杜洛克猪与梅山猪副性腺组织学比较及精浆蛋白mRNA表达分析

旨在探讨杜洛克公猪和梅山公猪精囊腺和尿道球腺的组织学特征及精浆蛋白mRNA的表达,运用石蜡切片并通过HE染色,分别在光学显微镜下对75日龄、270日龄梅山公猪与270日龄杜洛克公猪的精囊腺和尿道球腺进行组织学观察;并用实时荧光定量PCR对精浆蛋白mRNA的表达进行分析。270日龄梅山公猪与75日龄梅山公猪相比,精囊腺的腺小叶增多,小叶间结缔组织减少;270日龄杜洛克公猪精囊腺较270日龄梅山公猪有更多的腺泡黏膜褶皱和小叶间结缔组织。梅山公猪和杜洛克公猪尿道球腺被结缔组织分隔成小叶,腺泡密集且腺腔小;梅山猪在成年时尿道球腺腺小叶增大,小叶间结缔组织变少;270日龄杜洛克公猪尿道球腺腺小叶比270日龄梅山公猪小,而腺泡较大,且结缔组织更多。精浆蛋白PSP-Ⅰ和PSP-Ⅱ在精囊腺中高表达,在成熟的杜洛克和梅山公猪精囊腺中其mRNA表达水平相近(P0.05)。但随着日龄的增长,梅山公猪PSP-Ⅰ和PSP-ⅡmRNA表达水平显著升高(P0.05)。杜洛克公猪更多的精囊腺黏膜褶皱和更大的尿道球腺腺泡,增加了上皮分泌面积,有助于增加精浆体积,提高精子代谢率和存活率。另外,性成熟公猪中精浆蛋白PSP-Ⅰ和PSP-Ⅱ的高表达可能参与维持精子的代谢和活力等生理过程。     

藏猪生殖器官生长发育研究

选择舍饲条件下日龄、体重相近的藏公猪、母猪进行生殖器官生长发育测定。结果表明：藏公猪尿道球腺、精囊腺的发育早于其它生殖器官,而成熟要晚于其它生殖器官;藏公猪适宜配种时间为5-6月龄,藏母猪适宜配种时间为6月龄。     

梅山猪垂体中GH、PRL及TSHβ基因发育性表达规律的研究

生长激素(GH)、催乳素(PRL)及促甲状腺激素β亚基(TSHβ)是动物生长发育过程中的重要调控因子。本研究我们通过RT-PCR检测了GH、PRL及TSHβ基因在不同生长阶段梅山猪垂体中mRNA的表达水平。结果发现:GH和TSHβ基因在公、母猪的生长发育过程中的表达量总体上都呈现先升高后降低的趋势;而PRL在公猪中的表达量在不同发育阶段相对稳定,而在母猪中呈现先下降后升高的趋势。总体上GH、PRL及TSHβ3个基因在公、母猪的生长发育过程中的表达呈现性别二相性。其中GH基因的表达在1、30、90和150日龄公、母猪间呈现出显著差异(P0.05);而PRL基因的表达在1、60和90日龄的公、母猪表现显著差异(P0.05);而TSHβ基因的表达在60和150日龄的公、母猪表现显著差异(P0.05)。     

Tektin3基因在猪生殖系统发育中的表达规律研究

TEKTIN是附着于精子尾部基因丝的重要组分,对于基因丝的稳定和结构的复杂性起重要作用。Tektin3是其家族成员之一。研究采用RACE方法,获得梅山猪Tektin3基因全序列,利用生物信息学方法分析其结构和功能,采用半定量的方法研究150日龄梅山公母猪23个不同组织Tektin3基因的表达特征,并用Realtime PCR方法分析2、30、60、90和150日龄梅山公猪睾丸的表达规律。结果表明:(1)Tektin3基因全序列1 761bp,包含5'UTR 95bp,完整的CDS序列1 473bp和3'UTR 193bp,编码490个氨基酸,相对分子质量为56 481.3,理论等电点为7.94。(2)Tektin3基因在睾丸中高丰度表达,在下丘脑和子宫角中表达丰度较低。(3)Tektin3基因从梅山猪60日龄开始表达,到90日龄时显著下降(P0.05),150日龄时显著上升(P0.05)。结果表明该基因编码氨基酸除与精子发生有关,可能还与部分组织或器官的纤毛发生及公猪的性发育有关。     

梅山猪UBE2b基因的克隆及组织表达特征的研究

UBE2b基因编码的泛素结合酶参与的泛素-蛋白酶通路(UPP)在哺乳动物精子发生中起着重要作用。本研究根据相关ESTs拼接的序列采用PCR扩增鉴定的方法获得梅山猪UBE2b基因cDNA全序列,对其序列进行生物信息学分析,利用半定量RT-PCR的方法研究150日龄梅山公猪UBE2b基因的组织表达特征和不同日龄(2、30、60、90、150日龄)公猪睾丸的表达规律。结果表明:梅山猪UBE2b基因cDNA全长1 329 bp,编码153个氨基酸,具有UBCc家族的保守结构域。该基因编码氨基酸序列在不同物种间具有较高的保守性,根据编码氨基酸序列构建的分子进化树显示猪与牛的亲缘关系最近,与斑马鱼的关系最远。UBE2b基因在梅山公猪各组织中广泛表达,其中睾丸为高丰度表达。该基因在梅山猪睾丸中的表达随日龄增加呈显著上升趋势,与睾丸发育显著相关(r=0.82,P<0.01),推测UBE2b可能在梅山猪精子发育和性发育中起重要作用。     

合作猪生长曲线分析和拟合研究

对在异地舍饲的高原型小型猪种——合作猪的生长发育规律进行了研究,并利用Logistic、Gompertz和R ichards三种曲线拟合了其生长模型。研究结果表明,三种模型中,Gompertz模型拟合效果最理想(R2=0.999 5);公、母猪在150日龄前生长基本一致,但150日龄后母猪明显快于公猪;公、母猪的生长拐点分别为118.32、146.75日龄,母猪的成年体重明显快于公猪,但达到生长拐点的日龄比公猪迟。     

二花脸猪和大白猪睾丸IGF-I和IGF-IR基因表达发育变化的研究

为了研究生长轴激素对猪繁殖性能发育的影响,随机选取0、3、20、30、90、120、180日龄纯种二花脸公猪和大白公猪各4头,屠宰并采取睾丸组织样,以18S rRNA为内标,用相对定量RT-PCR法研究睾丸IGF-I和IGF-IRmRNA的表达及发育性变化。结果表明,二花脸猪与大白猪睾丸IG-FI mRNA表达的发育模式在30日龄前完全相同,即随着日龄的增加而呈极显著增加(P<0.01);二花脸猪睾丸IG-FI mRNA相对表达量在30~180日龄无显著变化;大白猪睾丸IGF-I mRNA相对表达量在90日龄有所下降,而在120和180日龄又恢复到30日龄水平。二花脸猪睾丸IG-FI mRNA相对表达量显著高于大白猪(P<0.05)。二花脸猪与大白猪睾丸IGF-IR mRNA表达的发育模式不同。二花脸猪睾丸IG-FIR mRNA相对表达量在90~120日龄呈显著下降趋势(P<0.05);大白猪IG-FIR mRNA相对表达量在0日龄较高,随后显著下降(P<0.05),并在观察期内持续保持较低水平。二花脸猪IG-FIR mRNA相对表达量显著高于大白猪(P<0.05)。睾丸IGF-I mRNA和IG-FIR mRNA相对表达丰度呈极显著正相关(r=0.575,P<0.01)。结论:(1)不同品种猪睾丸IGF-I和IG-FIR mRNA表达具有特定的发育模式;(2)猪睾丸中IGF-I和IG-FIR mRNA的协同表达可能对猪繁殖性能的发育有重要调节作用。     

猪GPR54基因在下丘脑、垂体、卵巢发育性表达变化的研究

分别随机选取处于初生、60日龄、120日龄、初情期、180日龄的苏姜猪母猪各4头,进行屠宰,采集下丘脑、垂体、卵巢,采用反转录多聚酶链式反应（RT-PCR）,以-βactin作内标,定量分析下丘脑、垂体、卵巢中GPR54mRNA发育性变化。结果显示：苏姜猪GPR54 mRNA在下丘脑、垂体、卵巢组织内从初生到初情期表达量逐渐上升,初情期后呈下降趋势,初情期GPR54 mRNA表达丰度与初生、180日龄差异显著（P〈0.05）;在不同组织器官中,GPR54 mRNA表达丰度在卵巢中最高,下丘脑中表达丰度最低,下丘脑的表达丰度与垂体、卵巢中的表达丰度均差异显著（P〈0.05）。     

甘肃合作猪的生长曲线分析和拟合研究

利用Logistic、Gompertz和Richards三种曲线拟合甘肃合作猪的生长模型。结果表明,三种模型中,Gompertz模型拟合效果最理想（R^2=0.9995）;合作猪公、母猪在150日龄前生长基本一致,150日龄后母猪的生长速度明显快于公猪;公、母猪的生长拐点分别为118.32日龄和146.75日龄,母猪的成年体重明显大于公猪,但达到生长拐点的日龄较公猪迟。     

后备母猪的饲养管理要点

后备母猪的饲养管理主要注意以下几个方面. (1)膘情控制和公猪诱导.后备母猪在60kg前应让其充分发育,60kg后到配种前半个月要适当限制饲养以防其过肥,每天喂料约2kg,日喂2次.后备母猪可使用生长肥育猪饲料.后备母猪长到160日龄时,最好每天将母猪赶入成年公猪栏接触35min.成年公猪下颌腺分泌的外激素有助于诱导小母猪发情.     

Spatial and temporal gene expression of Fn-type II and cysteine-rich secretory proteins in the reproductive tracts and ejaculated sperm of Chinese Meishan pigs

Fibronectin type II and cysteine-rich secretory proteins have been well studied in the murine and human. The present study evaluated CRISP1, CRISP2, CRISP3 and Fn-type II (ELSPBP1 and pB1) gene expression patterns in ejaculated sperm and reproductive tracts of Chinese Meishan pigs from birth to day 150 of age. In ejaculated sperm, except for ELSPBP1, all others genes studied were detectable. In sexually mature boars and gilts, CRISP1 gene was expressed strongly in whole epididymides, moderate in prostate and weak in seminal vesicle. CRISP2 gene represented extensive distribution along reproductive tracts with highest abundance in testis. CRISP3 gene was expressed highly in prostate and bulbourethral gland, but weakly in testis. ELSPBP1 gene was expressed with highest abundance in cauda epididymides, moderate in corpus epididymides and weak in seminal vesicle and prostate. pB1 mRNA expression was also abundant along reproductive tracts. During the sexual development of boars after birth, these genes showed different expression patterns. CRISP1 and CRISP3 gene expression was high on day 1 and maintained until day 150, while CRISP2 expression was detectable on day 60 with high abundance and maintained until day 90 and dropped on day 150. ELSPBP1 showed low expression at birth and increased significantly on day 30 (p < 0.05) and then kept static until day 150. pB1 gene displayed moderate expression from birth to day 30 and increased significantly on day 60 (p < 0.05) and maintained at high level until day 150. Collectively, CRISP and Fn-type II genes were expressed extensively along genital tracts, and most of them showed mRNA signal in ejaculated sperm. The expression of CRISP1 and CRISP3 genes in Meishan boar was not age-dependent, while CRISP2 and pB1 gene expression was parallel with sexual development. Their unique gene expression patterns may shed light on the mechanism for the high prolificacy of Meishan pigs.     

A histological study of the seminal vesicle of the armoured catfish Corydoras aeneus

Most species of Corydoras exhibited a reproductive behaviour called 'T-position', and exhibited an accessory gland in the male genital tract, called the seminal vesicle. It appeared that both the structure and the composition of the fluid varied considerably between the species investigated. Consequently, different opinions were proposed regarding the possible role of seminal vesicle on this particular reproductive behaviour. Male adults of Corydoras aeneus were collected, anaesthetized, and samples of seminal vesicle were fixed in Bouin's solution. The sections were stained with haematoxylin-eosin and periodic acid Schiff. The seminal vesicle showed a system of anastomosed secretory tubules, forming a vesicular collective network, which gave rise to the vesicular ducts. The latter fused with the testicular efferent ducts and formed the spermatic ducts. Considering this fusion, when the sperm cells reached the spermatic ducts, the fluid produced at the seminal vesicle covered them. Histochemical studies evidenced the presence of neutral and acid glycosaminoglycans in the seminal fluid. Considering the reproductive behaviour of C. aeneus, it is believed that the protection associated with the immobilization of the sperm cells assures the sperm integrity during the passage through female's intestine until fertilization.     

Response of testes, epididymis, and seminal vesicle of rabbits to zinc deficiency.

It is the purpose of this study to determine the effects of Zn deficiency on the biochemical composition of testes, epididymis, and seminal vesicle of rabbits. An attempt is made to evaluate previous physiological studies and to correlate them with biochemical changes. 30 mature male Balady rabbits were used in this study. 1 group was fed a Zn-deficient diet, and 2 control groups were pair-fed or fed ad libitum a Zn-sufficient diet, all for a period of 120 d. There was significant reduction in the levels of hyaluronidase, alkaline phosphatase, acid phosphatase, lactic dehydrogenase, sialic acid, protein, and Zn of both testes and epididymis of Zn-deficient rabbits. Reduction in the level of glyceryl-phosphoryl choline in the epididymis of Zn-deficient rabbits was the best indicator of inhibition of epididymal secretory activity. In contrast, the cholesterol and glycogen contents of the testes were elevated. The results also showed in Zn-deficient rabbits significant reduction in androgen-sensitive parameters, namely fructose and citric acid in the seminal vesicle. Zn levels were decreased in the seminal vesicle. The results indicated that Zn deficiency caused inhibition of testicular, epididymal, and seminal vesicle function and, consequently, caused reductions in the biochemical composition of these organs.     

Immunohistochemical and Biomolecular Identification of Orphanin FQ,eNOS, Atrial natriuretic Factor and Oxytocin in Rat Seminal Vesicles

In previous studies performed on rodents, we detected the presence of adreno‐cholinergic and peptidergic innervation in seminal vesicles and other organs of the male genital system, such as prostate and deferent duct, in which we also investigated the expression of NOS and NADPH‐diaphorase. During this project, we focused our attention on the expression of some peptides involved in local control of smooth muscle relaxation, contractility, vasodilatation and control of blood flow in rat seminal vesicles. We investigated, through immunohistochemistry and RT‐PCR, the presence of four peptides: orphanin, eNOS, ANF and oxytocin. Immunohistochemistry was used to detect the presence of the proteins, whereas RT‐PCR analysis confirmed gene expression of orphanin, eNOS and ANF, but not oxytocin. In our opinion, orphanin, eNOS and ANF could have paracrine effects regulating the function of seminal vesicles, whereas oxytocin, which may reach this anatomical district through the blood flow, may have a hormonal action. This is a pilot study that, with further investigation, may allow to better clarify the role of these molecules in the control of seminal vesicle tissues’ homeostasis.     

Successful Treatment of Seminal Vesiculitis with Imipenem-Cilastatin in a Stallion

A 7-year-old thoroughbred stallion presented with severe colic, which was resolved surgically. On postsurgical palpation and rectal ultrasonography, the left seminal vesicle was enlarged, the left testicle and epididymis showed signs of inflammation with hydrocele. Antibiotics and anti-inflammatory treatment were administered. Fever and colic syndrome recurred, with severe inflammation of the left testis and epididymis, and unilateral orchiectomy was performed. Histopathological diagnosis confirmed chronic orchioepididymitis, and Klebsiella pneumoniae was identified and typed from all cultures. At breeding soundness examination 2 months after treatment with ceftiofur, K. pneumoniae was again isolated, and cytology revealed degenerated polymorphonuclear cells and red blood cells. Transurethral endoscopic examination was used to locate the infection in the left seminal vesicle, and ticarcillin-clavulanic was locally infused for 10 days. Nevertheless, the pathogen was once again isolated in the ejaculate, and the affected seminal vesicle was again treated locally for 5 consecutive days, using imipenem-cilastatin, a new antibiotic showing high sensitivity. Follow-up breeding soundness examination revealed an improvement in seminal characteristics and no pathogen isolation. Total pregnancy rate (31 of 36; 86%) and first cycle pregnancy rate (18 of 36; 50%) in the last breeding season reached levels similar to those prior to infection (65 of 82; 80% and 47 of 82; 57% respectively), suggesting that local treatment with imipenem-cilastatin in stallions with seminal vesicle infections is effective and, in this case, did not produce any adhesions or other undesirable effects, permitting the stallion to resume breeding with no apparent deleterious effect on fertility despite having only one testicle.     

Ultrastructure of the ducts of the reproductive tract of males of Melipona bicolor bicolor lepeletier (Hymenoptera,Apinae, Meliponini)

The present paper describes the ultrastructural features of seminal vesicle, post-vesicular vas deferens and ejaculatory duct of Melipona bicolor bicolor from newly emerged and mature males. Although the results do not show very consistent morphological signs of secretory activity by the epithelium of these organs, lipidic droplets and lamellar granules present in mature males' seminal vesicles and the vacuoles present in post-vesicular vas deferens are probably secretion. Besides, the spermatozoa in the lumen are immersed in a material of characteristic structure, which must be produced in superior regions of the reproductive system of immature males, not studied here. The presence of sperm cells, apparently in cytoplasm vesicles of seminal vesicle and post-vesicular vas deferens, suggests spermiophagy by their epithelium.     

Three‐Dimensional Study of the Terminal Portion in Sprague‐Dawley Rat Ejaculatory Ducts

In mammals, a pair of ejaculatory ducts exists in the urethra at the seminal colliculus. The detailed anatomical structures of the distal end of the ejaculatory ducts of Sprague‐Dawley rats were investigated by the computer‐assisted three‐dimensional reconstruction analysis using light‐microscopic serial sections. A three‐dimensional reconstruction revealed that in adult rats, the ejaculatory sinus pair consists of two parts: the cranial section – a compartment region composed of a fusion of the ampullary gland duct and the seminal vesicle duct, and the caudal section – a grooved region composed of a long slitlike ejaculatory ostium that extends into the urethra on both sides of the seminal colliculus. But the sphincter structure was not observed. The long axis of the compartment region was approximately 58 μm in length, and that of the groove region was approximately 495 μm. Although many epithelial glands ducts were distributed throughout the ejaculatory sinuses, the prostate and coagulation gland ducts did not open in these sinuses. The urethra was composed of transitional epithelium, while the ejaculatory sinuses were composed of single to stratified cuboidal epithelium. The ejaculatory ducts continued to the ejaculatory ostium in male adult Sprague‐Dawley rat were composed of the seminal vesicle ducts received the ampullary gland ducts.     

Distribution of Mycobacterium avium subsp. paratuberculosis in organs of naturally infected bull-calves and breeding bulls

Paratuberculosis, caused by Mycobacterium avium subsp. paratuberculosis, has particular importance in cattle due to the resulting chronic diarrhoea, weight loss, decreased production, infertility and eventual death. While faecal oral route of infection is generally recognised, reports about semen-derived infection are rare. The objective of this work was to assess whether M.a. paratuberculosis may disseminate from the gastrointestinal tract to reproductive organs, and compare this event between naturally infected bull-calves and breeding bulls. Ten bull-calves, aged 6-28 weeks and four breeding bulls were tested by serology, faecal and tissue culture, IS900 PCR and RFLP. In seven bull-calves M.a. paratuberculosis was isolated predominantly from mesenteric lymph nodes (75%); isolates from mucosa of the intestine constituted 25%. In three breeding bulls, M.a. paratuberculosis was isolated both from intestinal mucosa and mesenteric lymph nodes. Head and mediastinal lymph nodes, liver, spleen and semen of bull no. 1 (Holstein-Friesian); testes and epididymis of bull no. 2 (Piemonte); testes, epididymides and seminal vesicle of bull No. 3 (Hereford); and seminal vesicle of bull No. 4 (Simmental) tested positive by culture. Hot-start PCR revealed M.a. paratuberculosis in semen, seminal vesicle and intestinal tissue where culture isolation was difficult. Isolates from bull-calves and breeding bulls were of RFLP types B-C9 and B-C1, respectively. Bull-calves born in infected herd can be sources of infection when later used for natural mating or artificial insemination. Sub-clinically infected bulls release M.a. paratuberculosis into semen, consequently infecting the uterine environment of cows.