). Intrauterine infusion of 3.3% NAC was performed 12 hours before insemination, and endometrial cytology and endometrial biopsy samples were obtained at 12 and 60 hours after insemination. Endometrial biopsies were evaluated for the degree of inflammation present. Clinical signs of endometrial edema and intrauterine fluid volumes were assessed by transrectal ultrasound at 12 and then every 24 hours after breeding. Data were analyzed using repeated measures analysis of variance and a Mann Whitney Wilcoxon Test. Treatment with NAC did not improve clinical signs in PBIE-affected mares. However, endometrial biopsies from mares treated with NAC displayed more diffuse and severe neutrophil infiltration than control cycles. Further research using a larger population of mares is required to evaluate the effects of NAC treatment on the endometrium of PBIE-susceptible mares.

Analysis of the uterine flush fluid proteome of healthy mares and mares with endometritis or fibrotic endometrial degeneration.

The objective of this study was to evaluate the differences in the uterine flush fluid proteome between healthy mares and mares with endometritis or fibrotic endometrial degeneration (FED). Uterine flush fluid samples were collected from healthy mares (n=8; oestrus n=5 and dioestrus n=3) and mares with endometritis (n=23; oestrus n=14 and dioestrus n=9) or FED (n=7; oestrus n=6 and dioestrus n=1). Proteomic analysis was performed using label-free liquid chromatography-tandem mass spectrometry. Of 216 proteins identified during oestrus, 127 were common to all three groups, one protein was exclusively detected in healthy mares, 47 proteins were exclusively detected in mares with endometritis and four proteins were exclusively detected in mares with FED. Of 188 proteins identified during dioestrus, 113 proteins were common between healthy mares and mares with endometritis, eight proteins were exclusively detected in healthy mares and 67 proteins were exclusively detected in mares with endometritis. Quantitative analysis revealed a subset of proteins differing in abundance between the three groups during oestrus and between healthy mares and mares with endometritis during dioestrus. These results provide a springboard for evaluation of specific proteins as biomarkers of uterine health and disease and for investigation of their roles in the establishment and maintenance of pregnancy.

A comparison of the uterine proteome of mares in oestrus and dioestrus.

Proteomic analysis of mare uterine flush fluid provides a minimally invasive technique for studying protein changes associated with the oestrous cycle. The aim of this study was to identify differentially abundant proteins in the uterine flush fluid of mares in oestrus and dioestrus. In this study, uterine flush fluid samples were collected from eight reproductively healthy mares in either oestrus (n = 5) or dioestrus (n = 3). Proteomic analysis was performed using liquid chromatography-tandem mass spectrometry. Of 172 proteins identified, six proteins (immunoglobulin lambda-like polypeptide 1, haemoglobin subunit alpha, alpha-1B-glycoprotein, serotransferrin, apolipoprotein A-1, and haemoglobin subunit beta) were significantly more abundant in oestrus. These proteins may contribute to the endometrial defence system through roles in inflammation, immunity or antimicrobial activity. In other species, some of these proteins have been described as immunoglobulins, negative acute phase proteins or defence agents against micro-organisms. During dioestrus, immunoglobulin alpha-1 chain C region-related, complement factor I, CD 109 antigen and uterocalin, were significantly more abundant. Research in other species suggests that these four proteins contribute to the immune response through proposed immunoregulatory characteristics, complement system involvement or roles in B cell-T cell interactions. In conclusion, ten differentially abundant proteins were identified in the uterine flush fluid of mares in oestrus and dioestrus. Targeted studies on these proteins could elucidate their role in uterine defence mechanisms during the oestrous cycle in the mare.

Short communication: The effect of calcium supplementation at calving on neutrophil function.

Low total blood calcium concentration after calving has been demonstrated to be a risk factor for reduced neutrophil function. The objective of this study was to evaluate whether administration of an injectable calcium supplement product soon after calving increased neutrophil oxidative burst or phagocytosis capacity. Cows (n = 27) from 4 farms were blocked by parity and randomly assigned to receive either calcium gluconate (35% wt/vol) in combination with calcium glucoheptonate (10% wt/vol; Theracalcium, Vétoquinol Canada Inc., Lavaltrie, Quebec, Canada) or a placebo within 12 h after calving and again 24 h later. Each dose of 120 mL was injected subcutaneously over 2 sites. Total serum calcium concentration, neutrophil oxidative burst, and neutrophil phagocytosis capacity were measured from coccygeal blood samples before (time 0) and 72 h after first treatment. There was no difference between treatment groups in lactation number, total calcium concentration, oxidative burst, or phagocytosis at time of enrollment. There was no effect of treatment on oxidative burst or phagocytosis by neutrophils. This preliminary study does not support an effect of supplemental calcium to improve neutrophil oxidative burst or phagocytosis capacity of low-parity parturient cows.

The effect of prepartum feeding and lying space on metabolic health and immune function.

The determinants of metabolic and reproductive health disorders in the peripartum period and the degree to which feeding and lying space and management can influence health are only partially understood. The objective of this randomized controlled study was to determine whether providing noncompetitive feeding and lying access in the close-up dry period improves health and immune function. Forty-eight Holstein cows of all parities were randomly assigned to a treatment group of 6 to 10 cows in 1 pen with either 80% cows to stalls and 90 cm of feeding space per cow (understocked) or 120% stocking density and 45 cm of feeding space per cow (overstocked) for 3 wk before expected calving. All cows wore electronic data loggers to monitor daily standing and lying time. Video recordings representing d 7 to 9 after group formation were reviewed, and a competition index (C_Ind) was calculated for each cow by dividing the number of times a cow displaced another as an actor by its total number of actor and reactor displacements. Cows were categorized as high success (C_Ind ≥0.6), moderate success (0.4 ≤ C_Ind <0.6), or low success (C_Ind <0.4). Weekly blood samples measured nonesterified fatty acids, ß-hydroxybutyrate, calcium, glucose, albumin, aspartate aminotransferase, bilirubin, haptoglobin, insulin, and insulin-like growth factor-1 from 3 wk before to 5 wk after calving. Measures of innate immune function (neutrophil phagocytosis and oxidative burst) were assessed at -2, -1, 1, 2, 3, and 5 wk relative to calving. Liver biopsies were collected at wk 1 and 3. Cows in the understocked group spent significantly more time per day lying; the back-transformed least squares means and 95% confidence interval were 14.8 h (13.9-15.6) versus 12.8 h (12.0-13.7). Controlling for parity, there was no difference between treatments in ß-hydroxybutyrate, nonesterified fatty acids, glucose, insulin, insulin-like growth factor 1, aspartate aminotransferase, bilirubin, or haptoglobin concentrations. Throughout the study, cows in the understocked treatment had higher mean calcium and tended to have higher albumin and at 3 wk after calving tended to have lower mean liver triacylglycerol content. Overall, there was no treatment effect on phagocytosis, but cows with a higher C_Ind in the understocked treatment group had greater oxidative burst function. There was no effect of treatment on endometritis. Despite increased competition and lower lying time, the expected harmful effects of crowding and competition on metabolic indicators and innate immune function were mostly not observed. Although this does not refute the importance of access to feeding and lying space, these results indicate that metabolic and reproductive health is more complex than can be explained solely by exposure to what are understood to be best practices for space allowances.

Dose-dependent inhibition of uterine contractility by nitric oxide: A potential mechanism underlying persistent breeding-induced endometritis in the mare.

Nitric oxide (NO) may have a role in persistent breeding-induced endometritis in mares through an inhibitory effect on uterine contractility. The objectives of this study were to test the effect of NO on spontaneous uterine contractility in-vitro and to evaluate whether this effect varied between the longitudinal and circular muscle layers of the uterus. Reproductive tracts were collected from eight euthanized non-pregnant mares (age 4-19 years; body weight 405-530 kg). Transrectal examination of the reproductive tract was performed before euthanasia to evaluate stage of the estrous cycle and presence of any apparent abnormality. After euthanasia, one uterine tissue sample was collected for histological evaluation and four full-thickness uterine tissue strips (10-12 mm × 2 mm), two parallel to each muscle layer, were excised for in-vitro contractility evaluation. Strips were suspended in tissue chambers containing Krebs-Henseleit solution, with continuous aeration (95% O2-5% CO2; pH 7.4) at 37 °C. After equilibration, spontaneous contractility was recorded (pre-treatment) and strips excised in each direction were randomly allocated to each of two groups: 1) SNAP (S-nitroso-N-acetylpenicillamine, an NO donor); or 2) NAP (N-acetyl-d-penicillamine, vehicle and time-matched control). These were treated at 15 min intervals with increasing concentrations (10-7 M to 10-3 M) of SNAP and NAP, respectively. Contractility data was recorded throughout the experiment. An interaction effect of group-by-concentration was observed (P < 0.0001). The mean contractility after treatment with 10-4 M and 10-3 M SNAP were significantly lower than the pre-treatment contractility and the mean contractility after treatment with lower SNAP concentrations. In contrast, contractility did not change significantly in the NAP treated controls. The effect of treatment on uterine contractility was not influenced by age or weight of the mare, stage of estrous cycle, uterine histology grade, or muscle layer. Secondary findings included significant main effects of stage of estrous cycle (increased contractility in estrus compared to diestrus), uterine histology grade (decreased contractility in grade IIB compared to grade I) and age (decreased contractility in mares aged > 8 years compared to mares aged ≤ 8 years). In conclusion, results of this study indicate that NO has a dose-dependent inhibitory effect on spontaneous uterine contractility irrespective of the muscle layer in the mare.

Randomized clinical trial of a calcium supplement for improvement of health in dairy cows in early lactation.

Prophylactic Ca supplementation immediately after calving is a common strategy to prevent clinical and subclinical hypocalcemia in parturient dairy cows. The objective of this study was to evaluate the effect of prophylactic administration of an injected Ca supplement on blood Ca concentration at 24 and 48h after treatment, incidence risk of clinical disease and culling, milk production in early lactation, and probability of pregnancy at first insemination. Cows without signs of visible milk fever (n=984) from 7 farms were blocked by parity and randomly assigned to receive either Ca gluconate (35% wt/vol) in combination with Ca glucoheptonate (10% wt/vol; TheraCalcium, Vétoquinol Canada Inc., Lavaltrie, Quebec) or a placebo (medication vehicle solution with no Ca) at first contact with each cow after calving and again 12 to 24h later. Each dose was 120mL injected subcutaneously over 2 sites. Total serum Ca concentration (tCa) was measured from coccygeal blood samples before (time 0) and 24 and 48h after first treatment in a subsample of cows (n=129). Blood ß-hydroxybutyrate concentrations were measured from all cows twice between 3 and 16d in milk at weekly visits and cows were evaluated for vaginal discharge once between 28 and 42d in milk. Disease events, production data from the first 3 Dairy Herd Improvement milk tests, reproduction, and culling data were collected from each herd. For cows that had received 1 injection of Ca before the blood sample at 24h (n=95), tCa was significantly higher in the treated cows: mean ± standard error, 2.03±0.03 versus 1.90±0.03mmol/L, accounting for tCa at time of enrollment and a treatment by tCa at enrollment interaction. At 48h, no significant difference was found in tCa between treatment and control (mean ± SE, 2.12±0.02 and 2.10±0.03mmol/L, respectively). Cows treated with the Ca product were significantly less likely to have received intravenous, subcutaneous, or oral supplemental Ca for exhibiting clinical signs of hypocalcemia than control cows (5.0 vs. 8.4%). No effect was found of treatment on retained placenta, metritis, hyperketonemia, prevalence of purulent vaginal discharge, culling from the herd, early lactation production, probability of pregnancy to first artificial insemination, or time to pregnancy. With this subcutaneous prophylactic Ca treatment regimen, blood Ca levels were temporarily increased at 24h after treatment, but no effect was observed of supplemental Ca on the risk of disease or culling, milk production, or reproductive performance.

The dynamic steroid landscape of equine pregnancy mapped by mass spectrometry.

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) allowed comprehensive analysis of various steroids detectable in plasma throughout equine gestation. Mares (n=9) were bled serially until they foaled. Certain steroids dominated the profile at different stages of gestation, clearly defining key physiological and developmental transitions. The period (weeks 6-20) coincident with equine chorionic gonadotropic (eCG) stimulation of primary corpora lutea and subsequent formation of secondary luteal structures was defined by increased progesterone, 17OH-progesterone and androstenedione, all Δ4 steroids. The 5α-reduced metabolite of progesterone, dihydroprogesterone (DHP) paralleled progesterone secretion at less than half the concentration until week 12 of gestation when progesterone began to decline but DHP concentrations continued to increase. DHP exceeded progesterone concentrations by week 16, clearly defining the luteo-placental shift in pregnane synthesis from primarily ovarian to primarily placental. The period corresponding to the growth of fetal gonads was defined by increasing dehydroepiandrosterone and pregnenolone (Δ5 steroids) concentrations from week 14, peaking at week 34 and declining to term. Metabolites of DHP (including allopregnanolone) dominated the steroid profile in late gestation, some exceeding DHP by weeks 13 or 14 and near term by almost tenfold. Thus Δ4 steroids dominated during ovarian stimulation by eCG, inversion of the ratio of progesterone: DHP (increasing 5α-pregnanes) marked the luteo-placental shift, Δ5 steroids defined fetal gonadal growth and 5α-reduced metabolites of DHP dominated the steroid profile in mid- to late-gestation. Comprehensive LC-MS/MS steroid analysis provides opportunities to better monitor the physiology and the progress of equine pregnancies, including fetal development.

Pregnancy without progesterone in horses defines a second endogenous biopotent progesterone receptor agonist, 5α-dihydroprogesterone.

One of the most widely accepted axioms of mammalian reproductive biology is that pregnancy requires the (sole) support of progesterone, acting in large measure through nuclear progesterone receptors (PRs) in uterine and cervical tissues, without which pregnancy cannot be established or maintained. However, mares lack detectable progesterone in the latter half of pregnancy. Instead of progesterone, several (mainly 5α-reduced) pregnanes are elevated and have long been speculated to provide progestational support in lieu of progesterone itself. To the authors' knowledge, evidence for the bioactivity of a second potent endogenously synthesized pregnane able to support pregnancy in the absence of progesterone has never before been reported. The 5α-reduced progesterone metabolite dihydroprogesterone (DHP) was shown in vivo to stimulate endometrial growth and progesterone-dependent gene expression in the horse at subphysiological concentrations and to maintain equine pregnancy in the absence of luteal progesterone in the third and fourth weeks postbreeding. Results of in vitro studies indicate that DHP is an equally potent and efficacious endogenous progestin in the horse but that the PR evolved with increased agonistic potency for DHP at the expense of potency toward progesterone based on comparisons with human PR responses. Sequence analysis and available literature indicate that the enzyme responsible for DHP synthesis, 5α-reductase type 1, also adapted primarily to metabolize progesterone and thereby to serve diverse roles in the physiology of pregnancy in mammals. Our confirmation that endogenously synthesized DHP is a biopotent progestin in the horse ends decades of speculation, explaining how equine pregnancies survive without measurable circulating progesterone in the last 4 to 5 mo of gestation.