GnRH-A免疫对兔去势效果的实验研究

探讨促性腺激素释放激素类似物(GnRH-A)对动物免疫去势的效果和作用机理.用自制的GnRH-A抗原乳剂在30只兔(EG-Ⅰ与EG-Ⅱ组)的颈背部皮下分2～3点注射1.0 mL(100 μg·mL-1)GnRH-A抗原,EG-Ⅱ组3周后加强免疫1次.测定睾丸长度和质量,体质量变化,GnRH抗体效价与血清睾酮浓度.制备的抗原无菌性、安全性和物理性状良好,EG-Ⅱ组睾丸长度与EG-Ⅰ组差异极显著(P<0.01);EG-Ⅱ组的抗体水平也明显高于EG-Ⅰ组;免疫注射后2个试验组与对照组的血清睾酮浓度差异逐渐增加,102 d时EG-Ⅱ组血清睾酮浓度极显著低于对照组(P<0.01),且第28天后EG-Ⅱ组显著低于EG-Ⅰ组(P<0.05);EG-Ⅱ组雄兔的体质量和平均日增质量均最大,显著高于EG-Ⅰ组和对照组(P<0.05).GnRH-A免疫家兔对睾丸发育、血清GnRH抗体效价和睾酮浓度具有显著的影响,加强免疫效果更理想.     

用注射器直接对睾丸打点注射EGFP生产转基因兔的试验研究

为探讨直接用 1 mL 注射器对睾丸打点注射pIRES2-EGFP建立操作方便、效率高、成本低、可批量生产转基因兔的可行性.选择4只成年新西兰雄兔,双侧睾丸内分别打点注射 0.5～0.8 mg/mL浓度的质粒 0.25 mL,第 3 周和第 8 周分别取4#和3#雄兔睾丸做冰冻切片,置荧光显微镜下观察是否发绿色荧光.其它雄兔于第 3 周开始参与配种,最后采新生仔兔耳组织提取其基因组进行 PCR 和 Southern 杂交检测阳性率.结果表明,雄兔睾丸冰冻切片于荧光显微镜下可见绿色荧光,PCR 和 Southern 杂交检测表明在睾丸注射后的第 6 周和第 7 周进行配种所得后代阳性率最高.不同雄兔后代经 PCR 和 Southern 杂交检测平均阳性率存在差异(P<0.05),2#雄兔后代平均阳性率达到 56.46%,3#雄兔的最低达36.96%的阳性率.表明用注射器直接对睾丸打点注射外源基因生产转基因的方法操作简便、高效,为大规模制备一些大型家畜的转基因后代奠定了基础.     

丙酸睾酮在肉鸡组织中残留及其对机体健康的影响

本研究旨在探讨丙酸睾酮在肉鸡组织中的残留及其对肉鸡血清生化指标及睾丸生长发育的影响,为保障畜产品安全提供检测依据。选取初始体重为（2.2±0.1） kg的60日龄青脚麻公鸡160只,随机分为对照组与试验组,每组4个重复,每个重复20只。对照组饲喂基础日粮,试验组饲喂添加20 mg/kg丙酸睾酮的试验日粮,连续饲喂7 d,分别于休药期第2、6、11、17天每组选择8只鸡屠宰,采集鸡冠、肝脏、睾丸和肌肉等组织,用液相色谱-质谱联用仪（HPLC-MS/MS）检测丙酸睾酮残留量、睾酮含量;翅静脉采集血液样品,分离血清后用全自动生化分析仪测定血清生化指标的含量;采集睾丸样品,利用HE染色病理组织检查、甲苯胺蓝病理形态学镜检观测睾丸组织细胞形态、肥大细胞数等。结果显示,①血液中丙酸睾酮残留量最高,鸡冠中残留量次之,胸肌、腿肌、肝脏和睾丸中无残留;休药期长短对鸡冠中丙酸睾酮残留量无显著影响（P>0.05）,休药17 d后血清丙酸睾酮含量极显著低于2、6和11 d （P<0.01）,且休药期与鸡冠、血液中的丙酸睾酮残留量呈一元二次回归关系。②试验组睾丸中睾酮含量在休药2、6、11、17 d时较对照组分别降低了71.45%、63.46%、62.86%、73.61%（P<0.01）。③在休药2 d时,试验组睾丸相对重量显著低于对照组（P<0.05）;试验组肝脏相对重量与对照组无显著差异（P>0.05）。④肉鸡血清大部分指标（总蛋白（TP）、白蛋白（ALB）、白球比（A/G）、胆固醇（CHO）、甘油三酯（TG）及乳酸脱氢酶（LDH）、谷丙转氨酶（ALT）和谷草转氨酶（AST）酶）均无显著变化（P>0.05）。⑤丙酸睾酮对公鸡睾丸的影响较大,细胞失去了原有的椭圆形,生精细胞在管腔中排列紊乱。试验组脱颗粒数目、肥大细胞（MC）数分别比对照组多。综上所述,饲喂丙酸睾酮会导致公鸡睾丸萎缩,可通过血液和鸡冠取样检测到其残留量,这一结果可为保障畜产品安全提供检测依据。     

母鼠孕期暴露双酚A对子代断乳雄鼠生殖内分泌以及睾丸DNA甲基化的影响

为探究母鼠妊娠期暴露双酚A(bisphenol A,BPA)对子代断乳雄鼠生殖内分泌与睾丸DNA甲基化的影响。按0,2.5,5.0,10.0,20.0,40.0 mg·kg~(-1)·d~(-1 )BPA剂量在妊娠期(PND)1～17 d灌胃孕鼠,制备孕鼠BPA暴露模型。然后统计母鼠妊娠期流产率,仔鼠断乳时存活率,睾丸系数。放射免疫法检测血清中雌二醇(E_2)、睾酮(T)含量;酶联免疫吸附法检测血清中雌激素受体α(ERα)、雌激素受体β(ERβ)含量;实时定量荧光PCR法检测睾丸中DNA甲基转移酶1(Dnmt1)、DNA甲基转移酶3a(Dnmt3a)、DNA甲基转移酶3b(Dnmt3b)mRNA相对转录水平。结果显示,20.0 mg/kg BPA暴露可极显著增加断乳时仔鼠睾丸系数(P0.01);20.0,40.0 mg/kg BPA暴露可显著增加母鼠流产率(P0.05),极显著降低仔鼠存活率(P0.01);2.5,20.0 mg/kg BPA暴露可极显著提高仔鼠血清T和ERβ含量(P0.01);各BPA剂量均可极显著降低仔鼠血清雌二醇水平(P0.01),促进睾丸内Dnmt1转录,抑制Dnmt3a和Dnmt3b转录;血清中雌激素受体α含量与BPA剂量呈现非线性关系。表明BPA通过干扰子代雄鼠睾丸内DNA甲基转移酶(Dnmt)的转录,使睾酮、雌二醇及雌激素受体分泌紊乱,影响子代雄鼠的生殖机能。     

不同外源性激素对雄性牛蛙睾丸中肥大细胞的影响

为了解外源性激素对雄性牛蛙生殖器官睾丸的作用,试验将复方甲地孕酮、苯甲酸雌二醇、丙酸睾酮注入雄牛蛙体内,饲养牛蛙7 d,用Bouin氏固定液固定,最后用改良的甲苯胺蓝染色法显示肥大细胞。结果表明:睾丸中肥大细胞主要分布在睾丸组织的被膜下、睾丸间质和输出小管内;睾丸肥大细胞呈圆形、椭圆形、梭形和不规则形,大小不一,可见肥大细胞的胞浆中充满紫红色颗粒;接受复方甲地孕酮、苯甲酸雌二醇、丙酸睾酮处理的雄性牛蛙,睾丸肥大细胞数量增多,与对照组相比差异极显著(P0.01),用丙酸睾酮处理的睾丸肥大细胞数量变化与对照组相比差异极显著(P0.01)。     

磁水对动物影响的实验研究

经磁水喂养的小白鼠、大白鼠、兔,测定其部分血液指标发现,小白鼠血红蛋白、白细胞减少兔白细胞减少,血清钙下降;大白鼠和兔甘油三酯、低密度脂蛋白下降,高密度脂蛋白升高。兔睾丸重量增加。精细管直径变大。显示饮用磁水对以上三种实验动物都有明显生物效应。对预防动脉粥样硬化,促进雄性动物生殖器官的发育等有良性作用。     

GnRH二聚体和GnRH并列二聚体合成肽疫苗对大鼠的免疫去势效果

为了比较GnRH二聚体和GnRH并列二聚体单次和两次免疫对雄性大鼠的免疫去势效果。将上述两种GnRH疫苗,分组免疫SD大鼠,免疫后每两周检测大鼠血清中的GnRH抗体滴度和血清睾酮水平、睾丸重量和组织结构变化。结果显示,两次免疫试验组的抗体滴度显著高于单次免疫试验组的抗体滴度(P<0.05);GnRH二聚体抗原免疫效果优于GnRH并列二聚体抗原的免疫效果,且差异极显著(P<0.01)。免疫组大鼠血清睾酮水平及睾丸重量均极显著低于空白对照组(P<0.01)。免疫去势效果显著的大鼠只数统计显示,GnRH二聚体(5/6)和GnRH并列二聚体(4/6)两次免疫均多于单次免疫(4/6和3/6)。同时,组织学观察结果可见,免疫组大鼠睾丸组织结构萎缩,曲精小管管径变窄,内部精母细胞脱落,精子变少或者没有。表明GnRH二聚体疫苗与GnRH并列二聚体疫苗均能达到去势效果,前者优于后者,两次免疫优于一次免疫。     

17β—雌二醇主动免疫对公兔生殖的影响

以１７β－雌二醇－６－人血清白蛋白对新西兰公兔进行主动免疫，用ＲＩＡ检测抗体滴度和１７β－雌二醇及睾酮浓度，并检测睾丸重量和间质细胞面积，试验组各兔均不同程度地产生了１７β－雌二醇抗体，其血浆１７β－雌二醇浓度极显著的低于对照组〔（２０．３±２１．６）ｎｇ／Ｌ，（１６７．７±４９．６）ｎｇ／Ｌ，Ｐ〈０．００１〕，睾酮浓度极显著地高于对照组〔（７．３±７．１）μｇ／Ｌ，（０．５２±０．２９）μｇ／Ｌ     

小鼠Wip1基因的表达及其对受精能力的影响

本研究旨在探讨Wip1基因的表达及其对精子受精能力的影响,为阐明Wip1基因对雄性繁殖的影响提供新的着眼点。选取相同饲养环境、同一遗传背景的8周龄左右的野生型雄鼠,利用实时荧光定量PCR技术检测Wip1基因在心脏、肝脏、脾脏、肺脏、肾脏、脑组织及雄性生殖器官中的表达情况,利用免疫荧光技术检测Wip1蛋白在睾丸组织及精子细胞中的分布情况。以Wip1敲除型雄鼠为试验组,野生型雄鼠为对照组,利用ELISA试剂盒检测两组小鼠血清中睾酮水平;通过体外受精试验比较两组的2-细胞率及囊胚率。结果显示,Wip1基因mRNA在野生型雄鼠各组织中广泛表达,且在雄性生殖器官中表达量较高,其中睾丸中表达量最高,附睾体、附睾头、附睾尾次之;Wip1蛋白主要定位于睾丸组织中的长形精子细胞及附睾成熟精子细胞的头部。ELISA检测结果发现,与野生型雄鼠相比,Wip1敲除型雄鼠血清内睾酮含量极显著下降(P0.01)。体外受精结果表明,Wip1敲除后2-细胞率及囊胚率均极显著下降(P0.01)。结果表明,Wip1基因敲除能够引起雄性小鼠受精能力降低,这可能与Wip1基因在精子发生过程中发挥作用相关。     

促性腺激素释放激素二聚体对公猪繁殖机能的影响

应用促性腺激素二聚体(GnRH-TD)对12头4月龄公猪进行主动免疫,观测内源促性腺激素(GnRH)的变化.注射GnRH-TD与卵清蛋白(OVA)的偶联物3次,结果发现GnRH-TD能降低血清睾酮浓度和睾丸重量.组织切片显示,曲细精管的精原细胞有少数退化.实验结果表明:公猪接种GnRH-TD-OVA能诱发免疫反应,中和内源GnRH的生物活性,且抑制睾酮的合成,从而导致性器官发育受阻,达到去势的目的.     

GnRH并列体二聚体主动免疫对公兔性腺的影响

用GnRH并列体二聚体主动免疫公兔，观察了免疫对公兔性腺及体重的影响。结果表明，免疫不影响公兔体重的工，而使公兔睾丸重量降低，就纵长缩短，表明性腺有萎缩的趋势。     

Reducing the length of time between slaughter and the secondary gonadotropin-releasing factor immunization improves growth performance and clears boar taint compounds in male finishing pigs

The objective of this study was to evaluate whether altering the timing of the secondary anti-gonadotropin-releasing factor (GnRF) immunization closer to slaughter in male finishing pigs would reduce the increase in P2 fat depth (6.5 cm from the midline over the last rib), while still limiting the incidence of boar taint. Entire male pigs are immunized against GnRF to reduce the concentration of testicular steroids that in turn limits the incidence of boar taint. Additionally, testicle measurements and color measurements were taken to examine whether they could be used to differentiate nonimmunized entire males from immunized male pigs. A total of 175 Large White × Landrace entire male pigs aged 16 wk (59 kg of BW) were used in a completely randomized design with 5 treatment groups based on the time that pigs received the secondary immunization before slaughter. Pigs were housed in groups of 7 and randomly allocated to 1 of 5 treatments with 5 replicates per treatment. The treatment groups were as follows: no secondary immunization before slaughter, and the secondary immunization given at 2, 3, 4, or 6 wk before slaughter. The P2 fat depth levels were reduced (P = 0.054) with the secondary immunization closer to slaughter (11.7, 11.3, 12.8, 12.6, and 13.7 mm for no secondary immunization, secondary immunization at 2, 3, 4, and 6 wk before slaughter, respectively). Androstenone concentration did not exceed the generally accepted industry sensory threshold of 1.0 μg/g of fat, and both androstenone concentration in the adipose tissue and testosterone concentrations in the blood were suppressed (P < 0.001) in all immunized pigs regardless of timing of the secondary immunization compared with pigs that did not receive the secondary immunization. Skatole concentration of all pigs in the experiment did not exceed the generally accepted industry sensory threshold of 0.2 μg/g. Testes weight was reduced (P < 0.001) with increased time between slaughter and the secondary immunization. Immunized pigs, regardless of time before slaughter, had greater L* (lightness) and b* (yellowness) color of the testicle surface (P < 0.001 and P = 0.020, respectively), and less a* (redness) color compared with entire males (P < 0.001). The study provides further evidence of the efficacy of the anti-GnRF immunization and indicates that the secondary immunization can be moved closer to slaughter, while still limiting the incidence of boar taint. Testicle measurements and color measurements together could provide a method of discrimination between carcasses from immunized entire males clear of boar taint and tainted carcasses.     

应用重组肌肉抑制素C-端重组蛋白提高仔鼠出生质量的研究

为探讨利用重组肌肉抑制素促进肌肉生长的可能性,本研究原核表达并纯化了肌肉抑制素C-端重组蛋白.将雌性小鼠同雄性小鼠合笼,妊娠后分笼饲养.应用重组蛋白免疫雌性昆明白小鼠.利用间接ELISA法检测抗血清效价.每周测母鼠及仔鼠体质量1次.结果表明:(1)免疫后对照组和试验组母鼠体质最差异不显著(P>0.05).在妊娠后期(免疫后第11周),两组间体质量出现差异(P<0.05),试验组为(56.23±3.37)g,而对照组为(64.11±3.18)g.试验组体质量在第12周达到(79.16±3.72)g.而对照组为(75.23±3.44)g,差异极显著(P<0.01).分娩后,这种差异消失(P>0.05).(2)仔鼠出生体质量在2处理组间差异极显著(P<0.01),试验组仔鼠出生体质量比对照组增加25.8%左右;但在生长阶段仔鼠体质量差异有逐渐缩小的趋势.结论:应用肌肉抑制素C-端重组蛋白免疫母鼠可以显著提高仔鼠出生体质量.     

抑制素研究进展

抑制素是一种主要由性腺分泌的糖蛋白激素,对ＦＳＨ起负反馈调节物作用。由于其具免疫原性,外源抑制素通过免疫调节可降低体内抑制素的生物活性,导致ＦＳＨ浓度升高,使排卵率和产仔数增加,近年来其作为多胎疫苗的角色已越来越为人们所重视。本文介绍了抑制素的生物学特性,还回顾了抑制素的几种主要测定方法,着重综述了抑制素对雌、雄动物生殖机能的调节作用。     

生长抑素基因工程苗对绵羊外周血液某些代谢物浓度和激素水平的影响

选择4～5月龄体重相近的杂种雄性去势绵羊16只,随机分成试验组和对照组,对照组平均体重为21.25kg,试验组平均体重为21.54kg。试验组绵羊一次性皮内多点注射生长抑素基因苗(SS苗)1mL,对照组绵羊皮内多点注射生理盐水1mL。试验共持续63d。试验结束时对照组平均体重27.79kg,试验组平均体重29.71kg。注射SS苗和生理盐水当天(0d)、28d、51d、63d,早晨放牧前颈静脉采血制备血浆,分析代谢物及激素含量。结果显示,SS苗免疫对动物葡萄糖和非酯化脂肪酸(NEFA)和尿素氮浓度无明显影响;SS苗免疫后28d时,IGF-I、GH浓度分别比对照高10.81%和5.23%,在51d时高57.36%(P<0.01)和35.73%(P<0.05)。     

Immunization of pigs against chicken gonadotropin-releasing hormone-II and lamprey gonadotropin-releasing hormone-III: effects on gonadotropin secretion and testicular function

The objective of this experiment was to evaluate the effects of active immunization against 2 GnRH isoforms on gonadotropin secretion and testicular function in pigs. Synthetic chicken (c) GnRH-II and lamprey (l) GnRH-III peptides, with the common pGlu-His-Trp-Ser sequence at the N-terminal omitted, were conjugated to BSA. Forty-eight male piglets were randomly assigned to 1 of 4 treatments. Pigs on treatment 1 were actively immunized against cGnRH-II, whereas pigs on treatment 2 were actively immunized against lGnRH-III. Control pigs on treatment 3 were actively immunized against the carrier protein (BSA), and pigs on treatment 4 were castrated and actively immunized against BSA. The BSA conjugate was emulsified in Freund's Incomplete Adjuvant and diethylaminoethyldextran. Primary immunization was given at 13 wk of age (WOA) with booster immunizations given at 16 and 19 WOA. Body weight and plasma samples were collected weekly beginning at 11 WOA. Treatments did not affect BW during the experimental period. Antibody titers were increased in animals immunized against cGnRH-II and lGnRH-III (P < 0.001). Cross-reactivity of the antibodies to mammalian GnRH or between cGnRH-II and lGnRH-III was minimal. Concentrations of testosterone were maximal in control boars (treatment 3) and minimal in control barrows (treatment 4) and immunized pigs (treatment x week; P < 0.01). Immunized animals had concentrations of LH (P < 0.001) and FSH (treatment x week; P < 0.03) that were less than control barrows and similar to control boars. At the end of the experiment, intact (noncastrated) pigs were exsanguinated. Testes were removed immediately; Leydig cells were isolated and treated with 0, 1, or 10 ng/mL of LH. There was an LH x GnRH treatment effect on testosterone concentrations (P < 0.03), indicating that Leydig cells were sensitive to the immunization protocol and doses of LH. Taken together, these data suggest that immunization against GnRH isoforms decreased gonadotropin secretion compared with control barrows. Additionally, immunization against cGnRH-II and lGnRH-III reduced the ability of Leydig cells to respond to LH challenges.     

Effects of somatostatin immunoneutralization on growth and endocrine parameters in chickens

The effects of somatostatin immunoneutralization on growth rate, growth hormone (GH) secretion and circulating insulin-like growth factor I (IGF-I) concentrations were investigated in chickens through the use of passive and active immunization techniques. Intravenous bolus injection of goat-antisomatostatin stimulated a significant (P less than .05) increase in plasma GH levels for one hour post-injection in four and six week old male broiler chickens. The GH response to an intravenous bolus injection of hGRF44NH2 was similar in the antisomatostatin treated chicks and normal goat serum treated controls. Despite the presence of circulating somatostatin antisera after 28 hours, plasma GH levels were not different between control and antisomatostatin-treated chicks at that time. Continuous administration of somatostatin antisera by Alzet pump over a two-week period resulted in significant (P less than .05) elevations in plasma GH levels at one week post-implantation and in circulating IGF-I concentrations after two weeks of administration. Chicks which developed antibodies against somatostatin following active immunization exhibited a 7.1% increase in growth rate which was associated with a significant decrease in abdominal fat. However, neither GH nor IGF-I concentrations were elevated in the chicks which developed somatostatin antibodies. Thus, the benefits gained from somatostatin immunoneutralization may be exerted through mechanisms other than GH.     

鸡群免疫失败原因分析

为查明内蒙古乌兰察布市地区鸡群免疫效果不佳及免疫失败的原因,对部分养鸡场和养鸡户进行了调查,经分析,发现引起免疫鸡群发病的主要影响因素有疫苗质量、鸡群健康情况、免疫方式和免疫程序等。     

猪繁殖与呼吸综合征病毒核衣壳蛋白刺激小鼠产生抗体的研究

为比较传统免疫、脾内免疫、胶粒免疫和硝酸纤维素膜皮下包埋等4种方法制备的猪繁殖与呼吸综合征病毒(porcine reproductive and respiratory syndrome virus,PRRSV)核衣壳蛋白抗原免疫小鼠后刺激机体产生特异性抗体的水平,比较不同方法对于特定抗原刺激机体产生免疫应答的影响,筛选一种最有效的能够用于PRRSV核衣壳蛋白多克隆抗体和单克隆抗体制备的免疫方法。用PRRSV核衣壳蛋白作免疫原,分别用传统皮下免疫法、脾内免疫法、胶粒免疫和硝酸纤维素皮下包埋法免疫BABL/c小鼠,间接ELISA检测血清效价,用Western blotting检测其特异性。三免后,常规免疫小鼠血清效价为1.28×105,脾内免疫法为1.28×105,胶粒免疫为3.2×104,NC膜皮下包埋法为6.4×104。结果表明,4种免疫方法均获得了高滴度、高特异性的多克隆抗体,与传统免疫法相比,其他3种方法有其明显的优势,即节省抗原,其中脾内免疫法为优先选用的免疫方法,其免疫效果好,可节省大量抗原和时间。     

Evaluation of dietary stevioside supplementation on anti-human serum albumin immunoglobulin G, Alpha-1-glycoprotein, body weight and thyroid hormones in broiler chickens

Sixty male broiler chickens fed a diet supplemented with 130 mg/kg stevioside (S group) or an unsupplemented diet (C group) from day 1 of age onwards. On day 21 of age, ten birds from either the S (SH) or C (CH) group were injected subcutaneously with 100 μg human serum albumin (HSA) and ten others from either S (SP) or C (CP) group injected with 100 μl phosphate-buffered saline (PBS) in the same way. There were no significant effect of supplementation nor interaction with age on average body weights, T(3) and T(4) concentrations of non-injected chickens. After the primary immunization, α(1) -glycoprotein concentrations increased in all treatment groups except the CP group, and were significantly higher in the CH group in relation to the other groups. Fourteen and 18 days after the primary immunization, HSA injected chickens of both dietary treatments had significantly higher anti-HSA immunoglobulin G (IgG) levels than their PBS injected controls. No effect of stevioside supplementation was observed for IgG level. In conclusion, dietary stevioside inclusion can attenuate the pro-inflammatory response after stimulation of the innate immune response in broiler chickens.