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1.
Sci Rep ; 14(1): 3151, 2024 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-38326534

RESUMO

Embryo-maternal crosstalk is essential to establish pregnancy, with the equine embryo moving throughout the uterus on days 9-15 (ovulation = day 0) as part of this interaction. We hypothesized that the presence of a mobile embryo induces local changes in the gene expression of the endometrium. On Day 12, the endometrial transcripts were compared among three groups: uterine horn with an embryo (P+, n = 7), without an embryo (P-, n = 7) in pregnant mares, and both uterine horns of nonbred mares (NB, n = 6). We identified 1,101 differentially expressed genes (DEGs) between P+ vs. NB and 1,229 DEGs between P- vs. NB. The genes upregulated in both P+ and P- relative to NB were involved in growth factor pathway and fatty acid activation, while downregulated genes were associated with oxytocin signaling pathway and estrogen receptor signaling. Comparing the transcriptome of P+ to that of P-, we found 59 DEGs, of which 30 genes had a higher expression in P+. These genes are associated with regulating vascular growth factors and the immune system, all known to be essential in early pregnancy. Overall, this study suggests that the mobile embryo influences the endometrial gene expression locally.


Assuntos
Endométrio , Útero , Gravidez , Cavalos , Animais , Feminino , Endométrio/metabolismo , Embrião de Mamíferos/metabolismo , Ocitocina/metabolismo , Ovulação
2.
Equine Vet J ; 2023 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-37344926

RESUMO

BACKGROUND: Abnormal or undesired mare behaviours are often assumed to be associated with ovarian abnormalities. OBJECTIVES: We aimed to determine the incidence of abnormal behaviours and their association with concentrations of one or more ovarian hormones associated with a granulosa cell tumour (GCT). STUDY DESIGN: Retrospective descriptive. METHODS: A total of 2914 hormonal profile samples submitted with the words behave, behaviour, or behaving in the submission history were analysed. The association between reported abnormal behaviours and concentrations of testosterone, anti-Müllerian hormone (AMH), inhibins and inhibin-B were assessed. Statistical analysis was performed using a Chi-squared test of association. RESULTS: Of the 2914 cases that were submitted due to behaviour issues, 2506 (86%) did not have any of the measured hormones reach GCT-like concentrations. The remaining 408 cases had either one (63%), two (25.5%), or three (11.5%) hormones with concentrations consistent with those from confirmed GCT cases. Testosterone had the lowest percent of GCT-like values among the cases (7.7%), compared with AMH (9.4%), inhibins (9.6%) and inhibin B (8.7%). Stallion-like behaviour was significantly associated with increased concentrations of all four hormones. In contrast, aggression, oestrous and other abnormal behaviours were significantly less likely to be associated with increased concentrations of the hormones. MAIN LIMITATIONS: Retrospective study, using sample submission history. CONCLUSION: Overall, the abnormal behaviours among mares, except the stallion-like behaviour, were not associated with increased ovarian hormones. These results highlight the common misassumption about the involvement of the ovaries in 'abnormal behaviours' or 'undesirable behaviours' of mares.

3.
J Vet Intern Med ; 37(2): 713-717, 2023 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-36692189

RESUMO

BACKGROUND: Cerebrospinal fluid (CSF) is highly labile and delayed processing might alter results of analysis. HYPOTHESIS/OBJECTIVES: To determine the effects of time and addition of autologous serum on cytological evaluation of CSF. ANIMALS: Ten client-owned adult horses requiring euthanasia. METHODS: Prospective study. Serum and CSF were collected from each horse before and within 10 minutes after euthanasia. CSF samples were divided into 15 aliquots (2 mL each); 1 aliquot was submitted for routine CSF analysis within 60 minutes of collection. Four drops of autologous serum were added to 7 of the aliquots, and stored at 4°C (serum group); the remaining 7 samples were stored unaltered at 4°C (control group). Total nucleated cell count (TNCC) and cell morphology score were done at T4, T8, T12, T24, T48, T72, and T96 hours after collection. Protein concentration was measured in the control group at T0 and T96 hours. RESULTS: The cell morphology scores were significantly different in the control group at T48 (median 2, range 0-4), T72 (2, 0-4), and T96 (3, 0-4) in comparison to T0 (1). No change was observed in the serum group. TNCC remained stable over time in both groups. No statistically significant difference in CSF protein concentration was found between T0 and T96. CONCLUSIONS AND CLINICAL IMPORTANCE: The addition of autologous serum to an aliquot of CSF sample before shipping improves the preservation of cell morphology up to 96 hours after collection.


Assuntos
Líquido Cefalorraquidiano , Cavalos , Animais , Estudos Prospectivos , Fatores de Tempo , Contagem de Células/veterinária
4.
Int J Mol Sci ; 22(22)2021 Nov 09.
Artigo em Inglês | MEDLINE | ID: mdl-34829994

RESUMO

A sufficient vascular network within the feto-maternal interface is necessary for placental function. Several pregnancy abnormalities have been associated with abnormal vascular formations in the placenta. We hypothesized that growth and expansion of the placental vascular network in the equine (Equus caballus) placenta is regulated by estrogens (estrogen family hormones), a hormone with a high circulating concentration during equine gestation. Administration of letrozole, a potent and specific inhibitor of aromatase, during the first trimester (D30 to D118), decreased circulatory estrone sulfate concentrations, increased circulatory testosterone and androstenedione concentrations, and tended to reduce the weight of the fetus (p < 0.1). Moreover, the gene expression of CYP17A1 was increased, and the expression of androgen receptor was decreased in the D120 chorioallantois (CA) of letrozole-treated mares in comparison to that of the control mares. We also found that at D120, the number of vessels tended to decrease in the CAs with letrozole treatment (p = 0.07). In addition, expression of a subset of angiogenic genes, such as ANGPT1, VEGF, and NOS2, were altered in the CAs of letrozole-treated mares. We further demonstrated that 17ß-estradiol increases the expression of ANGPT1 and VEGF and increases the angiogenic activity of equine endothelial cells in vitro. Our results from the estrogen-suppressed group demonstrated an impaired placental vascular network, suggesting an estrogen-dependent vasculogenesis in the equine CA during the first trimester.


Assuntos
Estrogênios/genética , Cavalos/genética , Letrozol/farmacologia , Neovascularização Fisiológica/genética , Androstenodiona/genética , Angiopoietina-1/genética , Animais , Aromatase/genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Cavalos/crescimento & desenvolvimento , Relações Materno-Fetais/efeitos dos fármacos , Neovascularização Fisiológica/efeitos dos fármacos , Placenta/irrigação sanguínea , Placenta/efeitos dos fármacos , Gravidez , Primeiro Trimestre da Gravidez , Receptores Androgênicos/genética , Esteroide 17-alfa-Hidroxilase/genética , Testosterona/genética , Fator A de Crescimento do Endotélio Vascular/genética
5.
Am J Reprod Immunol ; 85(5): e13363, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33098605

RESUMO

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.


Assuntos
Doenças dos Cavalos/imunologia , Interleucina-6/imunologia , Doenças Placentárias/imunologia , Infecções Estreptocócicas/imunologia , Streptococcus equi , Líquido Amniótico/imunologia , Animais , Endométrio/imunologia , Feminino , Doenças dos Cavalos/sangue , Doenças dos Cavalos/genética , Cavalos , Interleucina-6/sangue , Interleucina-6/genética , Placenta/imunologia , Doenças Placentárias/sangue , Doenças Placentárias/genética , Doenças Placentárias/veterinária , Gravidez , Receptores de Interleucina-6/genética , Receptores de Interleucina-6/imunologia , Infecções Estreptocócicas/sangue , Infecções Estreptocócicas/genética , Infecções Estreptocócicas/veterinária
6.
Reproduction ; 160(6): 819-831, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33112764

RESUMO

The endometrium, the inner uterine lining, is composed of cell layers that come in direct contact with an embryo during early pregnancy and later with the fetal placenta. The endometrium is responsible for signals associated with normal reproductive cyclicity as well as maintenance of pregnancy. In the mare, functionally competent in vitro models of the endometrium have not been successful. Furthermore, the ability to study various reproductive processes in vitro may allow critical evaluation of signaling pathways involved in the reproductive diseases of animals that cannot be handled frequently, such as various wildlife species. Here we report the establishment of organoids, 3D structures, derived from fresh and frozen-thawed equine endometrium (Equus ferus caballus and E. f. przewalskii). Although organoids from domestic mares responded to exogenous hormonal stimuli, organoids from Przewalski's horse failed to respond to exogenous hormones. The present study represents a 'first' for any large animal model or endangered species. These physiologically functional organoids may facilitate improved understanding of normal reproductive mechanisms, uterine pathologies, and signaling mechanisms between the conceptus and endometrium and may lead to the development of novel bioassays for drug discovery.


Assuntos
Endométrio/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Hormônios/farmacologia , Organoides/efeitos dos fármacos , Animais , Animais Selvagens , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/metabolismo , Endométrio/metabolismo , Receptor alfa de Estrogênio/genética , Receptor alfa de Estrogênio/metabolismo , Feminino , Cavalos , Organoides/metabolismo , Receptores de Progesterona/genética , Receptores de Progesterona/metabolismo
7.
Reprod Fertil Dev ; 32(16): 1239-1249, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-33108747

RESUMO

High blood urea nitrogen (BUN) in cows and ewes has a negative effect on embryo development; however, no comparable studies have been published in mares. The aims of the present study were to evaluate the effects of high BUN on blastocoele fluid, systemic progesterone and Day 14 equine embryos. When a follicle with a mean (±s.e.m.) diameter of 25±3mm was detected, mares were administered urea (0.4g kg-1) with sweet feed and molasses (n=9) or sweet feed and molasses alone (control; n=10). Blood samples were collected every other day. Mares were subjected to AI and the day ovulation was detected was designated as Day 0. Embryos were collected on Day 14 (urea-treated, n=5 embryos; control, n=7 embryos). There was an increase in systemic BUN in the urea-treated group compared with control (P<0.05), with no difference in progesterone concentrations. There were no differences between the two groups in embryo recovery or embryo size. Urea concentrations in the blastocoele fluid tended to be higher in the urea-treated mares, with a strong correlation with plasma BUN. However, there was no difference in the osmolality or pH of the blastocoele fluid between the two groups. Differentially expressed genes in Day 14 embryos from urea-treated mares analysed by RNA sequencing were involved in neurological development, urea transport, vascular remodelling and adhesion. In conclusion, oral urea treatment in mares increased BUN and induced transcriptome changes in Day 14 equine embryos of genes important in normal embryo development.


Assuntos
Nitrogênio da Ureia Sanguínea , Desenvolvimento Embrionário/efeitos dos fármacos , Progesterona/sangue , Transcriptoma/efeitos dos fármacos , Ureia/administração & dosagem , Animais , Feminino , Cavalos , Inseminação Artificial/veterinária , Indução da Ovulação/veterinária , Gravidez
8.
Anim Reprod Sci ; 216: 106464, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32414463

RESUMO

An intravenous large dose of protein led to an increased blood urea nitrogen (BUN), resulting in a lesser uterine pH and altered uterine gene expression in mares. The objective of the present study was to evaluate effects of a more physiological methodology to increase BUN on the endometrium of mares. Mares were fed hay and a treatment or control diet (n = 11 mares/treatment) in a crossover design starting at time of ovulation detection (D0) and continuing until D7. Mares of the treated group were fed urea (0.4 g/kg BW) with sweet feed and molasses, and those of the control group were fed sweet feed and molasses. Blood samples were collected daily, 1 hour after feeding, for BUN determination. Uterine and vaginal pH were determined after the last feeding on D7, and endometrial biopsies were performed. The RNA sequencing of the endometrium of a subset of mares (n = 6/treatment) was conducted. Differentially expressed genes (DEGs) between treatments were calculated (FDR-adjusted P-value<0.1). Urea-treated mares had greater BUN (P < 0.05), with no differences in uterine and vaginal pH compared to control mares. A total of 60 DEGs were characterized, those with largest fold change were SIK1, ATF3, SPINK7, NR4A1 and EGR3. Processes related to necrosis and cellular movement were predicted with the DEGs. Dietary administration of urea resulted in transcriptomic changes in the endometrium of mares related to necrosis, tissue remodeling and concentration of lipids. The observed changes in gene expression after an increased BUN might result in a disruption to the endometrium.


Assuntos
Dieta/veterinária , Endométrio/efeitos dos fármacos , Cavalos/metabolismo , Transcriptoma/efeitos dos fármacos , Ureia/farmacologia , Ração Animal/análise , Animais , Nitrogênio da Ureia Sanguínea , Estudos Cross-Over , Suplementos Nutricionais , Endométrio/metabolismo , Feminino , RNA Mensageiro , Reação em Cadeia da Polimerase em Tempo Real , Ureia/administração & dosagem
9.
Placenta ; 93: 101-112, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32250734

RESUMO

INTRODUCTION: Hydrallantois is the excessive accumulation of fluid in the allantoic cavities during the last trimester of pregnancy, leading to abdominal wall hernias, cardiovascular shock, abortion, and dystocia. It has been postulated that hydrallantois is associated with structural and/or functional changes in the chorioallantoic membrane. In the present study, we hypothesized that angiogenesis is impaired in the hydrallantoic placenta. METHOD: Capillary density in the hydrallantoic placenta was evaluated in the chorioallantois via immunohistochemistry for Von Willebrand Factor. Moreover, the expression of angiogenic genes was compared between equine hydrallantois and age-matched, normal placentas. RESULTS: In the hydrallantoic samples, edema was the main pathological finding. The capillary density was significantly lower in the hydrallantoic samples than in normal placentas. The reduction in the number of vessels was associated with abnormal expression of a subset of angiogenic and hypoxia-associated genes including VEGF, VEGFR1, VEGFR2, ANGPT1, eNOS and HIF1A. We believe that the capillary density and the abnormal expression of angiogenic genes leads to tissue hypoxia (high expression of HIF1A) and edema. Finally, we identified a lower expression of genes associated with steroidogenic enzyme (CYP19A1) and estrogen receptor signaling (ESR2) in the hydrallantoic placenta. DISCUSSION: Based on the presented data, we believe that formation of edema is due to disrupted vascular development (low number of capillaries) and hypoxia in the hydrallantoic placenta. The edema leads to further hypoxia and consequently, causes an increase in vessel permeability which leads to a gradual increase in interstitial fluid accumulation, resulting in an insufficient transplacental exchange rate and accumulation of fluid in the allantoic cavity.


Assuntos
Doenças dos Cavalos , Neovascularização Patológica/patologia , Doenças Placentárias , Placenta/irrigação sanguínea , Poli-Hidrâmnios/patologia , Prenhez , Alantoide/metabolismo , Alantoide/patologia , Animais , Feminino , Doenças dos Cavalos/genética , Doenças dos Cavalos/patologia , Doenças dos Cavalos/fisiopatologia , Cavalos , Densidade Microvascular , Neovascularização Patológica/genética , Neovascularização Patológica/fisiopatologia , Placenta/metabolismo , Placenta/patologia , Placenta/fisiopatologia , Doenças Placentárias/genética , Doenças Placentárias/patologia , Doenças Placentárias/fisiopatologia , Doenças Placentárias/veterinária , Poli-Hidrâmnios/etiologia , Poli-Hidrâmnios/fisiopatologia , Poli-Hidrâmnios/veterinária , Gravidez , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo
10.
Genes (Basel) ; 10(8)2019 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-31366166

RESUMO

High blood urea nitrogen (BUN) decreases fertility of several mammals; however, the mechanisms have not been investigated in mares. We developed an experimental model to elevate BUN, with urea and control treatments (7 mares/treatment), in a crossover design. Urea-treatment consisted of a loading dose of urea (0.03 g/kg of body weight (BW)) and urea injections over 6 hours (0.03 g/kg of BW/h). Control mares received the same volume of saline solution. Blood samples were collected to measure BUN. Uterine and vaginal pH were evaluated after the last intravenous infusion, then endometrial biopsies were collected for RNA-sequencing with a HiSeq 4000. Cuffdiff (2.2.1) was used to identify the differentially expressed genes (DEG) between urea and control groups (false discovery rate-adjusted p-value < 0.1). There was a significant increase in BUN and a decrease of uterine pH in the urea group compared to the control group. A total of 193 genes were DEG between the urea and control groups, with five genes identified as upstream regulators (ETV4, EGF, EHF, IRS2, and SGK1). The DEG were predicted to be related to cell pH, ion homeostasis, changes in epithelial tissue, and solute carriers. Changes in gene expression reveal alterations in endometrial function that could be associated with adverse effects on fertility of mares.


Assuntos
Dieta Rica em Proteínas/efeitos adversos , Endométrio/metabolismo , Cavalos/genética , Transcriptoma , Animais , Nitrogênio da Ureia Sanguínea , Feminino , Fertilidade , Cavalos/sangue , Cavalos/fisiologia
11.
Reprod Fertil Dev ; 31(6): 1144-1156, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30947806

RESUMO

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.


Assuntos
Membrana Corioalantoide/metabolismo , Endométrio/metabolismo , MicroRNAs/metabolismo , Doenças Placentárias/metabolismo , Placenta/metabolismo , Animais , Corioamnionite/sangue , Corioamnionite/genética , Corioamnionite/metabolismo , Feminino , Cavalos , Inflamação/sangue , Inflamação/genética , Inflamação/metabolismo , MicroRNAs/sangue , MicroRNAs/genética , Doenças Placentárias/sangue , Doenças Placentárias/genética , Gravidez
12.
Theriogenology ; 86(6): 1550-1556, 2016 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-27341773

RESUMO

The aim of the present study was to compare endometrial cytology with histopathology to diagnose subclinical endometritis (SCE) in dairy cows. Endometrial cytology samples were collected from Holstein-Friesian cows (n = 32) just before slaughtering. Half of them were obtained by in vivo cytobrush (IV-CB), whereas the other half by in vivo low-volume lavage (IV-LVL). After slaughtering, reproductive tracts were collected, and the endometrium was sampled at eight locations. At each location, both a ex vivo cytobrush sample (EV-CB) and a tissue sample for histopathologic examination were taken. In the histopathology slides, polymorphonuclear (PMN) cell counts were differentiated as PMN cells in direct contact with the epithelial cells of the endometrium (PMN-EP), and PMN cells present in the deeper stratum compactum (PMN-SC). Summation of both countings was referred to as PMN-total. Pearson's correlation and Cohen's kappa coefficient were used to assess the correlation and agreement between both sampling methods (in vivo cytology [IV-CB and IV-LVL] with EV-CB and PMN-total). A Poisson mixed effect model was used to analyze the PMN cells' distribution. The prevalence of SCE was 18.75% (n = 6/32) for in vivo cytology. The SCE prevalence based on EV-CB analyses and on the assessment of PMN-total was determined both at the sample (n = 256) as well as at the cow level (n = 32): EV-CB 25% (n = 64/256) and 35.5% (n = 12/32), and PMN-total 37.11% (n = 95/256) and 59.38% (n = 19/32). Correlation and agreement between IV-CB and EV-CB were r = 0.81 and k = 0.97, whereas between IV-CB and PMN-total r = 0.15 and k = 0.23, respectively. In vivo low-volume lavage correlation and agreement were r = 0.52 and k = 0.66 with EV-CB, and r = 0.45 and k = 0.44 with PMN-total. Moreover, correlation and agreement between EV-CB and PMN-total were r = 0.60 and k = 0.50, respectively. More PMN cells (P < 0.05) were detected in PMN-SC when compared to PMN-EP and EV-CB. A higher SCE prevalence was found using histopathology, rendering the latter as a more sensitive method to diagnose SCE in comparison to in vivo and ex vivo cytology. Although cytology had low and/or moderate sensitivity to diagnose SCE when compared with histopathology, its specificity is 100%, implying that all cows that were indicated to suffer from SCE using in vivo cytology were confirmed to do so by histopathologic examination. There is an uneven distribution of PMN cells throughout the endometrium, generally more PMN cells being found in the deeper stratum compactum than in contact with the superficial layers of the endometrium.


Assuntos
Doenças dos Bovinos/patologia , Endometrite/veterinária , Endométrio/patologia , Animais , Bovinos , Citodiagnóstico/métodos , Citodiagnóstico/veterinária , Indústria de Laticínios , Endometrite/patologia , Feminino , Contagem de Leucócitos , Neutrófilos/patologia
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