Escherichia coli Lipopolysaccharide-Induced Immunomodulation Along With Oxytocin Administration After Mating as a Treatment Protocol for Persistent Endometritis in Mares

Endometritis is accepted as a major hindrance to achieve optimal reproductive efficiency in mares. The objective of the present study was to evaluate the therapeutic efficacy of combined therapy of immunomodulator and ecbolic as an alternative stand-alone therapy for mares with persistent endometritis. On the basis of history, culture, endometrial cytology, and per rectal and/or ultrasonographic genital examinations, 76 subfertile mares were selected and assigned to three age groups and four treatment (G-1, 2, 3) and control (G-4) groups. At estrus, all the mares were bred once naturally. Thereafter, the mares of G-1 (n = 28) were aseptically treated at 6-hours after natural service with intrauterine infusion (in 50 mL normal saline solution) of 100 μg of Escherichia coli lipopolysaccharide (LPS). Additionally, these mares received two injections of 20 IU of oxytocin (IV) at 12 and 24-hours after infusion. Mares in group G-2 (n = 11) were treated with LPS as mares of G-1, whereas mares in G-3 (n = 12) received oxytocin injections only. The mares of G-4 (n = 23) did not receive any treatment. Pregnancy rates at day 21 and foaling rates were higher (P < .001) in group G-1 than in G-4. In G-1, higher percentage of mares at ages 6–10 years conceived and foaled than mares aged ≥16 years. On re-swabbing of mares that remained nonpregnant, the majority of G-1 and G-2 mares demonstrated sterile cultures and negative cytology, whereas uterine inflammation persisted in mares of G-3 and G-4. In conclusion, the combined therapy was effective for the elimination of persistent endometritis and improved reproductive performance of subfertile mares.     

Use of a Mycobacterial Cell Wall Extract (MCWE) in Susceptible Mares to Clear Experimentally Induced Endometritis With Streptococcus zooepidemicus

The ability of an immunomodulator, mycobacterial cell wall extract (MCWE), to clear uterine infection in susceptible mares after an experimental challenge withStreptococcus zooepidemicus was evaluated. Thirty mares susceptible to endometritis, based on the presence of uterine fluid during both diestrus and estrus, were selected from a herd of 896 and inoculated with a live culture of 5 × 10⁶ CFU of S. zooepidemicus on day 1 of estrus. Twenty-four hours later, mares were evaluated by ultrasonography, bacteriology, exfoliative cytology, and uterine biopsy to confirm infection. Forty-eight hours after inoculation, and on confirmation of uterine infection, mares were randomly assigned to one of four unbalanced experimental treatments to receive 1500 μg MCWE IU (n = 10) or IV (n = 10), or placebo IU (n = 5) or IV (n = 5). Mares were examined at ovulation and 7 days post-ovulation for uterine fluid via transrectal ultrasonography and for bacteriology, exfoliative cytology, and uterine biopsy. Efficacy was based on the ability of the mare to clear endometritis as determined by negative bacteriology and reduced numbers of polymorphonuclear cells (PMNs) on uterine biopsy. Because no statistical difference was detected between routes of administration on day 7 post-ovulation, the data sets were combined and re-analyzed to evaluate overall efficacy. Endometritis was observed in all placebo-treated mares 7 days post-ovulation, whereas treatment with MCWE resulted in the elimination of endometritis in 35% of the mares by the time of ovulation, and 70% of the mares by 7 days post-ovulation. Treatment with MCWE, compared with the placebo group, resulted in a significant decrease in the number of mares positive for endometritis at ovulation based on exfoliative cytology and bacteriology (P < .01) and at 7 days post-ovulation based on biopsy, exfoliative cytology, and bacteriology (P < .001). Results indicate that MCWE was an effective treatment for the elimination of endometritis caused by S. zooepidemicus in mares.     

溶菌酶治疗奶牛产后子宫内膜炎试验

经临床136例病例观察，溶菌酶对奶牛子宫内膜炎的总治愈率、溶菌酶治疗后各组子宫恢复程度均较抗生素治疗组和空白对照组明显好，对治疗子宫内膜炎的治愈率在82.35%-88.24%，明显好于在临床中常规使用药物金霉素的治愈率56.25%。溶菌酶试验组平均受胎率为75.38%，明显高于抗生素对照组的56.25%，空白对照组的15.63%。治愈牛产后至初配平均天数也较短。结果表明溶菌酶是一种取代抗生素药物治疗子宫内膜炎的生物类药品。     

An Investigation of Uterine Nitric Oxide Production in Mares Susceptible and Resistant to Persistent Breeding‐Induced Endometritis and the Effects of Immunomodulation

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

Randomized Comparative Trial of Acupuncture and Exercise Versus Uterine Ecbolics in the Treatment of Persistent Postbreeding Endometritis in Mares

Although exercise and acupuncture are frequently used therapies to treat persistent postbreeding endometritis, their efficacy to date is unproven. The objective of this study was to determine if exercise and acupuncture are effective methods to reduce intrauterine fluid and compare the effectiveness of these treatments to the use of uterine ecbolics. Twelve mares susceptible to postbreeding endometritis were enrolled in the study with a randomized cross-over design using both positive and negative controls. During each estrous cycle, mares were randomized into one of six treatment groups, including stall rest (SR), oxytocin, cloprostenol, exercise, electroacupuncture, and oxytocin and exercise. Each mare was challenged with an insemination dose of 500 × 10⁶ dead sperm at time 0 hours. Intrauterine fluid measurements were taken at 0, 4, 24, 48, 72, and 96 hours postbreeding. Associations between treatment efficacy and fluid clearance were investigated using a random-effects logistic regression model that controlled for positive uterine culture. Compared with the SR negative control, exercise was the most effective treatment and had 29.7 times increased odds of fluid clearance. The second most effective treatment was oxytocin alone, with 16.9 times increased odds of fluid clearance. This was followed by cloprostenol that had 10.6 greater odds of fluid clearance, and finally, the treatment that combined exercise with oxytocin had 8.4 times greater odds of fluid clearance. Results from this study confirm that exercise and exercise combined with oxytocin are effective methods to clear intrauterine fluid.     

Comparison of Clinical Signs,Endometrial Culture,Endometrial Cytology,Uterine Low-Volume Lavage,and Uterine Biopsy and Combinations in the Diagnosis of Equine Endometritis

Endometritis is the most common cause of infertility in mares; however, many mares fail to be diagnosed despite availability of many diagnostic tests. Our objective was to compare different diagnostic methods and establish a cutoff value for the number of polymorphonuclear cells in cytology samples. Fifty-four mares were classified positive for endometritis based on endometrial biopsy (“gold standard”), and in a later analysis, the mare was reclassified as positive for endometritis if two or more of the following five criteria on a checklist were present (“new gold standard” [NGS]): (1) abnormal clinical findings, (2) abnormal gross character of low-volume lavage (LVL) fluid, (3) positive endometrial cytology, (4) bacterial growth on culture of the LVL pellet, and (5) histologic evidence of inflammation on endometrial biopsy. Kappa (κ) coefficient and percentages were calculated for sensitivity and positive predictive value (PPV) using SAS 9.3 software. Endometritis was diagnosed in 35/44 (79.5%) mares by biopsy. Based on the endometritis checklist, 33/51 (64.7%) mares were positive for endometritis. The character of LVL was 45% sensitive, whereas culture was 22% sensitive, when compared with endometrial biopsy. One percent of neutrophil-to-epithelial cell ratio was the most sensitive cutoff value (93.3%) when using a guarded swab (κ = 0.64). Endometrial biopsy was the most sensitive diagnostic method when compared against the NGS (sensitivity = 86%). Abnormal clinical findings and positive cytology showed moderate agreement with the NGS (κ = 0.41 and κ = 0.38, respectively). These studies demonstrate the importance of combining clinical findings and laboratory data when evaluating mares for endometritis.     

抗菌药物治疗奶牛子宫内膜炎后牛奶中药物残留及其对酸奶发酵的影响

用TTC法检测44例经过抗菌药物治疗的患子宫内膜炎荷斯坦奶牛。其中，41例采用子宫内灌注药物治疗，主要药物有土霉素、金霉素和庆大霉素；每个病例治疗1～5次不等，3例采用静脉注射，主要药物有磺胺嘧啶钠和头孢噻啶。结果表明，31例经土霉素子宫内灌注的奶牛，停药后第24h所采的奶样均可检出抗生素；其中6头奶牛在第6d仍然抗生素阳性，残留时间最长的可达8d。8例用金霉素子宫灌注治疗的奶牛，停药后第48h所采的奶样，抗生素检出率100％，其在牛奶中的残留时间为2～5d不等。2例经庆大霉素子宫灌注治疗的患病牛，药残时间分别为1～2d。3头经静脉滴注治疗的病牛，牛奶中的药残时间约达到9d以上。此外，从3头用金霉素子宫内灌注的奶牛采集牛奶用于乳酸菌发酵试验。结果表明，牛奶TTC阳性时，发酵产酸较低。在治疗后6h采集的牛奶都不能正常发酵产酸、凝固。3例试验牛牛奶发酵产生的酸度分别于治疗后第13、24和48h恢复到治疗前的水平。     

Treatment of Persistent Mating‐Induced Endometritis in Mares with the Non‐Steroid Anti‐Inflammatory Drug Vedaprofen

Recently, successful treatment of mares with a history of persistent mating‐induced endometritis (PMIE) with dexamethasone has been reported. As systemic treatment of horses with glucocorticoids should be handled with caution, we tested the hypothesis that treatment with the non‐steroid anti‐inflammatory drug (NSAID) vedaprofen, an inhibitor of cyclooxygenase‐2, may have comparative, positive effects on fertility. Barren mares with a history of repeated PMIE were treated with vedaprofen (n = 8; initially 2 mg/kg bodyweight followed by 1 mg/kg orally twice daily) from 1 day before the first insemination to 1 day after ovulation or left untreated (n = 9). All mares received oxytocin (20 I.E. s.c.) thrice daily. Uterine swabs were collected for bacteriology and cytology. The day after ovulation, fluid accumulation was detected in three control mares and four treated mares (n.s.). The percentage of neutrophils in uterine cytology was significantly increased in comparison to the day before ovulation irrespective of treatment. Pregnancy was confirmed in two of nine mares in the control group and seven of eight mares in the treatment group (p < 0.05). NSAIDs may positively affect fertility in mares with a history of PMIE.     

盐酸土霉素及清宫液治疗奶牛子宫内膜炎试验

对７７头患子宫内膜炎的奶牛进行治疗试验，其中４６头患牛采用盐酸土霉素治疗，３１头患牛采用清宫液治疗，治疗结果：土霉素治疗组治愈率８０．４３％，好转率１０．８６％，无效８．６％；清宫液治疗组治愈率８７．０９％，好转率６．４５％无效６．４５％。试验结果表明：盐酸土霉素及清宫液治疗奶牛子宫内膜炎的效果显著，可在生产实践中推广应用。     

抗生素与“清宫促孕宝”治疗奶牛子宫内膜炎疗效报告

[目的]本研究利用抗生素联合中成药治疗子宫内膜炎,为该病有效治疗探索新方法。[方法]使用＂清宫促孕宝＂＋土霉素作为治疗药物,采用子宫内灌注法治疗患牛78例。[结果]试验组78头,有效70头,有效率为89.74%,治愈67头,治愈率为86.0%。[结论]临床治疗表明,＂清宫促孕宝＂＋土霉素治疗各类子宫内膜炎疗效显著,体现了中西医结合的优势。     

稀释液中添加半胱氨酸对绵羊冷冻精液品质的影响

在绵羊冷冻精液稀释液中添加0mmol／L、2．5mmol／L、5．0mmol／L、7．5mmol／L4个不同梯度的半胱氨酸,通过测定解冻后的精子活率、畸形率、顶体完整率、质膜完整率以及各项酶（SOD、GSH、GSH-PX、CAT和MDA）的活性来确定最适添加量。结果表明,添加5．0mmol／L的半胱氨酸组的精子活率和顶体完整率均极显著高于对照组和其他组（尸〈0．01）,而畸形率显著低于对照组和其他试验组（P〈0．05）;各项酶指标中,5．0mmol／L半胱氨酸组SOD和GSH浓度与其余各组间差异不显著（P〉0．05）,MDA浓度极显著低于对照组和7．5mmol／L试验组（P〈0．01）,CAT和GSH-PX活性极显著或显著高于对照组和其他试验组（P〈0．01,P〈0．05）。说明冷冻稀释液中添加5．0mmol／L的半胱氨酸可有效保护精子免受过氧化物的损害。     

Treatment of Intraoperative Persistent Penile Erection in a Stallion

This case report describes the occurrence of persistent penile erection in a breeding stallion that occurred while the horse was under inhalant anesthesia for a carpal arthroscopy. The horse had no history of breeding problems, and no abnormalities were detected on physical examination, complete blood count, or serum chemistry tests performed prior to surgery. Anesthesia was induced with guaifenesin and ketamine after sedation with xylazine and was maintained with isoflurane in 100% oxygen. Penile erection developed approximately 35 minutes after induction and persisted for over 2 hours despite various physical and pharmacological attempts to alleviate it (massage, cold compresses, intravenous benztropine administration, and intracavernosal phenylephrine). Successful resolution of the erection was obtained by cannulation and drainage of blood from the corpus cavernosum and subsequent irrigation with heparinized sterile saline and infusion of phenylephrine in the corpus cavernosum. The detumescent penis was placed back into the sheath, and purse string sutures were placed in the sheath to ensure the penis would remain inside the sheath during recovery. The stallion's recovery from anesthesia was uneventful, the sutures were removed, and the horse was fitted with a penile sling to prevent additional edema or trauma. The stallion recovered completely from the persistent penile erection. Semen was collected 6 days after the event, and he returned to normal pasture breeding 6 weeks after surgery.     

加味柴胡汤对子宫内膜炎奶牛血液白细胞的影响

本试验选子宫内膜炎患牛10头,随机分为中药治疗组和患病对照组（每组5头）,5头健康奶牛作为正常对照组.中药治疗组子宫灌注复方中药剂加味柴胡汤,连用6d,其他两组给予生理盐水.在用药前1d和用药2d、4d、6d采血,用血液自动分析仪检测.结果：（1）治疗前,中药治疗组和患病对照组白细胞数量显著（P＜0.05）高于正常对照组,治疗6d后,中药治疗组显著（P＜0.05）低于治疗前和患病对照纽.（2）治疗前,中药治疗组和患病对照组中间细胞数量和比率均显著（P＜0.05）高于正常对照组,用药6d后,中药治疗组显著（P＜0.05）或极显著（P＜0.01）低于患病对照组和治疗前.（3）治疗前,中药治疗组和患病对照组嗜中性粒细胞数量比率显著（p＜0.05）高于正常对照组.用药6d后,中药治疗组与正常对照组均显著（P＜0.05）低于患病对照组,低于治疗前.（4）治疗前,中药治疗组和患病对照组淋巴细胞数量显著（P＜0.05）高于正常对照组,比率显著（P＜0.05）低于正常对照组.用药6d后,中药治疗组和正常对照组淋巴细胞数量和比率显著（P＜0.05）高于患病对照纽.结论：加味柴胡汤可降低子宫内膜炎奶牛的炎症反应,提高免疫力.     

乙酰半胱氨酸对大鼠饮水镉暴露所致肾功能损伤的保护效应

采用饮水对大鼠进行镉染毒(CdCl2,50mg/L)和NAC保护(CdCl250mg/L+NAC1g/L)试验,为期8周,通过检测尿液中碱性磷酸酶(UALP)、N-乙酰-β-D-氨基葡萄糖苷酶(UNAG)、γ-谷氨酰基转移酶(UGGT)活性的动态变化及观察肾小管上皮刷状缘超微结构,探讨镉对大鼠肾脏功能损伤以及NAC的保护作用。结果显示,镉染毒2周后UNAG,UGGT,UALP活性即显著或极显著高于对照组(P0.05或P0.01),且升高幅度与染毒时间呈正相关;与染毒组比较,保护组尿液中酶活性从试验后4周开始显著或极显著下降(P0.05或P0.01)。超微结构观察发现,镉染毒使大鼠肾小管上皮刷状缘不同程度的断裂和脱落,保护组刷状缘基本完整。结果表明,50mg/LCdCl2饮水暴露对肾功能和结构造成了明显损伤,NAC对这种损伤有显著的保护作用。     

The Effects of Selenium Source on Measures of Selenium Status of Mares and Selenium Status and Immune Function of Their Foals

The effect of organic and inorganic sources of selenium (Se) on measures of Se status of mares and their foals, Se concentrations of colostrum and milk, and indices of immune function of foals was studied. Twenty late-gestation Standardbred mares were randomly assigned to one of two groups. All mares received an identical balanced ration, except for the source of supplementary Se: one group received organic Se (Se yeast) and the other group received inorganic Se (sodium selenite), fed to deliver 0.3 mg/kg supplementary Se based on total ration dry matter. Mares received the experimental diet from 2 months before estimated due date until 1 month after foaling. Source of Se did not affect Se concentrations in maternal plasma, red blood cells, colostrum, or milk. At 1 month of age, foals from mares fed organic Se had higher red blood cell Se concentration than foals from mares fed inorganic Se (P < .05). Measures of immunity included serum immunoglobulin G concentration, lymphocyte proliferation in response to concanavalin A, and relative cytokine gene expression of stimulated lymphocytes (interferon gamma [IFNγ], interleukin [IL]-2, IL-5, IL-10, tumor necrosis factor alpha [TNFα]) and neutrophils (IL-1, IL-6, IL-8, IL-12, TNFα). Relative gene expression of IL-2, TNFα, and IFNγ by foal lymphocytes was associated with the source of Se supplementation provided to the mares. We conclude that the source of dietary Se provided to mares may influence the immune function of foals at 1 month of age through changes in relative gene expression of certain lymphocyte cytokines.     

Comparison of the Cytology Brush and Cotton Swab in the Cytological Evaluation of the Endometrium in Mares with Regard to Fertility

The aim of this study was to compare the cytology brush (CB) and cotton swab (CS) in the cytological evaluation of the endometrium in mares with regard to fertility. The study was conducted on 26 mares displaying spontaneous estrus. Samples for cytological evaluation were taken from each mare by using commercially available CS and CB. After sample collection, all mares were mated in the same estrus, and pregnancy diagnosis was performed 14-18 days after last mating. No vaginal cells were found in smears, and the CB technique yielded significantly more cells/high-power field (HPF) than the CS technique. Additionally, more cases of endometritis were diagnosed using the CB than the CS. It was also shown that the degree of inflammation is more important in diagnosis of infertility in the mare than the mere presence or absence of inflammation. In conclusion, further studies are needed to establish more precise criteria for the interpretation of inflammation, especially if samples are collected using the CB.     

母马大、小卵泡的卵泡液代谢组学分析及其对卵泡发育的影响

本实验旨在研究母马大、小卵泡液代谢物的差异,从卵泡液代谢组层面初步探讨差异物对卵泡发育的影响及卵泡发育机制。选用8匹山东即墨地区母马的卵泡液进行代谢组学分析,大卵泡直径设为45～60 mm、小卵泡直径设为20～30 mm,采用超高液相色谱-高分辨质谱联用技术,通过主成分分析法(PCA)、偏最小二乘法-判别分析法(PLS-DA)得到大小卵泡液差异性物质质荷比。结果表明:在大卵泡液中含量多的代谢物有睾酮、1-棕榈酰溶血磷脂酸、β-硫酸雌醇、硫酸雌酮,小卵泡液中含量多的代谢物有硫酸吲哚酚、木子塘、水苏糖和3-脲基丙酸酯。综上可知,在大卵泡液中,上调物质主要是类固醇,比较单一集中,但小卵泡中不存在上调的类固醇物质,说明在母马中类固醇激素是促使卵泡发育的主要代谢物;在小卵泡液中,上调物质种类复杂,当优势卵泡出现时,可能有一些代谢物会抑制其余的小卵泡继续发育。     

The Role of Equine Chorionic Gonadotropin in the Stimulation of Luteal Steroidogenesis in Mares Carrying Horse or Mule Pregnancies

The stimulatory role of equine Chorionic Gonadotropin (eCG) in the production of steroid hormones was evaluated during the first 4 months of pregnancy in mares impregnated by either stallions or jack donkeys. Twenty mares were divided in two groups: Mares in the first group were inseminated with stallion semen (horse pregnancies), and those in the second group were inseminated with donkey semen (mule pregnancies). Blood samples were collected twice weekly from day 30 to day 120 of pregnancy to determine the concentrations of eCG, progesterone, androstenedione, and testosterone. Analysis of variance for repeated measures was used to compare the concentrations of each hormone between groups. Linear regression models that considered the linear and quadratic effects of week of gestation as well as the linear and quadratic effects of the concentrations of eCG on the production of each steroid hormone were carried out. Concentrations of eCG, progesterone, and androstenedione were higher in horse than in mule pregnancies (P < .01 for eCG and P < .05 for progesterone and androstenedione). Testosterone concentrations were also higher in horse pregnancies than in mule pregnancies at weeks 7, 9, and 10 (P < .05). Regression analysis indicated that eCG had considerable stimulatory effects on the secretion of progesterone and androstenedione and weaker effects on the secretion of testosterone. The results suggest that eCG stimulates luteal production of progesterone, androstenedione, and testosterone in horse and mule pregnancies, these effects being more evident in horse pregnancies than in mule pregnancies due to the higher concentrations of eCG in horse pregnancies.     

丹连花子宫灌注液治疗奶牛子宫内膜炎的实验性临床试验

为考察不同剂量的丹连花子宫灌注液对奶牛子宫内膜炎的临床治疗效果,选择患子宫内膜炎的患病奶牛50头,随机分为5组,每组10头,分别为丹连花子宫灌注液高、中、低剂量组,以及药物对照组和疾病对照组;另选健康奶牛10头作为健康对照。结果表明,丹连花子宫灌注液不同剂量组治疗后患病奶牛的临床症状明显改善,其中高、中剂量组奶牛子宫复旧完全,子宫颈黏液清亮,白细胞数明显减少,pH6.5~7.0,尿兰母检查阴性,治愈率60%,有效率90%,显著高于药物对照组(P<0.05),且血液白细胞数显著减少,其中中性粒细胞数极显著低于疾病对照组(P<0.01);不同剂量组血液生化指标无异常变化。表明丹连花子宫灌注液对奶牛子宫内膜炎有显著治疗效果,且使用安全,推荐临床使用剂量为100mL/次,子宫灌注给药,隔天1次,连用3次。