A 1-year intact, phenotypic female Doberman Pinscher dog, was evaluated for suspected sexual development disorder. Patient had a history of white mucoid vaginal discharge with no estrous signs or behavior. Clitoral hypertrophy with a palpable os clitoris were noted. Transabodminal ultrasonography revealed a right gonad and tubular structures (possibly uterus). Exploratory laparotomy revealed 2 underdeveloped gonads in caudodorsal abdomen and a markedly underdeveloped right uterine horn. Histopathology confirmed bilateral ovotestes. Karyotyping (number and morphology of chromosomes) and fluorescence in situ hybridization (XX and XY cells) results were normal. Samples were PCR positive for sex-determining region Y (SRY) and X-linked androgen receptor gene. We concluded that the patient was either a mosaic or chimera with normal female 78,XX and normal male 78,XY cells and genetically a mix of male and female. Final diagnosis was sex chromosome (78,XX/XY), SRY-positive, ovotesticular, disorder of sexual development with female phenotype.

Mobility of the equine conceptus is essential for maternal recognition of pregnancy (MRP) and is regulated by the conceptus's own derived prostaglandin secretion. Our objective was to compare prostaglandin-related genes and other genes of interest during the expected MRP time. Light breed mares were examined daily by transrectal ultrasound when in estrus until the day (D) of ovulation. Mares in the pregnant (P) group were bred by artificial insemination with >500 million progressively motile normal sperm from one fertile stallion until D0. Conceptuses were detected and measured via transrectal ultrasound and collected via uterine lavage using 0.9% NaCl through a transcervical bivona catheter. Endometrium was collected via a transcervical biopsy punch following conceptus retrieval. Endometrium of non-pregnant (NP) mares was collected at: D11 (NP11; n=6) and D13 (NP13; n=7), while endometrium from the pregnant horn and conceptuses was collected at D11 (P11; n=7), D13 (P13; n=8), and D16 (P16; n=6). Total RNA from trophoblast and endometrial samples was isolated and evaluated for expression of PTGS2, PTGES, mPGES2, PTGER4, PTGER2, PTGIS, PTGIR, HPGD, SLCO2A1, CRTC2, NR3C1, CBR1, CREB, NF-Kβ, SPP1, IGFBP1, ESR1 and DNTTIP2 using Real-time PCR. Mean threshold cycle was determined and normalized to the reference gene (GAPDH and ACTB) (ΔCT). Statistical analyses were performed using JMP Pro 16, and data was ranked, transformed if necessary, followed by a restricted maximum likelihood model with mare as arandom factor to test differences between Groups and Day. Regarding endometrial mRNA, NP11 had higher expression for NF-Kβ, PTGIS, PTGIR and CBR1 compared to NP13 (p<0.007). ESR1, mPGES2, PTGIS, and CBR1 mRNA were higher in P11 compared to P16 (p<0.05), while CRTC2 was higher in P16 compared to P11 (p<0.06). Lastly, NF-Kβ, PTGIR and CBR1 were higher in P11 compared to P13 (p<0.01), but NF-Kβ P13 was lower compared to P16 (p<0.04). Regarding the conceptuses, CREB and PTGFR expressions were higher in D16 compared to D11 (p<0.003), while CRTC2, NR3C1, NF-Kβ and SPP1 were higher in D16 compared to D11 and 13 (p<0.02). D13 conceptuses had lower expression of HPGD compared to D11 and 16 (p<0.02). Lastly, D11 conceptuses had higher expression of mPGES2, PTGIS and PTGER4 compared to D13 and D16 (p<0.03). Higher expression of D11 PTGIS from both tissues may increase nutrient and blood flow to the endometrium in support of the early conceptus. The upregulation of endometrial NF-Kβ at D11 may be a result of endometrial receptivity through the regulation of inflammation, and the up-regulation of HPGD may function to regulate PGE2 during early pregnancy. Understanding embryonic and endometrial prostaglandin regulation during MRP may help to develop future therapies to ameliorate pregnancy loss in the horse.

Cytokines and their receptor systems are present at the fetomaternal interface of some species and regulate trophoblast biology. The cytokine and chemokine systems are key in the communication between fetal derived trophoblast cells and the maternal tissue. However, evidence for the presence of cytokines and their receptors during early equine pregnancy is lacking. Our objective was to investigate interleukins, chemokine ligands, dendritic cells and TNFα in equine serum, conceptuses and endometrium during early pregnancy. Daily transrectal ultrasound examinations of the mares were performed when in estrus until the day (D) of ovulation (OV). An endometrial biopsy was taken from non-pregnant (NP) mares at: D11 (NP11; n=6) or D13 (NP13; n=7). Mares in the pregnant (P) group were bred by artificial insemination from a single fertile stallion, and transrectal ultrasound was performed to detect and measure conceptuses. On the day of sample collection, conceptuses were collected through a transcervical bivona catheter and uterine lavage using 0.9% NaCl, and this was followed by an endometrial biopsy. Pregnant mares’ conceptuses and endometrial biopsies were retrieved at D11 (P11; n=7), D13 (P13; n=8), or D16 (P16; n=6). A D16 sample was not obtained for NP group. A Bradford assay was performed on extracted endometrium and conceptus tissue to determine total protein concentration,followed by a bead-based equine validated multiplex assay (Luminex Corp. Austin, TX) to quantify concentration of the following equine specific inflammatory markers: IL-4, IL-17, IL-10, IL-1β, CCL2, CCL3, CCL5, CCL11, sCD-14 and TNFα. A restricted maximum likelihood model was performed to compare the protein concentration between groups and days in JMP Pro 16 (Cary, NC) with the mare as a random factor and significance set at p<0.05. Cytokine and chemokine serum concentrations were similar between groups and days of collection. Regarding the endometrium, IL-17 U/mg was higher (p<0.05) in group NP13 [median (interquartile)] [11.4 (8.2, 21.7)] than NP11 [4.1 (2.6, 5.3)]. In pregnant mares, endometrial IL-17 was higher in P13 group [6.3 (3.4, 13.9)] compared to group P16 [2.5 (2.1, 4.8)] (p<0.05). In conceptuses, D11 had significantly higher concentrations of CCL2 [1350.2 (793, 1839.5); p<0.03], CCL3 [1990.9 (998.6, 3083.8); p<0.01], CCL5 [363.2 (149.6, 480.6); p<0.006] and CCL11[3143.7 (1764.2, 5221.9); p<0.01 compared to D16 [66.7 (38.8, 644.8); 67.8 (43, 572.30); 9.1 (6.6, 96.3); 196.2 (150.9, 1436.6)], respectively. Higher chemokine concentrations in the earlier embryos may reflect the recruitment of endometrial leukocytes. Further investigation into the role of cytokines and chemokines in early equine pregnancy is warranted.

Screening methods for endometritis are often performed through endometrial culture and cytology with a variety of techniques including endometrial swab, cytobrush, low-volume lavage, or biopsy. With growing research interest in equine reproductive immunology and low sensitivity of current screening tools for endometritis, one area that would be of immediate benefit to clinicians and clients is inflammatory diagnostic development. Previous research detected inflammatory markers in low-volume lavage fluid in mares with chronic and acute endometritis (Lection et al. AAEP Proceedings. 2020; 66: 150-151). Therefore, our objective was to investigate the utility of the endometrial swab and cytobrush to screen mares for endometritis. Mares (n=84) of reproductive age ranging 3-20 years of age had an endometrial swab (n=103) and/or followed by cytobrush (n=94) taken once in the same estrous cycle and placed in Amies media (n=172) or PBS (n=25). Aerobic culture was performed in 139 of those samples. An endometrial biopsy was taken on a subset of mares (n=46) and graded by a board-certified veterinary pathologist. The media in which the cytobrushes and swabs were stored was used in a bead-based multiplex assay (Luminex Corp. Austin, TX) to quantify concentration of the following equine specific biomarkers: IFN-α, IFN-γ, IL-1β, IL-4, IL-10, IL-17, sCD14, TNF-α, CCL2, CCL3, CCL5, and CCL11. A Shapiro-Wilk test was used to check normality, followed by a Wilcoxon testperformed in JMP Pro 16 (Cary, NC) to assess for significant differences in inflammatory marker concentrations between healthy mares and those with either cytology-diagnosed endometritis (≥1 neutrophil/hpf), positive bacterial culture, or poor biopsy scores (IIB or III on the Kenney-Doig scale). IFN-γ (P≤0.02) and IL-17 (P≤0.04) were significantly increased in mares with poor biopsy scores in both swab and cytobrush compared to mares with biopsy I or IIA. Mares with inflammation on cytology showed increased concentration of IL-1β (P≤0.05) and decreased levels of IFN-γ (P≤0.02) and CCL5 (P≤0.03) in swab. Mares with intrauterine fluid on ultrasound had increased IL-17 (P≤0.05) on cytobrush. Bacterial growth was detected in 62/139 samples, with 31 of those being gram-positive. sCD14 and TNF-α tended to be increased in cytobrush samples from mares with a positive culture, and with gram-positive bacteria compared to gram-negative, respectively (P=0.06). Cytokine levels significantly differed between cytobrush and swab for IFN-γ (P≤0.004) with increased levels for cytobrush, likely due to the higher amount of cellular exfoliation. Several inflammatory markers show promise as an ancillary screening test for equine endometritis. In conclusion, as equine reproductive immunology expands as a field, our understanding of patterns of inflammatory cytokines and chemokines can allow for new diagnostic techniques for endometritis in mares.

Interferons (IFN) are involved in the maintenance and establishment of pregnancy in a variety of species. There are three types of IFNs, which include type I (IFN-α, β, ε, τ, and δ), type II (IFN-γ), and type III (IFN-λ1, λ2 and λ3). In the horse, IFNA, IFND and IFNO are expressed in the equine trophoblast, and IFNE is expressed in higher abundance in equine endometrium during the luteal phase. However, investigation of IFNs and their receptors during early equine pregnancy is still lacking. Our objective was to investigate the expression of IFN types I and II and their receptors, and other IFN related genes in equine conceptuses and endometrium during early pregnancy. Mares were examined daily by transrectal ultrasound when in estrus until the day (D) of ovulation (OV). Pregnant (P) group mares were bred using artificial insemination by a single fertile stallion. Conceptuses were detected and measured via transrectal ultrasound and collected via uterine lavage using 0.9% NaCl through a transcervical bivona catheter. Following conceptus retrieval, an endometrial biopsy was collected. Endometrial samples from nonpregnant (NP) mares were collected at D11 (NP11; n=6) and D13 (NP13; n=7), while endometrium and conceptuses were collected from pregnant mares at D11 (P11; n=7), D13 (P13; n=8), and D16 (P16; n=6). As the majority of nonpregnantmares would have undergone luteolysis, a D16 sample was not included in this study. Total RNA was isolated and evaluated for expression of interferon-related genes using Real-time PCR. The mean threshold cycle was determined and then normalized to the mean of the reference genes (GAPDH and ACTB) (ΔCT). A restricted maximum likelihood model was performed in JMP Pro 16 (Cary, NC) with mare as a random factor with significance set at P<0.05. There was no amplification for IFNB and IFND in the endometrium, and no amplification of IFNG, IFNB and IFNAR2 in the conceptus. In the endometrium, IFNAR1 and IFNGR1 had higher expression in P11 compared to P13 (P<0.02). Expression of endometrial IFNG mRNA was higher in P11 and P16 compared to P13 (P<0.004). There was no difference in the mRNA relative abundance of endometrial ISG15 and IFITM1 between groups or days. In the conceptus, IFNA, IFNO, and IFNGR2 expression were higher in D16 compared to other ages (P<0.05). D11 and D16 conceptuses had higher expression of IFNAR1 compared to D13 (P<0.0002), while IFNGR1 was higher in D16 compared to D11 (P<0.004). The role of interferons during maternal recognition of pregnancy in the horse is unknown. Unlike the pig, the horse embryo does not seem to express IFNG. Further investigation is underway.

Hydropsical conditions are exceedingly rare in the horse. However, when they occur, they are true emergencies due to the severe enlargement of the pregnant uterus, which can result in clinical signs, such as an enlarged round abdomen, dyspnea, reluctance to walk, and colic, and may lead to the development of abdominal wall disease. The pathogenesis of hydropsical conditions is not fully elucidated, but they have been associated with placentitis and fetal abnormalities. This report describes six cases of hydropsical conditions in mares with or without concurrent abdominal wall disease. Five out of six cases were hydrallantois, and of these five, two mares had abdominal wall disease; the remaining one out of six cases was hydramnios. All mares were treated by termination of the pregnancy through gradual fluid drainage transcervically over a number of hours, and their fetuses were delivered vaginally. All fetuses were euthanized immediately after vaginal delivery. Of the six mares, two had signs of placentitis, two were confirmed seropositive for leptospirosis, and two were euthanized (one because of a vaginal tear that communicated through the peritoneum and one mare that developed abdominal wall rupture and laminitis). The remaining 4 mares were available for follow-up; three mares were not rebred, and one mare became an embryo donor, with a successful embryo recovery. We reported the prevalence of leptospira involvement in two out of six cases of hydrallantois and also described the clinical outcome of the mares after treatment with slow fetal fluid drainage.