Effects of Hemicastration on Testes and Testosterone Concentration in Stallions

The endocrine system is critical to the maintenance of testicular function. The homeostasis of sex hormone levels is orchestrated by positive and negative feedback systems controlled by the hypothalamic-pituitary-gonadal axis. This study investigated the long-term effects of hemicastration on testicular size and function in stallions. Four Thoroughbred stallions, 4–6 years of age, were included in this study. Several parameters, including testicular weight and volume, plasma testosterone concentrations, VASA-positive germ cell populations and cross-sectional areas of the seminiferous tubules were compared in stallions that underwent two hemicastrations, approximately 11 months apart. The weights and volumes of testes harvested at the second hemicastration were significantly higher than those of testes collected at the first hemicastration. However, VASA-positive germ cell populations and the cross-sectional areas of seminiferous tubules were not significantly different between testes harvested at the first and second hemicastrations. Similarly, plasma testosterone concentrations measured weekly for 3 weeks before the first hemicastration, 3 weeks after the first hemicastration, and 3 weeks before the second hemicastration were not significantly different. Our results suggest that hemicastration results in compensatory enlargement of the remaining testis and compensatory steroidogenesis to maintain normal reproductive function in stallions.     

Effects of Intratesticular Injection of 70% Glycerin on Stallions

The removal of endogenous germ cells of recipient stallions is a key step to produce donor germ cell-derived sperm using the germ cell transplantation technique. Six Thoroughbred stallions were divided into a treatment (n = 3) and a control group (n = 3), and 70% glycerin (1, 2, 3-trihydroxypropane, 40 mL per testis) or phosphate-buffered saline, respectively, was locally injected into testes. General semen evaluation, libido, and testicular volume were performed weekly from 3 weeks before to 10 weeks after treatment. The number of round germ cells in the ejaculate was counted using a hemocytometer. The hematoxylin and eosin staining was performed on the cross sections of testicular tissue obtained 11th week of treatment. Plasma testosterone levels in blood collected weekly were measured using a colorimetric competitive enzyme immunoassay kit. The sperm number was significantly lower than that of the control group at 5 and 10 weeks after glycerin injection. No differences in the status of spermatogenesis in the cross sections of seminiferous tubules and testicular volume were found between the two groups. The 70% glycerin-treated stallions had reduced total and progressively motile sperm and exhibited a significantly higher population of round germ cells in the ejaculate. Testosterone levels, testicular volumes, and libido of stallions were not significantly different between the groups. In conclusion, although intratesticular injection of 70% glycerin may have caused disassociation of some germ cells in the seminiferous tubules for several weeks, it did not significantly ablate germ cells in the tubules at 11 week in stallions.     

Management Options for the Aged Breeding Stallion with Declining Testicular Function

In our experience, the testicular dysfunction that develops in aged stallions is typically progressive, contributing to a gradual deterioration in sperm output and quality over 2-4 years. As the ability to produce sufficient numbers of normal sperm in ejaculates declines, so do pregnancy rates until the stallion eventually becomes so subfertile that it is no longer commercially feasible to continue breeding. However, more intensive breeding management can sometimes result in pregnancy rates (per cycle and per season) that are sufficient to justify breeding of the aged stallion to a diminishing number of mares during the period of declining fertility.     

Quality of Raw and of Cold-Stored Semen in Icelandic Stallions

The aim of the present study was to evaluate the quality of raw and cooled semen in Icelandic stallions. Experiments were performed using seven stallions aged between 3 and 19 years. From each stallion, six ejaculates were collected, and semen quality was determined. Thereafter, the semen was split into eight equal parts and processed with and without centrifugation using the extenders INRA 82-egg yolk, INRA 96, GENT, and Equi-Pro to a final concentration of 30 × 10⁶ sperm/mL. The extended semen was then cooled in an Equitainer, where it was stored for 24 hours, and subsequently refrigerated for another 24 hours at 5°C. Immediately after dilution as well as after 24 and 48 hours storage, sperm motility was analyzed using computer-assisted sperm analyzer, and viability was assessed after dual DNA staining with SYBR-14 in combination with propidium iodide. The results show that the stallion had a significant (P < .05) influence on all variables evaluated in raw semen, and mean (±SEM) values of 43.4 ± 4.3 mL for the volume, 193.0 ± 17.0 × 10⁶ sperm/mL for the concentration, 6.7 ± 0.5 × 10⁹ for total sperm and 73.5 ± 2.1% for total sperm motility, 48.7 ± 2.0% for progressive motility, and 65.3 ± 2.0% for rapid cells were measured. In the cold-stored semen, all variables were significantly (P < .05) influenced by the stallion, extender, and storage time (48 hours). Except for Equi-Pro, all extenders examined were suitable for cooled semen preservation. For storage of more than 24 hours, centrifugation and removal of the seminal plasma were advantageous for all extenders with the exception of Equi-Pro.     

Midline Cysts of Colliculus Seminalis Causing Ejaculatory Problems in Stallions

Cystic structures are often seen during ultrasound examination of the internal genitalia of stallions. They are located between the ampullae of deferent ducts, either within the urogenital fold, or under the isthmus of the prostate (uterus masculinus). Occasionally, cystic dilatations are also found more caudally, behind the prostate, at the colliculus seminalis (urethral cyst, utriculus masculinus). These cysts are detected less frequently during routine examinations, possibly because of the fact that this area is screened less carefully for the pathologies than the more proximal portion of the internal reproductive tract of stallions. We have recently noticed that many stallions with ejaculatory problems have large cysts at the colliculus seminalis. This article describes the typical clinical presentation of these cases, diagnostic procedures, and management. In addition, we discuss the discrepancies in the currently used terminology pertinent to this condition, as well as introducing a new term, which seems to best describe the root cause of this disorder. Finally, this article presents new diagnostic and therapeutic options used in human medicine in similar cases, and proposes to investigate the applications of these methods in veterinary medicine.     

Post-thawing Sperm Quality in Chilean Purebred Stallions: Effect of Age and Seasonality

In this work, we investigated the influence of age and seasonality on sperm motility and DNA fragmentation in post-thawing semen from Chilean Purebred Stallions (CPS), a horse breed presenting the oldest genealogy record in South America with an interesting reproductive industry. Despite that semen from aged CPS is frozen all year round, there is a lack of studies characterizing the breed semen freezability in accordance with age and seasonality. Twenty fertile CPS were grouped into the young group, the middle group, and the aged group. Ten ejaculates from each stallion were obtained by using an artificial vagina during summer (December) and winter (July) and directly frozen. Subsequently, the frozen semen was thawed and analyzed by a computer-assisted semen analysis and flow cytometer assessing progressive motility, mean velocity, and DNA fragmentation spermatozoa. Kruskal–Wallis test and Pearson’s correlation were used to determine statistical differences among groups and correlation among variables (P ≤ .05). Both spermatozoa motility traits decreased progressively in accordance with age and seasonality, showing the lowest values in the aged group during winter and the highest values in the young group during summer. Deoxyribonucleic acid fragmentation increased significantly in accordance with age and seasonality being highest in the aged group during winter and lowest in the young group during summer. Post-thawing sperm quality showed a negative correlation with the age of the stallions and a positive correlation with the normal sperm morphology before freezing. These results allow the conclusion that age and seasonality are important factors that need to be considered during the selection of CPS for reproductive programs.     

Evaluation of Plasmid Delivery by Electroporation as a Means of Increasing Gonadotropin-Releasing Hormone Production in Stallions

A plasmid delivery system validated in other species was assessed for its potential for inducing long-term expression of gonadotropin-releasing hormone (GnRH) in stallions. The efficacy of this technique was demonstrated using two plasmids: pSEAP, expressing secreted embryonic alkaline phosphatase (SEAP), and pGnRH, expressing GnRH. In experiment 1, geldings were used as a model to test the effect of muscle of injection (splenius, pectoralis, and semitendinosus; n = 3 for each site) on the expression of the reporter plasmid, pSEAP. Concentrations of SEAP rose (P < .01) in jugular plasma samples, indicating uptake and expression of the pSEAP plasmid. Concentrations of SEAP were greatest (P < .05) and most consistent after pectoralis injection, and this site was chosen for injection and electroporation in the subsequent experiment. In ex-periment 2, stallions were treated with pGnRH (2 mg, n = 3; and 4 mg, n = 3) or 2 mg of pSEAP (control; n = 4) to determine the effects on the reproductive axis. Treatment with pGnRH (day 0) resulted in higher (P < .05) plasma testosterone concentrations from day 35 to 56 and increased the luteinizing hormone (LH) (P < 0.01) and testosterone (P < .1) responses to GnRH challenge on day 21. Daily semen characteristics from days 31 to 36 showed no effect (P > .1) of pGnRH treatment on seminal characteristics. It was concluded that delivery by electroporation of plasmids encoding peptide hormones may serve as a means of long-term in vivo production of peptides in the horse. Increases in LH and testosterone secretion after GnRH were observed in pGnRH-treated stallions; however, optimal conditions for expression need to be determined in future experiments.     

Presence of Bacteria on the External Genitalia of Healthy Stallions and its Transmission to the Mare at the Time of Breeding by Live Cover

The current field study used thoroughbred stallions and mares from central Kentucky to investigate the occurrence of potentially pathogenic bacteria on the stallion's external genitalia, based on cultures, and investigated the occurrence of bacteria and type of isolate in the mare's uterus after breeding by live cover to stallions with or without positive bacterial cultures. Fifteen thoroughbred stallions and 206 mares from two central Kentucky thoroughbred farms were used during the 2010 and 2011 breeding seasons. Samples for bacteriological evaluation were taken from the prepuce and postejaculate urethra (n = 201) of stallions. Uterine swabs (n = 264) were collected 12-18 hours postbreeding. For statistical analyses, a chi-squared test was used to test the relationship between stallion culture results and postbreeding uterine culture results, as well as the effect of bacterial types found on the stallion cultures with bacterial types found on the postbreeding uterine cultures. Of stallion cultures, 22.4% were positive for potentially pathogenic bacteria, with Streptococcus equi subspecies zooepidemicus (51.1%) being the most common isolate. Uterine cultures resulted in a 29.2% positive rate for potentially pathogenic bacteria, with S. equi subsp. zooepidemicus (90.9%) being the most common. There was no difference (P > .05) in the occurrence of bacteria or type of isolate found on uterine cultures after breeding stallions with or without positive cultures. In conclusion, potentially pathogenic bacteria found on the stallion's external genitalia did not affect the occurrence and type of bacterial isolate found in the mare's uterus after breeding by live cover.     

The Relationship of Mitochondrial Membrane Potential,Reactive Oxygen Species,Adenosine Triphosphate Content,Sperm Plasma Membrane Integrity,and Kinematic Properties in Warmblood Stallions

Equine sperm possesses a unique physiology because its energy supply is mostly dependent on oxidative phosphorylation of mitochondria as an aerobic source of adenosine triphosphate (ATP) generation. The present study was, therefore, conducted to investigate the relationship between sperm kinematic and functional variables in stallions. Semen samples were collected from five warmblood stallions (three ejaculates from each stallion), diluted with INRA96 and transferred to the laboratory. Next, sperm motility, mitochondrial membrane potential (MMP), production of superoxide anion (as a reactive oxygen species; ROS), ATP content, and plasma membrane integrity were assessed. Motion and functional characteristics differed among investigated stallions (P < .05). In addition, it was revealed MMP was positively correlated with the level of ROS and ATP content and progressive motility (P < .05). The level of ROS was positively correlated with ATP content and negatively correlated with plasma membrane integrity and straightness (P < .05). Adenosine triphosphate content was positively correlated with progressive motility, curvilinear velocity, average path velocity, and beat cross frequency and reversely correlated with plasma membrane integrity and straightness (P < .05). Plasma membrane integrity was positively correlated with straight line velocity, linearity, and straightness and negatively correlated with curvilinear velocity (P < .01). In conclusion, the present study substantiated that kinematic and functional characteristics varied among various warmblood stallions. Furthermore, the present study implicated although higher mitochondrial activity increases ATP synthesis, it leads to elevated superoxide anion production, which could culminate in disintegration of the sperm plasma membrane, thereby altering motion characteristics and swimming pattern of sperm.     

Characterization of Equine Phospholipase C Zeta: A Review and Preliminary Results on Expression Defects in Subfertile Stallions

In all mammalian species studied thus far, fertilization results in a series of intracellular Ca²⁺ ([Ca²⁺]_i) increases, referred to as oscillations, responsible for driving oocyte activation and embryonic development. Current evidence supports the notion that sperm-borne phospholipase C zeta (PLCZ) is responsible for the initiation of these [Ca²⁺]_i oscillations. Although this appears to be a highly conserved mechanism for oocyte activation, differences in PLCZ sequence, activity, and expression do exist among different species. Herein, we summarize the information supporting PLCZ as the oocyte-activating factor in mammals and present our current knowledge regarding the characterization of this protein in the horse. The equine sequence yielded a protein of high relative [Ca²⁺]_i-releasing activity. Equine PLCZ was expressed over the head region overlying the acrosome, equatorial segment, connecting piece between the head and midpiece, and on the principal piece of the flagellum of stallion sperm. Equine PLCZ expressed both over the head and tail sperm regions was catalytically active, with the latter representing a characteristic unique to the horse. We also present preliminary data in subfertile stallions displaying PLCZ expression defects, although further research is required to establish a clear association between these defects and fertility problems in the horse. In summary, the information presented raises the questions of whether equine PLCZ could play diverse roles in sperm physiology and/or become a marker for the evaluation of stallion fertility, both of which are worthy of further investigation.     

The Effects of Adjuvants on Autoimmune Responses Against Testicular Antigens in Mice

Experimental autoimmune orchitis (EAO) is a model of immunologic male infertility and pathologically characterized by lymphocytic inflammation, which causes breakdown of the testicular immune privilege with spermatogenic disturbance. Generally, murine EAO is induced by immunization with testicular homogenate (TH) from the testes of donor mice + complete Freund''s adjuvant (CFA) + Bordetella pertussigens (BP), and it has been considered that treatment with these two adjuvants is required to enhance the immune response against testicular antigens. However, there remains a possibility that CFA and BP may affect autoimmune responses against the testicular antigens without TH. In the present study, we examined this possibility using real-time RT-PCR, Western blotting and immunohistochemical staining. The results demonstrated that immunization with TH in combination with CFA and BP evoked more severe EAO than that with only TH. Real-time RT-PCR analyses revealed that Fas mRNA expression in TH+CFA+BP-induced EAO was significantly higher than that in TH-induced EAO. Interestingly, IL-6 mRNA expression dramatically increased in TH+CFA+BP-induced EAO; however, no apparent change in IL-6 mRNA expression occurred in TH-induced EAO. It was also noted that treatment with CFA and BP alone augmented autoimmune reactions against some testicular autoantigens. These results indicates that these adjuvants are helpful in evoking severe EAO, and treatment with the adjuvants alone can evoke autoimmune reactions against some testicular autoantigens despite the use of no TH.     

The Effects of Growth Factor on Testicular Germ Cell Apoptosis in the Stallion

Apoptosis is necessary for both initiation and control of spermatogenesis; however, an increase in apoptosis can lead to subfertility/infertility in stallions, causing substantial financial loss in the equine industry. The ability of stem cell factor (SCF), leukemia-inhibiting factor (LIF), granulocyte–macrophage colony-stimulating factor (GM-CSF), and estradiol (E₂), alone or in combination, to prevent apoptosis of germ cells in short-term equine testicular cultures was examined. Testicular tissue was sectioned into approximately 2-mm cubes and placed in media-filled culture chambers. Concentrations of SCF (100 ng/mL), LIF (10 ng/mL), GM-CSF (5 ng/mL), and E₂ (10⁻⁹ mol/l) were added alone or in combination to each well. After 6 hours in culture, the tissue was fixed and immunohistochemically (terminal deoxynucleotidyl transferase-mediated nick-end labeling; TUNEL) stained for apoptosis detection. Apoptotic cells per 100 Sertoli cell nuclei within seminiferous tubules were counted until the 500^th Sertoli cell nuclei was reached. This counting procedure was used for each slide. An analysis of variance (ANOVA) with a Tukey's test was used to compare apoptotic rates. In comparison with the control, GM-CSF alone lowered apoptosis by 34.77%. GM-CSF–treated tissue combined with SCF and LIF as well as GM-CSF combined with SCF, LIF, and E₂ reduced apoptosis when compared with the control (37.45% and 44.40%, respectively) or other treatment combinations. GM-CSF alone reduced apoptosis; results suggest possible synergy for the combinations of SCF and LIF with GM-CSF and for E₂ with SCF, LIF, and GM-CSF.     

纳米硒对波尔山羊睾丸发育的影响

为了研究日粮中添加纳米硒对公羊睾丸发育的影响,试验选用体重、日龄接近的20只波尔山羊公羔羊随机分2组,1组日粮中添加0.3mg/kg的纳米硒(以硒计),另一组饲喂基础日粮,预试2周,试验期每月测量睾丸周径、长度和高度,每组阉割1只公羔,按照常规方法制作石蜡切片,观察睾丸组织结构。结果显示,3月龄时纳米硒组睾丸间质组织增生发育,4月龄时纳米硒组睾丸曲精细管中精原细胞开始发育分化;5月龄时曲精细管中可见大量的精子,而对照组间质组织以及曲精细管发育、精母细胞发育分化较纳米硒组缓慢。试验表明,日粮中添加一定浓度的纳米硒能够促进睾丸间质组织及曲精细管的发育,促进精母细胞的分化。     

成年大鼠睾丸去精索上神经对睾丸功能及肾上腺素能受体表达的影响

试验旨在研究精索上神经（superior spermatic nerve,SSN）对睾丸功能的影响,25只成年Wistar大鼠随机分为2组,试验组左右睾丸切除精索上神经,手术30 d后两组大鼠同时处死取样分析。结果显示,睾丸去精索上神经不影响睾丸指数的大小但使附睾尾精子数极显著降低（P＜0.01）,平均日增重显著增加（P＜0.05）。增殖细胞核抗原（PCNA）免疫组织化学染色结果显示,睾丸去精索上神经不影响曲细精管精原细胞和初级精母细胞的增殖,但影响曲细精管的形态和生殖细胞的规则排列。RT-PCR结果显示,睾丸去精索上神经明显上调β1AR mRNA的表达,下调β2AR mRNA的表达;另外切除精索上神经不影响睾丸3β-羟胆固醇脱氢酶（3β-HSD）mRNA表达,但显著抑制类固醇快速调节蛋白（StAR）、胆固醇侧链裂解酶（P450scc）mRNA表达（P＜0.05）。结果表明,支配睾丸的精索上神经通过调节肾上腺素能受体β1AR和β2AR的表达及StAR和P450scc影响睾丸睾酮的合成和精子的发生。     

双峰驼肾动脉分布

应用大体解剖学和血管铸型方法,研究了12峰双峰驼肾动脉的分支分布。结果显示,肾脏的肾动脉在进入肾门之前均分成一背干和一腹干,在肾窦内,背干分为一前支,再由前、后支分别发出背部贤脏的各肾段动脉。腹干不分前、后支,而由主干直接发出腹部肾脏的各肾段动脉。不仅各肾段动脉之间未见吻合,背干及其分支与腹干及其分支之间也无吻合。左肾肾段动脉共13条,右肾肾段动脉12条。表明,双峰驼不仅左、右肾的肾动脉与肾段动脉不尽相同,各肾的背部和腹部的动脉供应也存在差异。肾动脉的背干和腹干是相对独立的。     

野外采集牦牛睾丸组织的冷冻保存及其生精细胞的复苏培养研究

采用玻璃化冷冻方法对野外采集的牦牛睾丸组织进行冷冻,通过HE(hematoxylin-eosin)染色分析发现玻璃化冷冻后的牦牛睾丸组织曲细精管结构保存较完好,曲细精管可见大量形态完好的各类生精细胞。采用台盼蓝染色检测细胞活率,发现冷冻复苏后细胞活率可达80.20%。分别通过生精细胞和精原干细胞标志蛋白DDX4和GFRA1免疫荧光染色发现复苏后培养14天后的生精细胞和精原干细胞的数量明显减少。通过RT-qPCR对复苏后不同实验处理组牦牛睾丸细胞标志基因的表达分析,发现培养30天的生精细胞中的精原干细胞标志基因Thy1和UCHL1的表达量显著增高。因此,玻璃化冷冻保存的牦牛睾丸组织中曲细精管及其生精细胞得到了较好的保护,该方法对于其他哺乳动物生精细胞的长久有效保存具有重要的参考价值。     

广东桑桑枝总黄酮含量测定及与体外抗氧化活性的相关性研究

为了探讨桑枝抗氧化作用的物质基础,进一步开发桑枝的药用功能,采用比色法对广东桑不同品种及不同生长季节桑枝的总黄酮含量、总抗氧化活性、.OH和O2-.清除作用进行了测定,结果表明:品种和生长季节对桑枝总黄酮含量和体外抗氧化活性有显著的影响(P<0.05)。分析结果还显示:染色体倍数性对桑枝总黄酮含量和体外抗氧化活性无显著的影响(P>0.05);桑枝总黄酮含量与抗氧化活性间呈现显著的正相关关系(P<0.01)。