【作者】 刘青；

【导师】 杨利国；

【作者基本信息】 华中农业大学 ， 动物遗传育种与繁殖， 2016， 博士

【摘要】 前期研究表明,抑制素基因免疫通过中和内源性抑制素,可促进FSH的分泌,进而促进卵泡发育和排卵,提高动物繁殖性能。本实验室构建了以减毒沙门氏菌为载体的抑制素基因疫苗C501(pVAX-asd-IS),其制备简便,便于扩大生产。在免疫小鼠后,提高了其大卵泡的数量和产仔数。且该疫苗没有抗生素抗性基因,不会造成抗生素危害,有利于在大动物上的推广应用。但在不同的物种中,抑制素基因免疫的效果也受到免疫接种途径、免疫剂量和免疫次数等因素的影响。且在临床应用和环境释放的安全性方面,特别是在水牛临床试验的安全性方面尚未研究。因此,本研究分别分析了抑制素基因疫苗肌注免疫的剂量和鼻饲免疫的天数,对杂交水牛在繁殖季节的卵泡发育、发情排卵和受胎的影响。挑选出免疫效果最佳的免疫剂量和天数,再比较以上两种免疫途径对杂交水牛在非繁殖季节的卵泡发育、发情排卵和受胎的影响。分析抑制素基因疫苗在繁殖季节和非繁殖季节应用于杂交水牛的促卵泡生长发育和排卵的效果,并从水牛临床应用和环境释放角度,分析抑制素基因疫苗的安全性,为促进抑制素基因疫苗的推广应用和优化提高杂交水牛繁殖性能的新技术奠定了基础,主要研究内容与结果如下:1.抑制素基因疫苗肌肉注射剂量对杂交水牛卵泡发育和发情排卵及受胎的影响选择158头杂交水牛分为四组,其中免疫组T1(n=41)、T2(n=37)和T3(n=37)分别注射1010、109和108 CFU/ml的抑制素重组菌C501(pVAX-asd-IS),10ml/头,对照组(n=43)注射PBS,初次免疫为第0天,加强免疫在第14天。所有牛只在第28天肌肉注射孕马血清促性腺激素(PMSG)1000IU,第30天注射氯前列腺素(PGF2ɑ)0.5mg,48小时后注射200μg促性腺激素释放激素(GnRH)。进行B超检测,记录卵泡发育和排卵情况。结果显示肌肉注射免疫抑制素重组菌C501(pVAX-asd-IS)后,各免疫组杂交水牛均能产生抗抑制素IgG抗体,以1010 CFU/ml剂量组的抗体水平最高。1010 CFU/ml剂量组的大卵泡(≥10mm)和排卵卵泡的数量与直径显著(P<0.05)高于对照组,且优势卵泡的生长速度显著高于108 CFU/ml剂量和对照组(P<0.05)。抗体阳性组的发情、排卵和受胎率显著高于抗体阴性组。以上结果说明以1010 CFU/ml的抑制素重组基因疫苗C501(pVAX-asd-IS)肌肉注射免疫杂交水牛,能显著促进卵泡的生长发育,促进杂交水牛的发情和排卵,提高其繁殖性能。2.抑制素基因疫苗鼻饲免疫天数对杂交水牛卵泡发育和发情排卵及受胎的影响选择134头杂交水牛分为4组:免疫5天组(n=34)、免疫3天组(n=35)和免疫1天组(n=31)分别用1010 CFU/ml C501(pVAX-asd-IS)疫苗连续鼻饲免疫5、3和1天,10ml/头,每天喷鼻两次。对照组(n=34)使用10ml PBS鼻饲免疫5天。所有牛只初次免疫为第0天,加强免疫在第14天。且在第28天注射促性腺激素释放激素(GnRH)200μg,第35天注射氯前列腺素(PGF2α)0.5mg,第37天再次注射促性腺激素释放激素(GnRH)200μg,在之后的18和24小时进行人工授精。B超检测记录卵泡发育和排卵情况。结果显示在初次免疫后的14和28天,鼻饲免疫C501(pVAX-asd-IS)能引起显著的免疫反应,免疫5天组的抗抑制素IgG抗体水平显著高于免疫1天组(P<0.01)。且加强免疫后抗体水平有上升的趋势。在二次注射GnRH后,免疫5天组的优势卵泡增长速度显著快于对照组和免疫1天组,且免疫5天组的大卵泡(≥10mm)和排卵卵泡的数量和直径高于其他各组,使得免疫5天组有较高的发情、排卵和受胎率。结果表明,以抑制素重组基因疫苗C501(pVAX-asd-IS)鼻饲免疫5天,能显著促进水牛的卵泡生长和发情排卵,与Ovsynch-TAI相结合,可提高杂交水牛在繁殖季节的受胎率。3.抑制素基因疫苗两种免疫途径对杂交水牛在非繁殖季节的卵泡发育和发情排卵及受胎的影响选取81头经产杂交水牛随机分为三组:肌肉注射组(n=29)肌注1010 CFU/ml抑制素重组菌苗C501(pVAX-asd-IS),10ml/头,每天一次,免疫3天;鼻饲免疫组(n=27)鼻饲1010 CFU/ml抑制素重组菌苗C501(pVAX-asd-IS),10ml/头,每天两次,免疫5天;对照组C(n=25)每头肌肉注射10ml PBS,每天一次,共注射5天。初次免疫为第0天,加强免疫在第14天。全体牛只在第28天肌肉注射孕马血清促性腺激素(PMSG)1000IU,第30天注射氯前列腺素(PGF2ɑ)0.5mg,48小时后注射200μg促性腺激素释放激素(GnRH)。B超检测记录卵泡发育和排卵情况。研究结果显示以肌肉注射和鼻饲免疫杂交水牛,均能诱导机体产生抗抑制素抗体,且两种免疫方式的抗体水平差异不显著。但肌肉注射组的抗体阳性率高于鼻饲免疫组(82.8%vs.74.1%)。两种免疫方式均能增加大卵泡和排卵卵泡的数量和直径,以肌肉注射的效果较强。在非繁殖季节,肌肉注射组杂交水牛的发情、排卵和受胎率均高于鼻饲免疫组和对照组,且与繁殖季节的发情、排卵和受胎率相当。抗体阳性牛的受胎率也显著高于抗体阴性牛(56.8%vs.27.0%;P<0.01)。结果表明,以肌肉注射和鼻饲免疫抑制素基因疫苗C501(pVAX-asd-IS)均能诱导良好的免疫反应,但以肌肉注射的免疫效果更优,能显著增加成熟卵泡的数量和直径,提高杂交水牛在非繁殖季节的发情,排卵和受胎率。4.抑制素基因疫苗的生物安全性评价选用81头经产杂交水牛随机分为三组:肌肉注射组(n=29)肌注1010 CFU/ml抑制素重组菌苗C501(pVAX-asd-IS),10ml/头,每天一次,免疫3天;鼻饲免疫组(n=27)鼻饲1010 CFU/ml抑制素重组菌苗C501(pVAX-asd-IS),10ml/头,每天两次,免疫5天;对照组C(n=25)每头肌肉注射10ml PBS,每天一次,共注射5天。初次免疫为第0天,加强免疫在第14天。对免疫后杂交水牛的体温和心率进行监测,并对粪便、水和土壤样本进行采集,用PCR方法检测inv A基因。结果表明各组杂交水牛在免疫后未出现临床症状,其体温和心率正常。在粪便、水和土壤样本中均未检测到inv A目的基因。说明以肌肉注射和鼻饲免疫抑制素重组菌苗,在杂交水牛上的运用是安全的,且重组菌苗不会通过牛体向外界传播,对环境不会产生不利影响。更多还原

【Abstract】 As previous researches indicated that immunization with inhibin DNA vaccines neutralize the endogenous inhibin could increase FSH secretions,improve the number of follicles,ovulation and conception rate.Our laboratory developed the attenuated S.choleraesuis-mediated inhibin DNA vaccine C501(pVAX-asd-IS),it has been proved to be a safe tool for increasing ovarian follicular development and improving reproductive traits in mice.Comparing with naked DNA vaccination,the production of the C501(pVAX-asd-IS)is easier and relatively low cost.Moreover,the vaccine was equipped with the asdˉ balanced lethal chromosome–plasmid system and without antibiotic resistance gene is safe for animals and human,depict that this vaccine may have direct application in large animal industry.But in different species,the efficacy of inhibin DNA vaccine was influenced by some factors such as vaccination approaches,the dose and times of immunization.Although the safety that using live bacterial as a DNA vaccine delivery vector has been confirmed in mice,there were few previous studies with regard to the safety using live bacteria as a DNA vaccine delivery vector for the surrounding environment.Therefore,in this study,we used the different dose of intramuscular injection and the different times of nasal immunization of inhibin DNA vaccine C501(pVAX-asd-IS),to evaluate the effect of inhibin DNA vaccine on follicular development,ovulation and fertility of crossbred buffaloes in breeding season.Subsequently,comparison on the safety and efficacy of intramuscular and nasal immunized with C501(pVAX-asd-IS)on ovarian responses and fertility of crossbred buffaloes in non-breeding season,and coupled with synchronization protocols,analyzed the pattern of follicular development and ovulation in crossbred buffaloes during the breeding and non-breeding season,which would provide a foundation for optimizing inhibin gene vaccine and increasing the fertility of crossbred buffalo.The main contents and results of this study are as follows:1.Intramuscular injection with inhibin DNA vaccine for improving follicular development and ovulation in crossbred buffaloesA total of 158 crossbred buffaloes divided into four groups and were intramuscular injected with 1010(T1,n=41),109(T2,n=37),108(T3,n=37)or 0(C,n=43)CFU /ml inhibin vaccine C501(pVAX-asd-IS)in 10 ml PBS on day 0 and 14,respectively.All animals were administered with 1000 IU PMSG on day 28,0.5mg PGF2α on day 30 and 200μg GnRH on day 32.The results showed intramuscular immunized with C501(pVAX-asd-IS)elicited significant immune responses,and group T1 have the highest anti-inhibin IgG antibody titers.The number and diameter of large follicles(≥10mm)as well as ovulatory follicles in group T1 was significantly greater than group C(P<0.05).The growth speed of dominant follicles in group T1 was significantly faster than groups T3 and C(P<0.05),resulting in a greater estrus,ovulation and conception rate in buffaloes with positive antibodies.These results demonstrate that intramuscular immunized with the bacterial delivered inhibin vaccine at high-dose of 1×1010 CFU /ml,could enhance follicular development,improve the proportion of estrus,ovulation and fertility in crossbred buffaloes.2.Nasal immunization with inhibin DNA vaccine for improving follicular development and ovulation in crossbred buffaloesA total of 134 crossbred buffaloes were divided into four groups: Group1(G1,n=34),group 2(G2,n=35)and group 3(G3,n=31)were nasal immunized twice a day with 10 ml of 1×1010 CFU /ml of the C501(pVAX-asd-IS)vaccine for 5,3 and 1 day,respectively.Control group(n=34)was nasal immunized with 10 ml PBS for 5 days.All animals were immunized twice with an interval of 14 days and administered with 200μg of a GnRH analogue on day 28,0.5mg PGF2α on day 35 and 200μg of the same GnRH analogue on day 37.TAI was performed at 18 and 24 h after the second-GnRH treatment.Nasal immunized with C501(pVAX-asd-IS)evidently induced antibodies against inhibin at 14 and 28 d after primary immunization,and the anti-inhibin IgG antibody titers in G1 were significantly higher(P < 0.01)than G3.Following booster immunization,the anti-inhibin antibody titers in G1 gradually increased.After the second-GnRH treatment,the growth speed of the dominant follicles in G1 was significantly faster(P < 0.05)than G3 and control group.The number and diameter of large follicles(≥10mm)as well as ovulatory follicles in G1 was the greatest in all groups,resulting in a greater estrus,ovulation and conception rate in G1.These results demonstrate that nasal immunization with C501(pVAX-asd-IS)for 5 days,coupled with the Ovsynch protocol,could be used as an alternative approach to improve reproductive performance in crossbred buffaloes.3.The efficacy of intramuscular and nasal immunized with inhibn DNA vaccine in improving ovarian responses and fertility of crossbred buffaloes in non-breeding seasonThe experiment was based on(n = 81)multiparous crossbred buffaloes(River ? Swamp)with body condition score ranging(BCS)between 3.0-4.0 were randomly divided into three groups: intramuscular immunization(IM,n=29)were intramuscular immunized with10 ml of 1010 CFU /ml inhibin vaccine C501(pVAX-asd-IS)for 3days;nasal immunization(NI,n=27)were nasal immunized twice a day with the same dose of the inhibin vaccine for 5 days;Group C(n=34)was intramuscular immunized with 10 ml PBS for 5 days.All animals were immunized twice with an interval of 14 days and administered with 1000 IU PMSG on day 28,0.5mg PGF2α on day 30 and 200μg GnRH on day 32.The results showed intramuscular and nasal immunized with the inhibn vaccine could induce effective immune responses in crossbred buffaloes,and the anti-inhibin IgG antibody titers have no significant difference between two immunized groups.But group IM have the greater positive rate of anti-inhibin antibodies than group NI(82.8% vs.74.1%).Compared to the control group,the number and diameter of large follicles(≥10mm)as well as ovulatory follicles in group IM were increased significantly.Intramuscular immunized with C501(pVAX-asd-IS)increased the proportion of estrus,ovulation and conception rate of crossbred buffaloes in non-breeding season.Buffaloes with positive anti-inhibin antibodies showed significantly greater conception rate compared with the buffaloes with negative anti-inhibin antibodies(56.8% vs.27.0%;P< 0.01).These results demonstrate that intramuscular and nasal immunized with the inhibin vaccine induce effective immune responses,but the effects induced by the intramuscular injection were better than the nasal immunization,increased the number and diameter of ovulatory follicular,enhanced estrus and ovulation of crossbred buffaloes and improved conception rate in non-breeding season.4.The safety of intramuscular and nasal immunized with inhibn DNA vaccine in crossbred buffaloes.A total of 81 crossbred buffaloes were divided into three groups: intramuscular immunization(IM,n=29)were intramuscular immunized with10 ml of 1010 CFU /ml inhibin vaccine C501(pVAX-asd-IS)for 3days;nasal immunization(NI,n=27)were nasal immunized twice a day with the same dose of the inhibin vaccine for 5 days;Group C(n=34)was intramuscular immunized with 10 ml PBS for 5 days.All animals were immunized twice with an interval of 14 days.Take the temperature and heart rate of crossbred buffaloes after immunization,and faecal,soil and water samples originating from immunized buffaloes and surrounding environment were collected.The results showed the temperature and heart rate of crossbred buffaloes in each group have no significant difference after immunization.The target gene(invA gene)was not detected by PCR amplification in faecal,soil and water samples,indicating that the surrounding environment was not contaminated by recombinant bacteria,and intramuscular and nasal immunization of C501(pVAX-asd-IS)was safe to crossbred buffalo.更多还原