Metagenomic analysis unravels novel taxonomic differences in the uterine microbiome between healthy mares and mares with endometritis.

BACKGROUND: The application of high throughput technologies has enabled unravelling of unique differences between healthy mares and mares with endometritis at transcriptomic and proteomic levels. However, differences in the uterine microbiome are yet to be investigated. OBJECTIVES: The present study was aimed at evaluating the differences in uterine microbiome between healthy mares and mares with endometritis. METHODS: Low-volume lavage (LVL) samples were collected from the uterus of 30 mares classified into healthy (n = 15) and endometritis (n = 15) based on their reproductive history, intrauterine fluid accumulation, gross appearance of LVL samples, endometrial cytology and bacterial culture. The samples were subjected to 16S rRNA sequencing. RESULTS: Notable differences in the uterine microbiome were observed between healthy mares and mares with endometritis at various taxonomic levels. In healthy mares, the most abundant phylum, class, order and family were Firmicutes, Bacilli, Bacillales and Paenibacillaceae, respectively. In contrast, the most abundant corresponding taxonomic levels in mares with endometritis were Proteobacteria, Gammaproteobacteria, Enterobacterales and Enterobacteriaceae, respectively. At the genus level, Brevibacillus and Paenibacillus were more abundant in healthy mares, whereas Escherichia, Salmonella and Klebsiella were more abundant in mares with endometritis. In healthy mares, Brevibacillus brevis was the most abundant species, followed by Brevibacillus choshinensis and Paenibacillus sp JDR-2. However, in mares with endometritis, Escherichia coli was the most abundant species, followed by Salmonella enterica and Klebsiella pneumoniae. CONCLUSIONS: These results confirmed the previously reported presence of a uterine microbiome in healthy mares and helped unravel some alterations that occur in mares with endometritis. The findings can potentially help formulate new approaches to prevent or treat equine endometritis.

Comparison of two pharmacological protocols for inducing ex copula ejaculation in donkeys.

This study aimed to compare the efficacy of two pharmacological protocols for inducing ex copula ejaculation in donkeys. Seven healthy jacks (male donkeys) aged 4 to 20 years (median 8 years) and weighing 136.2±4.17 kg (mean±SE) were enrolled. Using a crossover design, each jack was subjected in a random order to two treatment protocols (IX and IDO) with an interval of 7 days between the two protocols. Each jack was orally administered 3 mg/kg imipramine hydrochloride followed 2 hours later by intravenous (IV) administration of 1.1 mg/kg xylazine hydrochloride (IX protocol) or 0.02 mg/kg detomidine hydrochloride and 20 IU total dose oxytocin (IDO protocol). The jacks were monitored for behavioral changes and ejaculation up to 3 hours from the beginning of each protocol. A total of 22 ex copula ejaculation replicates were attempted. Both protocols resulted in deep sedation and partial to complete penile protrusion in all jacks. There was no difference in the efficacy with the IX protocol inducing ejaculation in 1 of the 11 replicates and the IDO protocol inducing ejaculation in none of the 11 replicates. The results suggest that neither of the two tested pharmacological protocols were effective in inducing ex copula ejaculation in donkeys.

A rare case of vulvar discharge associated with exogenous oestrogen exposure in a spayed Weimaraner bitch.

OBJECTIVE: This report documents a rare case of vulvar discharge associated with exogenous oestrogen exposure in a large-breed dog. CASE DESCRIPTION: A 4-year-old spayed Weimaraner bitch was presented for evaluation of inappetence and intermittent sanguineous vulvar discharge. Physical examination, vaginal cytology, haematological, and ultrasonographic findings were indicative of a uterine stump pyometra. A celiotomy was performed, and the uterine stump appeared grossly cystic and thickened. Histopathological evaluation of the removed uterine stump and ovarian pedicles revealed cystic endometrial hyperplasia and no ovarian tissue. Fifteen days after surgery, the patient presented again with a sanguineous vulvar discharge. Vaginal cytology revealed predominantly superficial cells, indicating oestrogen influence. Further questioning of the owner revealed the long-term use of a topical oestrogen cream by a member of the household. Serial examinations were performed, and the cytology remained uniform, with predominantly superficial cells, indicating continued oestrogen influence. Progesterone and anti-Müllerian hormone tests were negative, which made the presence of ectopic ovarian tissue unlikely. These results coupled with the history of topical oestrogen cream use in the household suggested that the patient's clinical signs were most likely due to exogenous oestrogen exposure. After the owner implemented various recommendations made to prevent the exposure, the clinical signs resolved completely. CONCLUSIONS: This case demonstrates that although rare, exogenous oestrogen exposure can be a cause of vulvar discharge in a large-breed spayed bitch. Therefore, regardless of the breed, exogenous oestrogen exposure must be included in the list of differential diagnoses for all clinical presentations associated with oestrogenic influence in dogs.

A retrospective study of canine transmissible venereal tumour in Grenada, West Indies.

BACKGROUND: Canine transmissible venereal tumour (CTVT) is a naturally occurring neoplasia affecting dogs worldwide. Previous CTVT studies in Grenada were limited to case records of dogs with neoplastic conditions at a veterinary diagnostic laboratory. OBJECTIVES: The present retrospective study aimed to determine the occurrence and risk factors of CTVT in a wider population of owned dogs presented to a university-affiliated veterinary hospital between 2008 and 2018. METHODS: Data on the age, breed, gender, and gonadectomy status were retrieved from an electronic database and analyzed using logistic regression. RESULTS: Of the 7180 dogs presented during the period, 102 dogs (1.4%) were diagnosed with CTVT. A higher predisposition was observed in Grenadian pothounds (odds ratio [OR] = 22.8, 95% confidence interval [CI] 10.3-50.4; p < 0.001) and mixed-breed dogs (OR = 9.2, 95% CI 4.1-20.7; p < 0.001) in comparison to the purebreds. Neutered dogs (OR = 2.2, 95% CI 1.4-3.3; p < 0.001) were at an increased risk of CTVT than intact dogs. Age and gender were not identified as significant risk factors. CONCLUSIONS: The percentage of dogs with CTVT in this study represents a crude estimate of the CTVT prevalence in the owned dog population in Grenada. Further studies including both owned and free-roaming dogs are required for a more accurate estimation of the CTVT prevalence in the region. Our results indicate that breed and gonadectomy status are significant risk factors for the occurrence of CTVT in Grenada.

Pharmacologically Induced Ex Copula Ejaculation in Horses and Donkeys.

Pharmacologically induced ex copula ejaculation is a method used for collection of semen when the traditional methods of semen collection are not feasible. Common indications for this method include health issues that either preclude the physical act of mating or result in impaired erection and ejaculation. The method also offers an alternative when there is a lack of equipment and facilities required for semen collection using the conventional artificial vagina method. A variety of pharmacological protocols for ex copula ejaculation in stallions have been evaluated in both research and clinical settings with no serious side-effects reported. In general, these protocols included tricyclic antidepressants, alpha-2 adrenergic receptor agonists, and smooth muscle stimulators, either individually or in combination. Although there is a lot of variation in the ejaculatory rates among studies, a combination of imipramine and xylazine appears to be an effective option for inducing ejaculation in stallions. In cases where this protocol is not successful, collections should be reattempted using a combination of imipramine, detomidine, and oxytocin. Regardless of the protocol used, a quiet environment with minimal disturbance is associated with a better outcome. In contrast to the body of literature available on pharmacologically induced ex copula ejaculation in horses, only a few studies have been published so far on this topic in donkeys. Further studies are warranted to investigate whether pharmacologically induced ex copula ejaculation is an effective method of semen collection in jacks and to compare different pharmacological protocols for inducing ejaculation in jacks.

Qualitative analysis and functional classification of the uterine proteome of mares in oestrus and dioestrus.

Quantitative analysis of the uterine flush fluid proteome of mares in oestrus and dioestrus has been previously reported. The objectives of this study were to: a) evaluate qualitative differences in the uterine flush fluid proteome between mares in oestrus and mares in dioestrus and b) perform a functional classification of proteins either unique to each stage or common between the two stages. Uterine flush fluid samples were collected from 8 light breed mares in either oestrus (n = 5) or dioestrus (n = 3). Proteomic analysis of the samples was conducted using liquid chromatography-tandem mass spectrometry. Proteins exclusively detected in oestrus or dioestrus and those common to both stages were identified using the Scaffold software (version 4.4.8, Proteome Software Inc., Portland, OR). The identified proteins were classified into gene ontology (GO) categories (cellular component [CC], molecular function [MF] and biological process [BP]) using the PANTHER (www.pantherdb.org) classification system version 14.0. Of 172 proteins identified, 51 and 28 were exclusively detected in mares in oestrus and dioestrus, respectively, and 93 proteins were common to both stages. The most represented terms in various GO categories were similar among the three subsets of proteins. The most represented CC terms were extracellular region and cell, the most represented MF terms were catalytic activity and binding, and the most represented BP terms were metabolic process and cellular process. In conclusion, proteomic analysis of the uterine flush fluid enabled the identification of subsets of proteins unique to oestrus or dioestrus, or common to both stages. The results of this study can serve as a baseline for future research focused on finding stage-specific protein markers or evaluating differences in the uterine flush fluid proteome between normal mares and those with uterine disease.

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.

Canine cryptorchidism: An update.

This review article is aimed at providing an update on canine cryptorchidism based on the research studies published in this area over the last 26 years. Although a majority of these research studies were directed at investigating the genetic and molecular basis of cryptorchidism, the mechanisms underlying this problem in dogs are still not clear. Future studies using advanced genomic and molecular approaches and including a larger number of dogs of multiple breeds are required to further our understanding of canine cryptorchidism. Diagnosis and treatment of cryptorchidism in dogs have not received much attention. Research advancements include investigation of ultrasonography and anti-Müllerian hormone assay as potential diagnostic methods. Advancements in treatment include the use of laparoscopic-assisted cryptorchidectomy. Based on the available literature, this method appears to be the best for removal of abdominal cryptorchid testes from dogs. However, future studies that compare laparoscopic-assisted cryptorchidectomy with the traditional approaches are required for a more conclusive determination of the best method for cryptorchidectomy in dogs.

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.

Regulation of pluripotency of inner cell mass and growth and differentiation of trophectoderm of the bovine embryo by colony stimulating factor 2.

Colony-stimulating factor 2 (CSF2) enhances competence of the bovine embryo to establish and maintain pregnancy after the embryo is transferred into a recipient. Mechanisms involved could include regulation of lineage commitment, growth, or differentiation of the inner cell mass (ICM) and trophectoderm (TE). Experiments were conducted to evaluate regulation by CSF2 of pluripotency of the ICM and differentiation and growth of the TE. Embryos were cultured with 10 ng/ml recombinant bovine CSF2 or a vehicle control from Days 5 to 7 or 6 to 8 postinsemination. CSF2 increased the number of putative zygotes that developed to blastocysts when the percent of embryos becoming blastocysts in the control group was low but decreased blastocyst yield when blastocyst development in controls was high. ICM isolated from blastocysts by lysing the trophectoderm using antibody and complement via immunosurgery were more likely to survive passage when cultured on mitomycin C-treated fetal fibroblasts if derived from blastocysts treated with CSF2 than if from control blastocysts. There was little effect of CSF2 on characteristics of TE outgrowths from blastocysts. The exception was a decrease in outgrowth size for embryos treated with CSF2 from Days 5 to 7 and an increase in expression of CDX2 when treatment was from Days 6 to 8. Expression of the receptor subunit gene CSF2RA increased from the zygote stage to the 9-16 cell stage before decreasing to the blastocyst stage. In contrast, CSF2RB was undetectable at all stages. In conclusion, CSF2 improves competence of the ICM to survive in a pluripotent state and alters TE outgrowths. Actions of CSF2 occur through a signaling pathway that is likely to be independent of CSF2RB.

Fructose-1,6-bisphosphate has anticonvulsant activity in models of acute seizures in adult rats.

A variety of observations suggest that decreasing glycolysis and increasing levels of reduced glutathione, generated by metabolism of glucose through the pentose phosphate pathway, would have an anticonvulsant effect. Because fructose-1,6-bisphosphate (F1,6BP) shifts the metabolism of glucose from glycolysis to the pentose phosphate pathway, it was hypothesized to have anticonvulsant activity. The anticonvulsant activity of F1,6BP was determined in rat models of acute seizures induced by pilocarpine, kainic acid, or pentylenetetrazole. The efficacy of F1,6BP was compared with that of 2-deoxyglucose (2-DG; an inhibitor of glucose uptake and glycolysis), valproic acid (VPA), and the ketogenic diet. One hour before each convulsant, Sprague Dawley rats received either saline (as seizure controls), F1,6BP (0.25, 0.5 or 1 g/kg), 2-DG (0.25 or 0.5 g/kg), or VPA (0.3 g/kg). Additional animals received the ketogenic diet (starting at 20 or 60 d old). Time to seizure onset, seizure duration, and seizure score were measured in each group. F1,6BP had dose-dependent anticonvulsant activity in all three models, whereas VPA had partial efficacy. 2-DG was only effective in the pilocarpine model. The ketogenic diet had no effect in these models. F1,6BP was also partially effective when given at the first behavioral seizure after pilocarpine. Administration of sodium lactate, which bypasses the block in the glycolytic pathway, abolished the anticonvulsant activity of 2-DG in the pilocarpine model, but only decreased the efficacy of F1,6BP. These data demonstrate that F1,6BP has significant anticonvulsant efficacy.