Equine endothelial cells in vitro

Certain in vitro culture conditions were determined for equine endothelial cells obtained from the aorta and pulmonary arteries. Cells were enzymatically isolated from the vessel lumen, using clostridial collagenase (2.5 mg/ml of Hanks's balanced salt solution) incubated at 37 C for 30 minutes. Cells were cultured in alpha minimum essential medium supplemented with plasma-derived and nonplasma-derived bovine fetal sera, endothelial cell-growth supplement, heparin, and antibiotics. Smooth muscle cell growth was not inhibited with nonplasma-derived animal sera, plasma-derived equine serum, or heparin. Heparin and a serum replacement were toxic to the cells used in the present study. Statistically significant differences were not found between the various media supplements.     

Recombinant Equine Luteinizing Hormone Stimulates Production of Progesterone from Murine Leydig, Ovine Small Luteal, and Equine Granulosal Cells

Recombinant equine luteinizing hormone (reLH) was evaluated for its ability to stimulate production of progesterone in cell lines from three species including murine Leydig tumor (MA-10), equine granulosal, and ovine small luteal cells (SLC). The response to reLH was compared with that obtained with human chorionic gonadotropin (hCG), equine chorionic gonadotropin, ovine luteinizing hormone, and equine luteinizing hormone (eLH). Media were collected hourly for 6 hours and assayed for progesterone content through radioimmunoassay. In MA-10 cells, production of progesterone was stimulated above baseline by reLH and hCG (P < .05). Ovine SLC responded to treatment with eLH, reLH, ovine luteinizing hormone, and hCG by increasing production of progesterone above that stimulated by vehicle control (P < .05). Production of progesterone in equine granulosal cells was maximally stimulated by treatment with hCG (P < .05), followed by reLH and eLH (P < .05). In conclusion, reLH elicited a progesterone response in MA-10, ovine SLC, and equine granulosal cells. Thus, reLH stimulates the production of progesterone in cell lines from three species.     

Myeloperoxidase in Equine Semen: Concentration and Localization during Freezing Processing

Myeloperoxidase (MPO) is a pro-oxidant enzyme contained in and released by neutrophils during degranulation or after lysis. Post-thaw semen contains MPO, and concentration of this enzyme is associated with decreased motility. The aim of this study was to determine kinetics of MPO concentration during freezing, its origin, and its impact on frozen-thawed semen. Forty ejaculates were used. Semen was frozen using the classical freezing procedure. MPO concentrations were assayed in fresh semen, after centrifugation, and after cooling down to 4°C. Post-thaw MPO assay results and spermogram characteristics were determined. MPO immunocytochemistry was performed on 4 different ejaculates at each step of freezing procedure. MPO concentration increased after cooling down to 4°C and thawing compared with fresh semen. As temperature decreased, MPO was higher or tended to be higher in post-thaw poor quality samples. Nonsperm cells showed various degrees of MPO immunostaining and were observed as epithelial cells with nuclear pyknosis and keratinization. MPO immunostaining increased in medium and decreased in nonsperm cells during freezing. Our study shows that MPO concentration in equine semen increases when temperature decreases. We hypothesize that nonsperm cells present in fresh semen could release MPO.     

Immunohistochemical Localization of the Spermadhesin AWN-1 in the Equine Male Genital Tract

Spermadhesins are proteins with various functions in sperm capacitation and zona pellucida binding. In this study the cellular localization of the spermadhesin AWN-1 has been examined in the equine male genital tract. Results obtained by immu-nohistochemical methods reveal that in the horse AWN-1 is synthesized in spermatogonia, in the rete testis, the ductus epi-didymidis and the seminal vesicles. These findings indicate that the cellular origin of spermadhesins is species-specific.     

Equine coronavirus induces apoptosis in cultured cells

Equine coronavirus (ECoV) was first isolated from a diarrheic foal and was found genetically similar to group II coronaviruses. However, its pathological characteristics were not adequately investigated. In our preliminary in vitro investigation, ECoV-induced cell death was observed in bovine kidney-derived MDBK cells. Based on this finding, we investigated whether the ECoV-induced CPE was apoptosis. Following ECoV infection, MDBK cells showed morphological changes such as cell rounding and detachment from the culture surface. Moreover, syncytium formation was observed as the other type of cytopathic effect in ECoV infection. Morphologic and biochemical features of apoptosis, such as nuclear fragmentation and DNA ladder formation, were also detected in ECoV-infected cells. Moreover, as is commonly observed in coronavirus infection in other animals, the activities of effecter caspases – caspase-3/7 – and initiator caspases – caspase-8 and caspase-9 – that are representative factors in the death receptor-mediated apoptotic pathway and mitochondrial apoptotic pathway, respectively, were increased in ECoV-infected MDBK cells. Therefore, it was suggested that ECoV can induce apoptosis in MDBK cells via a caspase-dependent pathway. Apoptotic death of infected cells is detrimental because it causes cell and tissue destruction and inflammatory responses. Although the pathological characteristics of ECoV are largely unknown, apoptosis may be the pathological basis of lesions of the digestive system in ECoV infection.     

Azoospermia of dogs with apoptotic germ cells and Leydig cells

Apoptotic cell death in the testes of 4 dogs with azoospermia was examined. Blood plasma luteinizing hormone (LH), testosterone (T), and estradiol-17beta (E2) concentrations, and testicular transferrin (Tf) concentration as a marker of Sertoli cell function were measured in the 4 azoospermic dogs and in 5 normal dogs. The spermatids in 2 of the 4 azoospermic dogs and the Leydig cells in 3 of them exhibited apoptotic cell death. Mean LH, E2, and Tf concentrations in the 4 azoospermic dogs were significantly higher than in the normal dogs (P<0.01). These findings suggested that the azoospermia in all 4 dogs might has been caused by abnormal functions of Sertoli cells as well as Leydig cells.     

玉米赤霉烯酮对小鼠睾丸间质细胞线粒体的损伤作用

将不同作用时间（时间梯度组）及不同质量浓度（质量浓度梯度组）的玉米赤霉烯酮（Zearalenone,ZEA）作用于体外培养的原代小鼠睾丸间质细胞,时间梯度组设0（对照组）、6、12、24h4个观察组,染毒质量浓度为2.5mg/L;质量浓度梯度组设0（对照组）、5、10、20mg/L ZEA 4个观察组,染毒时间为12h。采用流式细胞技术测定线粒体膜电位和透射电子显微镜观察细胞超微结构的方法观测ZEA对睾丸间质细胞线粒体的损伤作用。结果显示,睾丸间质细胞线粒体膜电位2.5mg/L ZEA暴露6h较对照组显著下降（P〈0.05）,暴露12h及24h较对照组都有极显著下降（P〈0.01）;ZEA质量浓度为5mg/L组较对照组显著下降（P〈0.05）,10mg/L组和20mg/L组较对照组都有极显著下降（P〈0.01）。细胞超微结构分析显示,线粒体的空泡化和内质网断裂的程度与ZEA的暴露存在时间-效应关系和质量浓度-效应关系。结果表明,ZEA可导致睾丸间质细胞线粒体损伤,这是ZEA抑制睾丸间质细胞分泌睾酮的一个重要原因。     

广州及其周边地区马梨形虫病血清流行病学调查

为广东从化无规定马属动物疫病区建设的顺利进行和了解周边地区马属动物马梨形虫病的存在情况,采用酶联免疫吸附试验(ELISA)和涂片镜检的方法对广州、东莞和深圳的马属动物进行了马梨形虫病(马巴贝斯虫和驽巴贝斯虫两种虫体)的流行病学调查。结果表明,上述三个地区的马属动物均存在不同程度的马梨形虫病两种虫体的抗体,且出现同一个马场和在同一匹马同时存在两种虫体抗体的情况。对所有抗体阳性马匹进行涂片镜检未发现虫体,也未发现有马梨形虫病临床症状的马属动物。     

Regulation of DNA synthesis in Leydig cells obtained from neonatal pig testes.

Three distinct waves of Leydig cell development are found in the pig testes, which occur during fetal, perinatal, and prepubertal periods. Proliferation of Leydig cells is primarily regulated by luteinizing hormone (LH); however, effects of LH on proliferation of immature rat Leydig cells are mediated by specific growth factors and cytokines such as transforming growth factor-alpha (TGFalpha), insulin-like growth factor-1 (IGF-1), interleukin-1beta (IL-1beta), steroidogenesis-inducing protein (SIP), and TGFbeta. The objective of the present study was to identify growth factors that could possibly be involved in the proliferation of Leydig cells in the neonatal pig testis. Leydig cells were isolated from 3- to 5-d-old pig testes, cultured for 48 hr in serum-free media, washed, and treated with hCG and/or IGF-1, epidermal growth factor (EGF), IL-1beta, SIP, and TGFbeta for 18 hr. Tritiated thymidine incorporation into DNA was assessed over a subsequent 4-hr period. Incorporation of [3H]-thymidine was stimulated by hCG treatment with a 2.3-fold increase over control cultures. SIP also induced a significant increase (P < 0.0001) in the incorporation of [3H]thymidine into Leydig cell DNA. Similarly, EGF and IGF-1 also increased DNA synthesis in neonatal porcine Leydig cells, whereas IL-1beta had no effect. TGFbeta had very little, if any, effect on DNA synthesis when added alone, but inhibited the stimulatory effects of other mitogens (SIP, hCG, EGF/TGFalpha, and IGF-1). Our results indicate that these growth factors may play a role in the LH/hCG-dependent proliferation of Leydig cells during the perinatal period of development.     

Prenatal and neonatal exposure to flutamide affects function of Leydig cells in adult boar

In this study, flutamide, an androgen receptor antagonist, was used as a tool to better understand the role of androgen receptor signaling and androgen signaling disruption during fetal and neonatal periods on porcine Leydig cell development and function. Flutamide, 50 mg kg(-1) d(-1) was administered into pregnant gilts during gestational days 20 to 28 and days 80 to 88 and into male piglets on postnatal days 2 to 10 (PD2). Leydig cells of flutamide-exposed boars, especially those of PD2 males, displayed morphologic alterations, increased size, and occupied increased area (P < 0.001) of the testes when compared with the control. Despite this, testosterone concentrations were reduced significantly in comparison with those of controls (P < 0.05, P < 0.001). Reduced testosterone production in response to flutamide exposure appeared to be related to changes in testosterone metabolism, as shown by increased aromatase mRNA (P < 0.05, P < 0.01), protein expression (P < 0.01, P < 0.001), and elevated estradiol concentrations (P < 0.001). Moreover, impaired Leydig cell responsiveness to LH was indicated by the decreased expression of LH receptor (P < 0.05, P < 0.001). No significant effect of flutamide was found on LH and FSH concentrations. Taken together, our data indicate that flutamide when administered during prenatal or neonatal period have a long-term effect on Leydig cell structure and function, leading to androgen-estrogen imbalance. Leydig cell failure was most evident in adult boars neonatally exposed to flutamide, suggesting that androgen action during neonatal development is of pivotal importance for the differentiation and function of porcine adult Leydig cell population.     

Equine keratomycosis in Japan

Objective To describe the incidence, clinical progress, visual outcome, and laboratory findings of equine keratomycosis in Japan. Procedure Retrospective study of the medical records of horses clinically and mycologically diagnosed with keratomycosis at the Equine Hospitals of the Japan Racing Association from 2005 to 2011. Results The diagnosis of keratomycosis was confirmed in eight horses (40.0% of the 20 horses with infectious keratitis from which fungi and/or bacteria were isolated). Fungi recovered from corneal swabs were identified as Aspergillus flavus (4), Aspergillus niger (1), Fusarium solani (1), and Mortierella wolfii (2). All horses were treated medically with topical antifungals, and one horse was also treated surgically. The median of treatment period was 40 days. Two horses were rendered blind in the affected eye and the others retained vision. Conclusions Equine keratomycosis comprises a considerable portion of infectious keratitis in Japan, and the causative fungi that we isolated had been isolated previously from horses with keratomycosis in other regions with the exception of M. wolfii. Culture and cytological examination of corneal lesions should be immediately performed on eyes with signs of keratitis, particularly on those not improving with antibacterial medication, as early initiation of aggressive antifungal treatment tended to result in better outcome and shorter treatment period.     

Equine glanders in Turkey

In the course of an epidemiological study of glanders on a number of Turkish islands in the Sea of Marmara, 1128 horses were examined by using the intracutaneous mallein test. Thirty-five (3-1 per cent) developed an increase in rectal temperature and a swelling at the point of injection. Ten of these horses were killed and glanders was confirmed in five cases by the presence of lesions and by the immunohistological demonstration of the causative agent, Burkholderia mallei. Clinical and pathological findings indicated that in all cases the infection was restricted to the mucous membrane of the nasal cavity with its parasinus, the nostrils and the upper lips. It was confirmed that equine glanders is endemic in Turkey.