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Contents In the horse, breeding induces a transient endometrial inflammation. A subset of mares are unable to resolve this inflammation, and they are considered susceptible to persistent mating-induced endometritis PMIE Select seminal plasma proteins cysteine-rich secretory protein-3 ( CRISP-3) and lactoferrin have been shown to affect the innate immune response to sperm in vitro. The objective of this study was to determine whether the addition of CRISP-3 and lactoferrin at the time of insemination had an effect on the mRNA expression of endometrial cytokines in susceptible mares after breeding. Six mares classified as susceptible to PMIE were inseminated during four consecutive oestrous cycles with treatments in randomized order of: 1 mg/ml CRISP-3, 150 μg/ml lactoferrin, seminal plasma (positive control) or lactated Ringer's solution ( LRS; negative control) to a total volume of 10 ml combined with 1 × 109 spermatozoa pooled from two stallions. Six hours after treatment, an endometrial biopsy was obtained for qPCR analysis of selected genes associated with inflammation (pro-inflammatory cytokines interleukin ( IL)-1β , IL-8, tumour necrosis factor ( TNF)-α , interferon ( INF)-γ, anti-inflammatory cytokines IL-1 RN and IL-10, and inflammatory-modulating cytokine IL-6). Seminal plasma treatment increased the mRNA expression of IL-1β ( p = .019) and IL-8 ( p = .0068), while suppressing the mRNA expression of TNF ( p = .0013). Lactoferrin also suppressed the mRNA expression of TNF ( p = .0013). In conclusion, exogenous lactoferrin may be considered as one modulator of the complex series of events resulting in the poorly regulated pro-inflammatory response seen in susceptible mares. [ABSTRACT FROM AUTHOR]

Contents The expression of 12 different aquaporin subtypes in equine endometrium was examined at the m RNA 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 m RNA 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. [ABSTRACT FROM AUTHOR]

Contents The first objective of this study was to evaluate intrauterine nitric oxide ( NO) and endometrial inducible NO synthase (i NOS) 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 i NOS expression. A second experiment evaluated the effect of treatment with dexamethasone or mycobacterial cell wall extract ( MCWE) on uterine NO production and endometrial i NOS m RNA 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 i NOS m RNA. In Experiment 1, resistant mares had an increase in i NOS m RNA 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 m RNA or protein expression of i NOS 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 i NOS m RNA expression. Susceptible mares had increased NO when compared to resistant mares (p = 0.008) and MCWE decreased NO (p = 0.047). [ABSTRACT FROM AUTHOR]

Contents 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. [ABSTRACT FROM AUTHOR]

Contents 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. [ABSTRACT FROM AUTHOR]

Contents 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-[14C] oleoyl phosphatidylcholine. The enzyme was shown to be calcium dependent, to have an optimum pH of 8 and an apparent Michaelis constant of 127 μ m. 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 thinsp;± thinsp;1.23 thinsp;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. [ABSTRACT FROM AUTHOR]

Contents During the second and third week of pregnancy, the equine conceptus is covered by an acellular glycoprotein capsule. This capsule contains glycoproteins resembling those of the mucin family with sialic acid making up a high proportion of the carbohydrate. Coinciding with conceptus fixation, a marked decline in sialic acid content of the capsule occurs, which has been proposed to contribute to cessation of conceptus mobility. Herein, we describe the expression of neuraminidase 2 (NEU2) by pre-implantation stages of equine conceptus development. NEU2 transcript abundance was examined in conceptuses obtained 8, 10, 12, 14 and 16 days after ovulation; highest levels were observed 16 days after ovulation. Transcript abundance observed in endometrial tissue was on average 474-fold lower than in conceptus tissue. Protein expression was localized to trophoblast cells and capsular material. Functionality of NEU2 was shown using an Amplex Red reagent-based assay. NEU2, formerly known as sialidase 2, belongs to a family of enzymes that cleave sialic acid from polysaccharide chains. The expression of NEU2 described herein provides a mechanism by which the conceptus can regulate the sialic acid content of its own capsule. The timely desialylation coinciding with conceptus fixation has been suggested integral for establishment of normal pregnancy. [ABSTRACT FROM AUTHOR]

Contents Ultrasound-guided follicular aspiration was performed in 26 Criollo crossbred mares, followed by the evaluation of ultrasonographic images of the Corpus luteum (CL) that was formed after puncture of follicles of different diameters (Group 25-29 mm; Group 30-35 mm and Group >35 mm). Serum progesterone (P4) concentrations were measured to determine CL function. The size of the CL was measured and the CL was classified based on the following echoscore: 1- anechoic tissue; 2- poorly defined luteal structure with low echogenicity; 3- echogenicity analogous to a luteal structure. The proportion of aspirated follicles that formed a functional CL (based on P4 concentration) 8 days after aspiration was 57.1% (4/7; CL size 25-29 mm), 75.0% (6/8; CL size 30-35 mm) and 72.7% (8/11; CL size >35 mm), respectively (p > 0.05). The echographic scores of aspirated follicles (indicating the presence or absence of a CL) were consistent with serum P4 concentrations (p < 0.0001). Of 26 aspirations, 18 resulted in luteal function confirmed by increased progesterone concentrations ([P4] > 1.0 ng/ml); 17 of these mares (94.4%) had an echoscore (2-3) compatible with luteinization (p = 0.0372). Eight days after aspiration, serum [P4] > 2.0 ng/ml was associated with high (p = 0.0056) CL echoscore (3) in 15 of 17 mares (88.2%). The echoscore used in this study was valuable as a screening test to detect the presence of a functional CL after aspiration. An echoscore of 3 served as a practical and efficient method to confirm luteinization. [ABSTRACT FROM AUTHOR]

The establishment of pregnancy involves a fine-tuned balance between protection and tolerance within the maternal immune system, as the female needs to accept a foreign antigen (the semi-allogenic fetus) while still being able to combat pathogens from the uterus. In the horse, the first uterine exposure to paternal antigens is during mating when sperm is introduced to the tissue and draining lymphatics of the uterus. Additionally, it has been suggested that seminal plasma and its proteins within it play an essential role in preparing the female tract for a suitable immunologic environment but this has not been confirmed in the horse. Therefore, the objective of this study was to evaluate the endometrial transcriptome following insemination either with seminal plasma or with reduced seminal plasma. We hypothesised that reduced seminal plasma would alter the endometrial transcriptome and affect transcripts relating to immunotolerance, antigen presentation and embryo growth and development. To do so, six (n = 6) mares were inseminated in a randomised switch-back design over the course of four oestrous cycles. Mares were rectally palpated and scanned via ultrasonography for the detection of a pre-ovulatory follicle (>35 mm) alongside increasing uterine oedema and relaxed cervix, and then treated with one of four treatment groups including (1) 30 mL lactated Ringers solution (LRS; NegCon), (2) 500 × 10 6 spermatozoa in conjunction with 30 mL seminal plasma (SP+), (3) 30 mL lactated Ringers solution (LRS; wash out) and (4) 500 × 10 6 spermatozoa with seminal plasma reduced via gradient centrifugation and resuspended in 30 mL LRS (SP-). Human chorionic gonadotropin (hCG) was administered to standardise the time to ovulation and endometrial biopsies were collected 7 days after insemination. RNA was isolated utilising Trizol, and RNA-Seq was performed by Novogene, with 97.79% total mapping and 40 million read depth. p value was set to <0.05. When comparing SP+ to SP-, 158 differentially expressed genes (DEGs) were identified. Biological processes impacted included antigen processing and regulation, cholesterol synthesis, and immune/inflammatory response. Gene ontology (GO) enrichment analysis using DAVID v6.8 revealed that many of these DEGs were involved in biological process such as antigen presentation (HLA-DM beta chain, HLA-DRB, HLA-DQA and RASGRP1), immune cell signalling (CXCL9, CXCL1, DEFB1 and MIP-2B), embryo growth and development (INHA, KLF2, RDH10, LAMA3 and SLC34A2) and embryo metabolism (ABCA1, ABCA2, APOA1, LDL, INSR, IGFBP2 and IGFBP3). Overall, reduction of seminal plasma from the insemination dose impacted the endometrial transcriptome at the time of early embryonic exposure to the uterine environment. Further work is justified to evaluate these alterations impact on embryo maturation, placental development, pregnancy outcome and development of offspring., (© 2024 The Author(s). Reproduction in Domestic Animals published by Wiley‐VCH GmbH.)

The equine industry holds substantial economic importance not only in the USA but worldwide. The occurrence of various infectious bacterial diseases in horses can lead to severe health issues, economic losses, and restrictions on horse movement and trade. Effective management and control of these diseases are therefore crucial for the growth and sustainability of the equine industry. While antibiotics constitute the primary treatment strategy for any bacterial infections in horses, developing resistance to clinically important antibiotics poses significant challenges to equine health and welfare. The adverse effects of antimicrobial overuse and the escalating threat of resistance underscore the critical importance of antimicrobial stewardship within the equine industry. There is limited information on the epidemiology of antimicrobial-resistant bacterial infections in horses. In this comprehensive review, we focus on the history and types of antimicrobials used in horses and provide recommendations for combating drug-resistant bacterial infections in horses. This review also highlights the epidemiology of antimicrobial resistance (AMR) in horses, emphasizing the public health significance and transmission dynamics between horses and other animals within a One Health framework. By fostering responsible practices and innovative control measures, we can better help the equine industry combat the pressing threat of AMR and thus safeguard equine as well as public health.

Background: Equine embryonic loss following the development of endometrial cups delays return to cyclicity due to the production of equine chorionic gonadotropin (eCG). Natural degradation of endometrial cups coincides with an influx of immune cells at 100-120 days of gestation, but therapeutic stimulation of reduced eCG production has been relatively unsuccessful. Recently, we observed an increase in pro-inflammatory cytokine production following the use of the immunostimulant mycobacterium cell wall fraction (MCWF)., Objectives: To evaluate the efficacy of hysteroscopic-guided injection of MCWF on the accelerated decline of eCG secretion., Study Design: In vivo experiment., Methods: Mares were pharmacologically aborted at 40-45 days of gestation, and then divided into groups: MCWF-treated (6 mg MCWF suspended in 20 mL LRS; n = 10) and Control (20 mL LRS; n = 6). Five days after abortion, hysteroscopic-guided injection of endometrial cups was performed, with 1 mL of volume placed into each visible endometrial cup. This was repeated 7 days later. Trans-rectal ultrasonography was performed to monitor ovarian activity, and serum was obtained to assess eCG and cytokine concentrations., Results: Concentrations of eCG decreased in the MCWF-treated group (p < 0.01) with a significant suppression noted as early as 14 days after onset of treatment and remained suppressed for the duration of the study. This coincided with an increase in peripheral IFN-γ (p < 0.01) and IL-1β (p < 0.01) concentrations. Eight out of ten MCWF-treated mares (80%) developed pre-ovulatory follicles, in comparison to 2/6 controls (33%). A pre-ovulatory follicle was noted 23 ± 4 days after onset of treatment., Main Limitations: No pregnancy data was obtained following treatment., Conclusions: This is the first report of a treatment for the accelerated reduction of eCG following abortion. Stimulation of this process allowed mares to develop a pre-ovulatory follicle within a month of MCWF treatment onset, granting repeat attempts at breeding within the confines of a single breeding season., (© 2024 The Authors. Equine Veterinary Journal published by John Wiley & Sons Ltd on behalf of EVJ Ltd.)

Introduction: The interleukin (IL)-6 family of cytokines is grouped by a common receptor subunit (gp130), but functions in distinct but overlapping physiological activities, including regulation of acute phase reaction and the balance between effector and regulatory T cell populations-both of which play a role in successful pregnancy maturation., Methods: Here, we aim to assess the expression profiles of members of the IL-6 cytokine family throughout equine gestation. To do so, 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). Expression levels of members of the IL-6 cytokine family including ciliary neurotrophic factor (CNTF), cardiotrophin 1 (CT-1), cardiotrophin-like cytokine factor 1 (CLCF1), galectin-10, oncostatin M (OSM), and IL-6, -11, and -27 were evaluated in addition to the receptors for IL-6 (IL-6R) and the common receptor subunit gp130. Additionally, peripheral concentration of IL-6 was assessed., Results: In the chorioallantois, differential expression of IL-6, IL-11, CNTF, CLCF1, OSM, and CT-1 was noted. In the endometrium, the gestational age of pregnancy impacted the expression of IL-11, CNTF, and CT-1. Circulatory IL-6 concentrations reached their highest concentrations at 120 days, with lesser concentrations noted at 45, 180, 300, and 330 days. Both IL-6R and gp130 altered in expression throughout equine gestation., Conclusion: In conclusion, members of the IL-6 cytokine family appear to fluctuate constantly throughout equine pregnancy, with varying expression profiles noted when comparing individual members. Additionally, different expression profiles were noted when comparing chorioallantois, endometrium, and circulation, indicating that the function of the cytokine is tissue-specific., (© 2024 The Author(s). American Journal of Reproductive Immunology published by John Wiley & Sons Ltd.)

Breeding-induced endometritis is a physiological reaction to clear the uterus from excess spermatozoa and bacteria after breeding. Cysteine rich secretory protein 3 in seminal plasma (spCRISP3) protects spermatozoa from binding and destruction by uterine PMNs, but it is not clear if this involves all sperm and bacteria, or if it is selective to a sub-population of live sperm. The objective of this report was to determine if spCRISP3 (1) is selective in its suppression of PMN-binding to sperm based on viability of spermatozoa, (2) protects bacteria from binding to PMNs, and (3) to determine the localization pattern of spCRISP3 on viable and dead sperm. Semen was collected from five stallions and each ejaculate was divided into (1) live and (2) snap frozen (dead) sperm. Two distinct sperm populations were confirmed by DNA fragmentation and membrane integrity assays. CRISP3 was purified from pooled seminal plasma, and binding of PMNs (isolated from peripheral blood) to the two sperm populations and E. coli was evaluated with flow cytometry in the presence of spCRISP3. In addition, localization of spCRISP3 on live and dead spermatozoa was determined by immunocytochemistry. Comparisons between treatments were analyzed using a one-way-ANOVA and Bonferroni's comparison test, or Kruskal-Wallis ANOVA if not normally distributed. spCRISP3 significantly suppressed binding of PMNs to live spermatozoa (p < 0.0001) but had no effect on dead sperm or bacteria (p > 0.05). Immunocytochemistry confirmed binding of spCRISP3 to live, but not dead spermatozoa. It was concluded that a selective interaction between spCRISP3 and live spermatozoa may be part of a biological mechanism that allows safe transport of viable spermatozoa to the oviducts, while enabling dead spermatozoa and bacteria to be eliminated in a timely fashion after breeding., Competing Interests: Declaration of competing interest The authors declare no conflict of interest. The research was completed prior to Dr. Doty's employment with Parse Biosciences, and the results will bring no benefit to this company., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Equine endometrial degeneration is diagnosed by microscopy of an endometrial biopsy but it is uncertain if findings in a single biopsy represent the state of the entire endometrium. Previous studies have compared samples from multiple sites but conclusions are inconsistent. Further clarification is therefore needed. In this study, presence and characteristics of endometrial degeneration were compared in two full thickness specimens from the ventral base of both uterine horns, obtained post mortem from cyclic mares (n = 82). Hematoxylin and eosin-stained sections were blinded evaluated for periglandular fibrosis, glandular nests, dilated glands, glandular dilation, excessive lymphatic vessels, and lymphatic lacunae. Each section was also assigned to a modified Kenney and Doig categorization. Statistical analysis included multiple logistic regression models for fraction of mares with disagreement in the modified Kenney and Doig category and fraction of mares with disagreement for each parameter, multiple generalized linear regression models for fraction of parameters with disagreement, and equivalence tests for agreement of a cumulative score of degenerative parameters. Possible effect of age, mare type and estrous cycle stage were included in the analyses. For the Kenney and Doig categorization, 49% of the mares had good agreement, 46% had moderate agreement and 5% had poor agreement between the two biopsies. Disagreement in the Kenney and Doig categorization between the two biopsies increased with age (OR = 1.1, P = 0.009). Disagreement within each parameter ranged from 10% of mares (glandular nests), to 31% (periglandular fibroblasts) and 37% (lymphatic lacunae). Disagreement for changes in endometrial glands increased with age (OR = 1.15-1.16, P < 0.040). Several mares (33%) had disagreement in at least two parameters, and the number of parameters with disagreement increased with age (HR = 1.04, P = 0.010). When tested for equivalence, the cumulative scores for paired endometrial samples did not differ for mares younger than 18 years (P = 0.011). In conclusion, the degenerative changes were not uniformly distributed, revealed by a disagreement between paired endometrial samples for individual parameters and for the Kenney and Doig categorization, which increased with age. This suggests that conclusions based on a single biopsy, particularly in mares over 17 years of age, should be interpreted with caution, and evaluation of two biopsies should be considered., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Sperm protein at 22 kDa has been associated with fertility., Objectives: The objectives of this study were to determine (1) the localization pattern of SP22 on ejaculated and caudal epididymal equine spermatozoa and in epididymal fluid, and to (2) characterize SP22 protein and mRNA expression in testicular and epididymal tissues in response to heat-induced testicular degeneration., Materials and Methods: Semen was collected before and after hemi-castration, as well as prior to and following insulation of the remaining testes, and tissue specimens were collected for analysis., Results: Histopathology confirmed degeneration in insulated testes. Ejaculated and epididymal spermatozoa from samples collected prior to insulation of the testicles had a predominant staining pattern of SP22 over the equatorial region. However, the equatorial pattern in the pre-insulation epididymal semen samples was significantly lower than in the pre-insulation ejaculated semen samples (68 ± 3, 81 ± 2.6, respectively). Ejaculated and epididymal samples collected after insulation of the testicles showed a complete loss of staining as the predominant pattern. Western blot analysis verified the presence of SP22 on fresh ejaculated spermatozoa prior to and following heat-induced degeneration, on epididymal spermatozoa after testicular insulation, and in testicular and epididymal tissues. Heat insulation significantly reduced messenger RNA expression in the head of the epididymis and testicular tissues. Immunohistochemistry of the testicular and epididymal tissues pre-heating showed considerably weaker staining than the same tissues post-heating., Discussion and Conclusion: It was concluded that heat-induced testicular damage causes both loss and relocation of SP22 on the sperm membrane. Future studies are warranted to determine the diagnostic value of these findings., (© 2023 American Society of Andrology and European Academy of Andrology.)

Competing Interests: Declaration of competing interest None.

Galectins are proteins that bind to glycans in targeted cells and function in cell-to-cell signaling throughout the body. Galectins have been found to be involved in various reproductive processes, including placental dysfunction, but this has not been investigated in the horse. Therefore, the objective of this study was to assess alterations in galectin expression of the abnormal placenta in pregnant mares. Next-generation RNA sequencing was performed on the postpartum chorioallantois of two placental pathologies following clinical cases of ascending placentitis (n = 7) and focal mucoid placentitis (n = 4), while chorioallantois from healthy postpartum pregnancies (n = 8; 4 control samples per disease group) served as the control. When evaluating ascending placentitis, both galectin-1 ( p < 0.001) and galectin-3BP ( p = 0.05) increased in the postpartum chorioallantois associated with disease, while galectin-8 ( p < 0.0001) and galectin-12 ( p < 0.01) decreased in the diseased chorioallantois in comparison with those in the control. In mares with focal mucoid placentitis, numerous galectins increased in the diseased chorioallantois, and this included galectin-1 ( p < 0.01), galectin-3BP ( p = 0.03), galectin-9 ( p = 0.02), and galectin-12 ( p = 0.04), in addition to a trend toward increases in galectin-3 ( p = 0.08) and galectin-13 ( p = 0.09). In contrast, galectin-8 expression decreased ( p = 0.04) in the diseased chorioallantois in comparison with that of the controls. In conclusion, galectins alter in abnormal placentae with variations observed among two forms of placental pathologies. These cytokine-like proteins may further our understanding of placental pathophysiology and warrant attention as potential markers of placental inflammation and dysfunction in the horse.

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.

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.

Background: Diarrhea remains an important cause of morbidity and mortality in neonatal foals, and correct identification of etiologic agents is essential for effective disease management., Objective: To examine the association between diarrhea and detection of Enterococcus durans or other enteropathogens in neonatal foals on 1 breeding farm in Kentucky, USA., Animals: Fifty-nine Thoroughbred foals and their broodmares., Methods: Prospective observational study. Study foals and broodmares were sampled and tested for E. durans and other enteropathogens during the first 10 days after foaling. The frequency of foals in which E. durans or other enteropathogens was compared between foals with or without diarrhea., Results: Seven of 59 foals developed diarrhea. The frequency of foals with E. durans infection was higher in foals with diarrhea 5/7 (71%), compared to foals without diarrhea 0/51 (0%; P < .01). Detection of E. durans in foals was associated with detection of E. durans in broodmares; in 2/7 (29%) foals with diarrhea, the 2 broodmares tested positive for E. durans, and, in 51/51 (100%) foals without diarrhea, all broodmares tested negative to E. durans (P = .01). Based on the spatial and temporal distribution of foals with diarrhea, 5 of 6 additional cases of diarrhea were attributed to lateral transmission of E. durans infection., Conclusions and Clinical Importance: Detection of E. durans was associated with diarrhea in foals. Implementation of enhanced biosecurity measures might mitigate disease transmission associated with E. durans infection in foals., (© 2022 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals LLC on behalf of American College of Veterinary Internal Medicine.)

Ascending placentitis is the leading cause of abortion in the horse. The pleiotropic cytokine tumor necrosis factor (TNF) is an upstream regulator of this disease, but little is understood regarding its function in pregnancy maintenance or placental infection. To assess this, RNA sequencing was performed on chorioallantois and endometrium of healthy pregnant mares at various gestational lengths (n = 4/gestational age), in addition to postpartum chorioallantois, and diestrus endometrium to assess expression of TNF, TNFR-1, and TNFR-2. Additionally, ascending placentitis was induced via trans-cervical inoculation of S. equi spp. zooepidemicus in pregnant mares (n = 6 infected / n = 6 control) and tissues and serum were collected to evaluate TNF-related transcripts. IHC was performed to confirm protein localization of TNFR-1 and TNFR-2. In healthy pregnancy, TNFR-1 appears to be the predominant TNF-related receptor. Following induction of disease, TNF concentrations increased in maternal serum, but expression did not alter at the tissue level. While both TNFR-1 and TNFR-2 increased following induction of disease, alterations in downstream pathways indicate that TNFR-1 is the dominant receptor in ascending placentitis, and is primarily activated within the chorioallantois, with minimal signaling occurring within the endometrium. In conclusion, TNF appears to be involved in the pathophysiology of ascending placentitis. An increase in this cytokine during disease progression is believed to activate TNFR-1 within the chorioallantois, leading to various pro-apoptotic and necroptotic outcomes, all of which may signal for fetal demise and impending abortion., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Problem: Ascending placentitis is the leading cause of abortion in the horse. Interleukin (IL)-6 is considered predictive of placental infection in other species, but little is understood regarding its role in the pathophysiology of ascending placentitis., Method of Study: Sub-acute ascending placentitis was induced via trans-cervical inoculation of S zooepidemicus, and various fluids/serum/tissues collected 8 days later. Concentrations of IL-6 were detected within fetal fluids and serum in inoculated (n = 6) and control (n = 6) mares. RNASeq was performed on the placenta (endometrium and chorioallantois) to assess transcripts relating to IL-6 pathways. IHC was performed for immunolocalization of IL-6 receptor (IL-6R) in the placenta., Results: IL-6 concentrations increased in allantoic fluid following inoculation, with a trend toward an increase in amniotic fluid. Maternal serum IL-6 was increased in inoculated animals, while no changes were noted in fetal serum. mRNA expression of IL-6-related transcripts within the chorioallantois indicates that IL-6 is activating the classical JAK/STAT pathway, thereby acting as anti-inflammatory, anti-apoptotic, and pro-survival. The IL-6R was expressed within the chorioallantois, indicating a paracrine signaling pathway of maternal IL-6 to fetal IL-6R., Conclusion: IL-6 plays a crucial role in the placental response to induction of sub-acute equine ascending placentitis, and this could be noted in amniotic fluid, allantoic fluid, and maternal serum. Additionally, IL-6 is acting as anti-inflammatory in this disease, potentially altering disease progression, impeding abortion signals, and assisting with the production of a viable neonate., (© 2021 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Preterm labor and/or abortion causes considerable economic impact on the equine industry. Unfortunately, few experimental models exist for the induction of various pregnancy-related complications, and therefore extrapolations are made from the experimental model for ascending placentits, although inferences may be minimal. Certain steroid hormones (progestogens, estrogens) and fetal proteins (alpha-fetoprotein; AFP) might improve the diagnostics for abnormal pregnancy, but the utility of these markers in the field is unknown. To assess this, thoroughbred mares (n = 702) were bled weekly beginning in December 2013 until parturition/abortion. Following parturition, fetal membranes were assessed histopathologically and classified as either ascending placentitis (n = 6), focal mucoid placentitis (n = 6), idiopathic abortion (n = 6) or no disease (n = 20). Weekly serum samples were analyzed for concentrations of progesterone, estradiol-17β, and AFP. Samples were analyzed retrospectively from the week of parturition/abortion in addition to the preceding four weeks. For both ascending and focal mucoid placentitis, a significant increase in progesterone and AFP was noted, alongside a significant decrease in estradiol-17β and the ratio of estradiol-17β to progesterone in comparison to controls. In contrast, idiopathic abortions experienced a decrease in progesterone concentrations alongside an increase in AFP, and this was only noted in the week preceding parturition/abortion. In conclusion, spontaneous placental infection in the horse altered both endocrine and feto-secretory markers in maternal circulation, while minimal changes were noted preceding noninfectious idiopathic abortion. Additionally, this is the first study to report an alteration in steroid hormones and AFP during the disease process of focal mucoid placentitis, the etiology of which includes Nocardioform placentitis., (Copyright © 2021. Published by Elsevier Inc.)

Ascending placentitis is a leading cause of abortion in the horse, but adaptive immune response to this disease is unknown. To evaluate this, sub-acute placentitis was experimentally-induced via trans-cervical inoculation of S. zooepidemicus, and endometrium and chorioallantois was collected 8 days later (n = 6 inoculated/n = 6 control). The expression of transcripts relating to Th1, Th2, Th17, and Treg maturation was assessed via RNASeq. IHC of transcription factors relating to each subtype in the same tissues (Th1: TBX21, Th2: GATA3, Th17: IRF4, Treg: FOXp3). An immunoassay was utilized to assess circulating cytokines (Th1: IFNg, IL-2; Th2: IL-4, IL-5; Th17: IL-17, IL-6; Treg: IL-10, GM-CSF). An increase in Th1 and Th17-related transcripts were noted in the chorioallantois, although no alterations were seen in the endometrium. Th2 and Treg-related transcripts altered in a dysregulated manner, as some transcripts increased in expression while others decreased. Immunolocalization of Th1, Th2, and Th17 cells was increased in diseased chorioallantois, while no Treg cells were noted in the diseased tissue. Secreted cytokines relating to Th1 (IFNg, IL-2), Th17 (IL-6), Th2 (IL-5), and Treg (IL-10) populations increased in maternal circulation eight days after inoculation. In conclusion, the Th1/Th17 response to ascending placentitis occurs primarily in the chorioallantois, indicating the adaptive immune response to occur in fetal derived placental tissue. Additionally, ascending placentitis leads to an increase in the helper T cell populations (Th1/Th17/Th2) while decreasing the Treg response. This increase in Th17-related responses alongside a diminishing Treg-related response may precede or contribute to fetal demise, abortion, or preterm labor., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Equine abortions are attributed to both infectious and noninfectious causes. Clinical extrapolations are often made from the experimental model for ascending placentitis towards other causes of fetal compromise, including various markers of inflammation, including the cytokines IL-2, 5, IL-6, IL-10, IFNγ, and TNF. It is unknown if these cytokine changes are noted under field conditions, or if they increase preceding other pregnancy related complications. To assess this, Thoroughbred mares (n = 702) had weekly blood obtained beginning in December 2013 and continuing until parturition. Fetal membranes were submitted to the UKVDL for complete gross and pathologic assessment and classified as either ascending placentitis (n = 6), focal mucoid placentitis (n = 6), idiopathic abortion (n = 6) or control (n = 20). Weekly serum samples were analyzed via immunoassay for concentrations of IL-2, IL-5, IL-6, IL-10, IFNγ, and TNF. For both focal mucoid placentitis and ascending placentitis, an increase (P < .05) in the concentrations of IL-2, IL-5, IL-6, IL-10, IFNγ, and TNF was noted preceding parturition in comparison to controls. Cytokine profiles preceding idiopathic abortion did not differ from controls. In conclusion, serum cytokines may be considered potential biomarkers for the prediction of placental infection, while no changes in cytokine profiles were noted when noninfectious causes of abortion occurred. Additionally, this is the first study to report an increase in cytokines during the disease process of focal mucoid placentitis, the etiology of which includes Nocardioform placentitis., (Copyright © 2021. Published by Elsevier Inc.)

Reproductive steroids testosterone (T) and estrone sulfate (E1S) are used as diagnostic markers for cryptorchidism in horses. The human chorionic gonadotropin (hCG) stimulation test is used as a diagnostic aid because administration of this hormone results in greater incremental differences in circulating steroid concentrations. Thoughts regarding optimal sampling times following hCG administration, however, are inconsistent. Additionally, determination of half-life of these steroids is important in postsurgical samples to confirm complete removal of testicular tissue. Objectives of this study, therefore, were to determine optimal sampling periods for peak T and E1S after hCG administration and half-life of these steroids after castration. Eight pony stallions were randomly assigned to control or treatment groups (5000 IU hCG). Blood samples were collected following hCG administration. Subsequently, stallions were castrated and blood samples were collected post-castration. The T concentrations were greatest at 72 h after hCG and were greater (P < 0.02) in samples from hCG-treated than control animals: 9,903.4 ± 384 and 784.0 ± 192 pg/mL, respectively (Mean ± SEM). The T concentrations were also greater at 1, 12, 24, 48 and 96 h. The E1S concentrations did not change after administration of hCG. The T response to hCG administration was biphasic with a maximal response between 48-96 h after administration. Half-lives of T and E1S were 1.1 and 0.7 h, respectively, and concentration of T and E1S was similar to that of geldings at 24 h post-castration, which, therefore, should be considered an optimal time to ensure complete castration has occurred., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Assessment of pregnancy viability in the equine patient is currently based on gestational profiles of limited prognostic value. In recent years Doppler technology has been applied to uterine and umbilical arteries of pregnant mares to monitor fetal responsive haemodynamics, suggestive of compromise. To date, uterine artery Doppler indices failed to provide sufficient evidence of pregnancy viability and the umbilical cord of the equine fetus is inconsistently visualized past 250 days gestation. The objectives of this study were to: i) evaluate intracranial blood flow impedance by Doppler examination of the fetal carotid artery and ii) establish reference values for healthy, uncomplicated pregnancies. The middle cerebral artery (MCA), a branch of the carotid artery, is commonly employed to assess intracranial hemodynamics in the human fetus, exhibiting high resistance to circulation to the fetal brain in uncomplicated pregnancies. For the purpose of this study, 12 pregnant mares were examined at 2-3 weeks interval by B mode and Doppler ultrasonography until delivery and a novel technique was developed for Doppler evaluation of the carotid artery in the equine fetus, in order to provide objective evidence of the hemodynamic status of the equine fetus. Additional biophysical and biochemical parameters were collected to demonstrate appropriate pregnancy development. In this study Doppler waveform analysis of fetal intracranial vasculature demonstrated an elevated blood flow impedance, showing a significant correlation of carotid Doppler indices with gestational age. Results were comparable to human fetal trends for the middle cerebral artery (MCA) from mid gestation to term. Biochemical data showed expected patterns of uncomplicated pregnancies., Competing Interests: Declaration of competing interest No disclosures nor conflicts of interest pertain to the authors of this study., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Maintaining yearly foal production is important for the economic success of the broodmare, and this requires breeding to occur as quickly postpartum as possible. The initial postpartum estrus occurs within 5-20 days postpartum, whereas the uterus is still undergoing repair from tissue alterations during pregnancy and parturition, a process known as involution. Attempts have been made to hasten this process, but with minimal success. Mycobacterium cell wall fraction (MCWF) is an immunomodulator that has been shown to reduce bacterial growth and alter aspects of the immune response to breeding, but it is unknown if MCWF hastens the process of involution. Therefore, the objectives of this study were to (1) investigate the effect of MCWF on tissue remodeling, (2) assess the effect of MCWF on the local immune system of the uterus, and (3) determine the optimal treatment interval needed for these processes to occur. We hypothesize that repeated treatments of MCWF postpartum will hasten the process of involution. To study this, 16 pregnant mares of mixed breeds were evaluated postpartum. Control mares (n = 4) received 1.5 mL lactated Ringer's solution intravenously on Day 1 (Day 0 = day of parturition) postpartum and again on Day 7, whereas treated mares either received 1.5 mL Settle intravenously on Day 1 and Day 7 (TX1; n = 6) or 1.5 mL Settle intravenously on Day 1 and then every 3 days until ovulation was detected (TX2; n = 6) and then evaluated until 15 days postpartum. Mares were assessed every 3 days for clinical, immunologic, and histologic parameters. Clinical parameters were assessed with transrectal ultrasonography and included ovarian activity, uterine fluid retention, and measurement of the uterine diameter, in addition to endometrial culture. Immunologic parameters included endometrial biopsies for quantitative polymerase chain reaction for expression of various cytokines (interleukin [IL]-1β, IL-1RN, IL-4, IL-6, IL-8, IL-10, tumor necrosis factor [TNF], interferon [IFN]-γ, and granulocyte-macrophage colony-stimulating factor) in addition to endometrial cytology. Formalin-fixed endometrial biopsies were histologically assessed for the retention of microcaruncles, dilation of endometrial glands, and inflammation of the mucosa, stratum compactum, and spongiosum. Statistics were performed using SAS 9.4, using a mixed model for repeated measures with mare and treatment as a random effect. All post-hoc analysis was done using a Tukey's honestly significant difference test. Involution was considered complete by Day 15 postpartum in all mares, and the day postpartum had a significant effect on almost all parameters investigated, indicating the immunologic process of involution. Treatment with MCWF decreased the magnitude of bacterial growth in addition to time to negative culture. In addition, MCWF increased the expression of IL-1β, IFNγ, and TNF. Although minimal treatment effect was noted histologically, a decrease in mucosal inflammation was seen in MCWF-treated mares. In conclusion, involution appears to be influenced by the immune system. In addition, MCWF appears to have a bactericidal effect on the postpartum mare, and this may be because of an increase in proinflammatory cytokines. It is unknown if this bactericidal property will improve fertility on the first estrous cycle postpartum, and future studies are needed to determine this., (Copyright © 2020 Elsevier Inc. All rights reserved.)

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., Competing Interests: Declaration of competing interest None., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Problem: Ascending placentitis is one of the leading causes of abortion in the horse. Minimal work has focused on its effect on fetal fluids or the antenatal immune response of the fetus., Methodology: Placentitis was induced via transcervical inoculation of Streptococcus equi ssp Zooepidemicus, and fluids/serum/tissues were collected 4-6 days later following euthanasia. Cytokine concentrations were detected using a multiplex immunoassay within fetal fluids (amniotic and allantoic) and serum (maternal and fetal) in inoculated and control mares. In addition, tissues from fetal (spleen, liver, lung, umbilicus, amnioallantois) and maternal (spleen, liver, lung, chorioallantois, endometrium) origin were analyzed in inoculated and control mares utilizing qPCR for expression of cytokines., Results: No difference in cytokine concentrations in maternal or fetal serum was noted between inoculated and control mares. Concentrations of IL-1β, IL-6, IL-10, and GRO were upregulated in the amniotic fluid following inoculation, with a trend toward higher IL-6 concentration in allantoic fluid. The amnioallantoic tissue separating the two fluids had higher expression of IL-1β and IL-6 following inoculation, while chorioallantois and endometrium upregulated IL-1β and IL-8 expression. IL-1β was upregulated in the maternal spleen following inoculation. Fetal spleens were upregulated in expression of IL-1β, GRO, and IL-6, while IL-6 was higher in fetal liver after inoculation than in controls., Conclusion: The maternal response to placentitis is primarily pro-inflammatory while the fetus appears to play a regulatory role in this inflammation. Additionally, amniotic fluid sampling may be more diagnostic of ascending placentitis than circulating cytokines., (© 2019 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Problem: Progestins are immunomodulatory in a variety of species. In the horse, the most commonly administered synthetic progestin is altrenogest (ALT), but its effect on the immune system of the non-pregnant mare is unknown., Methods: Peripheral blood mononuclear cells (PBMCs) from diestrous mares were incubated with varying concentrations of progesterone (P4) or ALT to assess intracellular production of IFNγ and the expression of select cytokines. Additionally, ten mares received either ALT or VEH daily utilizing a switchback design beginning on the day of ovulation and continuing for 7 days. Circulating PBMCs and endometrial biopsies were obtained to assess the production and expression of the same cytokines., Results: In vitro, both P4 and ALT caused a dose-dependent decrease in intracellular IFNγ in PBMCs. P4 caused a dose-dependent decrease in the expression of IFNγ, IL-10 and IL-4, while ALT caused an increase in the expression of IL-6 and IL-1β in PBMCs. In vivo, ALT suppressed the intracellular levels of IFNγ in PBMCs on d6. While control mares experienced a decrease in IL-1β expression from d0 to d6, ALT-treated mares did not. In the endometrium, ALT increased the expression of IL-1RN and IFNγ in comparison with VEH-treated mares., Conclusion: P4 and ALT appear to alter the immune system of the non-pregnant mare both systemically in addition to locally within the endometrium. Further research is necessary to determine the pathways through which this synthetic progestin functions on the immune system of the horse, and the consequences it may have., (© 2019 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Equine arteritis virus (EAV) is the causative agent of equine viral arteritis (EVA), a reproductive and respiratory disease of horses. Following natural infection, 10 to 70% of infected stallions can become carriers of EAV and continue to shed virus in the semen. In this study, sequential viruses isolated from nasal secretions, buffy coat cells, and semen of seven experimentally infected and two naturally infected EAV carrier stallions were deep sequenced to elucidate the intrahost microevolutionary process after a single transmission event. Analysis of variants from nasal secretions and buffy coat cells lacked extensive positive selection; however, characteristics of the mutant spectra were different in the two sample types. In contrast, the initial semen virus populations during acute infection have undergone a selective bottleneck, as reflected by the reduction in population size and diversifying selection at multiple sites in the viral genome. Furthermore, during persistent infection, extensive genome-wide purifying selection shaped variant diversity in the stallion reproductive tract. Overall, the nonstochastic nature of EAV evolution during persistent infection was driven by active intrahost selection pressure. Among the open reading frames within the viral genome, ORF3, ORF5, and the nsp2-coding region of ORF1a accumulated the majority of nucleotide substitutions during persistence, with ORF3 and ORF5 having the highest intrahost evolutionary rates. The findings presented here provide a novel insight into the evolutionary mechanisms of EAV and identified critical regions of the viral genome likely associated with the establishment and maintenance of persistent infection in the stallion reproductive tract. IMPORTANCE EAV can persist in the reproductive tract of infected stallions, and consequently, long-term carrier stallions constitute its sole natural reservoir. Previous studies demonstrated that the ampullae of the vas deferens are the primary site of viral persistence in the stallion reproductive tract and the persistence is associated with a significant inflammatory response that is unable to clear the infection. This is the first study that describes EAV full-length genomic evolution during acute and long-term persistent infection in the stallion reproductive tract using next-generation sequencing and contemporary sequence analysis techniques. The data provide novel insight into the intrahost evolution of EAV during acute and persistent infection and demonstrate that persistent infection is characterized by extensive genome-wide purifying selection and a nonstochastic evolutionary pattern mediated by intrahost selective pressure, with important nucleotide substitutions occurring in ORF1a (region encoding nsp2), ORF3, and ORF5., (Copyright © 2019 American Society for Microbiology.)

Intrauterine infection and inflammation remain a major cause of preterm labour in women and mares, with little known about small RNA (sRNA) expression in tissue or circulation. To better characterise placental inflammation (placentitis), we examined sRNA expression in the endometrium, chorioallantois and serum of mares with and without placentitis. Disease was induced in 10 mares via intracervical inoculation of Streptococcus equi ssp. zooepidemicus, either with moderate or high levels of inoculum; three uninoculated gestationally matched mares were used as controls. Matched chorioallantois and endometrium were sampled in two locations: Region 1, gross inflammation near cervical star with placental separation and Region 2, gross inflammation without placental separation. In Region 1, 26 sRNAs were altered in chorioallantois, while 20 were altered in endometrium. Within Region 2, changes were more subdued in both chorioallantois (10 sRNAs) and endometrium (two sRNAs). Within serum, we identified nine significantly altered sRNAs. In summary, we have characterised the expression of sRNA in the chorioallantois, the endometrium and the serum of mares with experimentally induced placentitis using next-generation sequencing, identifying significant changes within each tissue examined. These data should provide valuable information about the physiology of placental inflammation to clinicians and researchers alike.

The objectives of this study were to evaluate; 1) the stability of measured serum anti-Müllerian hormone (AMH) concentrations in samples after multiple freeze-thaw cycles, 2) the repeatability of AMH concentrations within mares during the same breeding season as well as across breeding seasons, and 3) the relationship between serum AMH concentrations and fertility (measured as first cycle pregnancy rates) in thoroughbred mares. For the first aim, AMH concentrations (n = 9) were examined across four freeze-thaw cycles with no significant change in measured AMH concentrations. For the second aim, serum AMH concentrations (n = 12) were examined over three successive estrous cycles and over two successive breeding seasons and AMH levels were significantly correlated for individual animals within (r; 0.71-82) or across breeding seasons (r = 0.81). For the third aim, Thoroughbred mares (n = 419) on farms in central Kentucky had blood samples taken during estrus. Pregnancy was determined with transrectal ultrasonography at Days 13-18 after mating and ovulation, and pregnancy outcome was recorded as open, pregnant or twins. The relationships between mare age, serum AMH concentrations and the interaction of age and AMH with pregnancy outcome was examined by nominal logistic regression, and the relationship between serum AMH concentrations and mare age, pregnancy outcome and the interaction of age and pregnancy outcome was examined by ANOVA. Data in this study were then stratified according to quartiles into lower (25%), mid-50% (second and third quartiles combined - 50%) and upper (25%) quartiles for age and serum AMH concentration for further analysis by logistic regression. There were significant effects of mare age and pregnancy outcome, but not their interaction on serum AMH concentrations which were higher (P = 0.04) in pregnant than in open mares (0.65 ± 0.03 vs 0.55 ± 0.04 ng/mL, respectively). Likewise, logistic regression revealed significant effect of mare age and AMH but not their interaction on pregnancy outcome on the first mated cycle. Mares in the lower AMH quartile were more likely to be open at Day 13-18 than mares in the middle (odds ratio (OR) = 1.87)=13 or upper quartile (OR = 2.62) for AMH concentrations. Mares in the mid-50% (OR = 3.91) or upper (OR = 4.97) age quartile were more likely to be open at Day 13-18 compared to mares in the young age quartile. Based upon a Chi-squared analysis, the proportion of pregnant mares differed across age quartiles (P < 0.0001) and was greater (P < 0.05) in the young mare quartile. The proportion of pregnant mares tended to differ across AMH quartile (P = 0.1), and when adjusted for age quartile using the Cochran-Mantel-Haenszel Test, the proportion of pregnant mares differed (P < 0.05) across AMH quartile. In conclusion, mares with peripheral AMH concentrations in the lowest quartile had lower fertility compared to mares in the mid-50% or upper quartile., (Copyright © 2018 Elsevier Inc. All rights reserved.)

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 × 10 6 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., (Copyright © 2018. Published by Elsevier Inc.)

MicroRNAs (miRNAs) have important posttranscriptional regulatory abilities, and there is considerable interest in evaluating their expression patterns in different pathophysiological states. The most common method of quantifying miRNA expression is quantitative reverse transcription PCR; however, the identification of tissue-specific and species-specific reference miRNA is a prerequisite for miRNA expression analysis. Currently, no reference genes have been described for evaluating miRNA expression in equine serum and chorioallantoic membrane (CAM) during pregnancy. The aim of the present study was to characterize reference genes for normalization of miRNA expression in CAM and serum in the pregnant equine. To identify the most stable miRNAs in serum, expression of potential candidates was evaluated in serum samples from diestrous mares, pregnant mares and geldings. To identify the most stable miRNAs in CAM, expression of potential candidates was evaluated in CAM, collected from mares at 4, 6 and 10 months of pregnancy and immediately postpartum. From a previously generated miRNA sequencing dataset, two separate lists of potential reference miRNAs were identified (serum and CAM) using the NormFinder program, in addition to the commonly used small RNA normalizers, 5S rRNA and U6 snRNA. The putative reference miRNAs were selected using geNorm and NormFinder. In case of a nonsignificant correlation between the results of ranking and stability value between these two programs, ranking from BestKeeper was also included. NormFinder and geNorm consistently identified eca-miR-21-5p, eca-let-7a-5p and eca-miR-10a-5p as the three most stable reference genes for the normalization of serum miRNAs. Within CAM samples, the average ranking obtained from the ranking of NormFinder, geNorm and BestKeeper identified eca-miR-8908a-1-5p, eca-miR-369-5p and eca-miR-106a-5p as the three most stable miRNAs. These observations provide information about equine-specific reference genes that can be used for normalizing miRNAs expression patterns in CAM and serum during the equine pregnancy.

Eleven pregnant pony mares (D270-326) were administered ceftiofur sodium intramuscularly at 2.2 mg/kg (n = 6) or 4.4 mg/kg (n = 5), once daily. Plasma was obtained prior to ceftiofur administration and at 0.5, 1, 2, 4, 8, 12, and 24 hr after administration. Eight pony mares were re-enrolled in the study at least 3 days from expected foaling to ensure steady-state concentrations of drug at the time of foaling. Mares were administered ceftiofur sodium (4.4 mg/kg, IM) daily until foaling. Parturition was induced using oxytocin 1 hr after ceftiofur sodium administration. Allantoic and amniotic fluid, plasma, and colostrum samples were collected at time of foaling. Serial foal plasma samples were obtained. Placental tissues were collected. Desfuroylceftiofur acetamide (DCA) concentrations were measured in samples by high-performance liquid chromatography (HPLC). Mean (±SD) peak serum concentrations of DCA were 3.97 ± 0.50 μg/ml (low dose) and 7.45 ± 1.05 μg/ml (high dose). Terminal half-life was significantly (p = .014) shorter after administration of the low dose (2.91 ± 0.59 hr) than after administration of the high dose (4.10 ± 0.72 hr). The mean serum concentration of DCA from mares at time of foaling was 7.96 ± 1.39 μg/ml. The mean DCA concentration in colostrum was 1.39 ± 0.70 μg/ml. DCA concentrations in allantoic fluid, amniotic fluid, placental tissues, and foal plasma were below the limit of quantification (<0.1 μg/ml) and below the minimum inhibitory concentration of ceftiofur against relevant pathogens. These results infer incomplete passage of DCA across fetal membranes after administration of ceftiofur sodium to normal pony mares., (© 2017 John Wiley & Sons Ltd.)

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., (Copyright © 2017 Elsevier Inc. All rights reserved.)

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. IMPORTANCE The major challenge for the worldwide control of EAV is that this virus has the distinctive ability to establish persistent infection in the stallion's reproductive tract as a mechanism to ensure its maintenance in equid populations. Therefore, the precise identification of tissue and cellular tropism of EAV is critical for understanding the molecular basis of viral persistence and for development of improved prophylactic or treatment strategies. This study significantly enhances our understanding of the EAV carrier state in stallions by unequivocally identifying the ampullae as the primary sites of viral persistence, combined with the fact that persistence involves continuous viral replication in fibrocytes (possibly including tissue macrophages) and T and B lymphocytes in the presence of detectable inflammatory responses, suggesting the involvement of complex viral mechanisms of immune evasion. Therefore, EAV persistence provides a powerful new natural animal model to study RNA virus persistence in the male reproductive tract., (Copyright © 2017 American Society for Microbiology.)