【作者】 张剑；

【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， 2010， 博士

【摘要】 繁殖力和泌乳性能是反映奶牛生产性能的重要因素,阐明奶牛生殖系统发育／恢复规律,对于最大限度发挥奶牛繁殖潜能、开发繁殖新技术具有重要的意义；而利用现代生物技术,提高奶牛泌乳水平,是提高奶牛养殖经济效益的关键。本研究首先利用B型超声波诊断技术研究不同胎次奶牛产后子宫复旧与卵巢机能恢复的规律,为提高奶牛产后繁殖力进而促进奶牛生产提供理论依据；之后利用基因免疫技术研究生长抑素基因免疫对奶牛生殖机能恢复和泌乳性能的作用,为开发提高奶牛繁殖力和泌乳力的基因疫苗奠定基础。在用新型生长抑素DNA疫苗——pVGS/2SS-asd免疫奶牛前,首先利用大鼠模型进行试验,探讨其对大鼠繁殖及泌乳性能的影响,并对其可能存在的安全性问题进行评价,为应用大动物(如奶牛)提供依据。然后将pVGS/2SS-asd生长抑素DNA疫苗免疫产后奶牛,研究其对奶牛产后繁殖及泌乳性能的影响。1.奶牛产后子宫复旧与卵巢机能恢复规律研究良好的子宫复旧和卵巢活动对产后奶牛再次受孕并缩短产犊间隔是非常重要的。本研究的目的是在产后营养和泌乳条件相对一致的情况下,评估胎次对中国荷斯坦奶牛产后子宫复旧和卵巢活动恢复的影响。选取46头中国荷斯坦奶牛,其中初产18头、产第二胎13头、经产15头。奶牛产后子宫和卵巢检测采用超声波诊断技术,干物质摄入量(DMI)、产奶量、体况评分(BCS)测定依据DHI(奶牛群体改良)标准方法,奶样中雌二醇浓度(E2)测定采用放射免疫测定(RIA)方法。结果显示,初产奶牛的子宫孕角恢复时间(天数)显著多于其他胎次的奶牛。就产后首次和第二次排卵的间隔天数及产后首次排卵前的卵泡波数目而言,初产奶牛也比其他胎次奶牛多。总之,中国荷斯坦奶牛在相似的营养和泌乳条件下,随着胎次的增长,奶牛产后子宫复旧与卵巢活动恢复时间明显缩短。2.生长抑素基因免疫对大鼠繁殖和泌乳性能的影响为了探讨新型生长抑素DNA疫苗——pVGS/2SS-asd对大鼠繁殖和泌乳性能的影响,同时分析疫苗剂量和注射方式对免疫效果的影响,将216只SD雌性大鼠随机分为9组,每组24只。试验组：T1～T3组大鼠按高(2×109 CFU)、中(2×108CFU)、低(2×107CFU)三种剂量进行股四头肌两点注射pVGS/2SS-asd重组菌200μL; T4～T6组所用疫苗及其用量对应于T1～T3组,进行背部皮下注射。对照组：C0组注射10%铝胶生理盐水(肌肉注射和皮下注射各半)；C1组肌肉注射2×109CFU不含质粒的C500菌200μL；C2组皮下注射2×109CFU不含质粒的C500菌200μL。各组在首次免疫后间隔4周,用同等剂量疫苗进行加强免疫2次。结果表明,这种新型pVGS/2SS-asd生长抑素DNA疫苗对大鼠有很好的免疫原性,能诱导高水平的免疫应答,而且通过加强免疫能在整个试验期保持较长时间(约11～13周)。在免疫方式上,肌肉注射在诱导产生抗体水平的绝对值(OD=0.823～0.075)和免疫应答的持久性(约13周)方面比皮下注射有优势。在免疫剂量上,并不存在剂量依赖关系,两种免疫方式均以中剂量优势明显。无论是肌肉注射或皮下注射,三种免疫剂量都对大鼠繁殖性能没有影响,但间接促进了产后泌乳期仔鼠的生长,改善了大鼠的泌乳性能。其中以肌肉注射的中剂量组的仔鼠断奶窝增重(215.8±18.8g)明显高于其他各组,与对照组相比差异显著。皮下注射的中剂量组次之(212.2±13.6g)。3.生长抑素基因疫苗染色体整合的安全性评价在用pVGS/2SS-asd生长抑素DNA疫苗肌肉注射和皮下注射SD大鼠试验结束时,采集各组母鼠的心、肝、脾、肺、肾、卵巢和肌肉以及断奶仔鼠的心、肝、脾、肺和肾等组织,提取基因组DNA,采用PCR方法检测质粒DNA与宿主染色体整合情况。结果表明,未发现质粒整合至母鼠和仔鼠基因组DNA中,揭示生长抑素DNA疫苗在染色体整合方面对大鼠是安全的。4.生长抑素基因免疫对奶牛繁殖和泌乳性能的影响为了探讨新型生长抑素DNA疫苗——pVGS/2SS-asd对产后奶牛繁殖和泌乳性能的影响,将40头产后奶牛随机分为4组,每组10头。3个试验组奶牛从奶牛产后第7d开始,分高(1×1011CFU)、中(1×1010CFU)、低(1×109CFU)三种剂量肌肉注射pVGS/2SS-asd重组菌1mL,对照组肌肉注射10%铝胶生理盐水1mL。首次免疫(注射当天0d)后间隔4周,用同等剂量疫苗加强免疫2次。结果表明,pVGS/2SS-asd生长抑素DNA疫苗对奶牛有很好的免疫原性,各剂量组均能诱导一定强度的免疫应答。高剂量组在诱导产生抗体最大峰值(OD=0.490±0.171)和抗体阳性牛比例的维持(100.0%持续7周左右)上优势明显。免疫后各组奶牛子宫颈和子宫孕角复旧时间、首次排卵时间和排卵卵泡最大直径等方面差异不显著,说明该疫苗对奶牛的繁殖性能没有不良影响,而对泌乳性能有一定的促进作用,其中以中剂量组奶牛优势明显,在首次免疫后有5周时间(第2～6周)产奶量明显高于其他组。更多还原

【Abstract】 Reproduction and lactation are important factors which reflect the performance of dairy cattle. The explanation of the development/resumption laws of cows’reproductive system plays a significant role in the full development of the potential for cows’ reproduction and new reproductive technologies. Modern biotechnology has been used to improve the milk level of Dairy cows, which is the key to improving the economic efficiency of dairy farms. The first part of this study is to investigate the law of uterine involution and ovarian function resumption of Chinese Holstein dairy cows with different parities by B-mode ultrasound during postpartum, so as to provide the research basis for improving the fertility of postpartum dairy cows. The second part is to study the role of somatostatin gene immunization on reproductive function recovery and lactation performance of dairy cows by genetic immunization technology, and to provide the basis for the development of genetic vaccine which improve the fertility and lactation of dairy cows. A novel somatostatin DNA vaccine—pVGS/2SS-asd was tested on the rat model prior to the dairy cow, and used to explore the effects on the fertility and lactation performance of rats. The safety issue of this DNA vaccine on rats was also evaluated. These results provided the basis for the application of large animals (such as dairy cows). Then postpartum dairy cows were immunized against the pVGS/2SS-asd somatostatin DNA vaccine, exploring the effects on the fertility and lactation performance of the postpartum dairy cows.1. Study of uterine involution and resumption of ovarian activities in postpartum dairy cows with different parities.Favorable uterine involution and ovarian activity are very important for the next pregnancy and the shorter calving interval of postpartum cows. The objective of this study was to evaluate the effect of parity on uterine involution and resumption of ovarian activity in Chinese Holstein dairy cows after calving under similar postpartum nutritional and lactational conditions.46 Chinese Holstein dairy cows in various parities (primiparous=18; biparous=13; multiparous= 15) were selected. The status of uterus and ovaries was detected by ultrasonography. According to DHI (Dairy Herd Improvement) standard method, dry matter intake (DMI), milk yield, body condition score (BCS) were determined, and estradiol (E2) concentration in milk samples were measured by radioimmunoassay (RIA). The results showed that days of the previous gravid uterine horn involution were significantly greater in primiparous dairy cows than in biparous and multiparous dairy cows. Days from calving to ovulation (first and second) and the number of follicular waves to first ovulation were also significantly greater in primiparous cows than in multiparous cows. In summary, under similar postpartum nutritional and lactational conditions, postpartum uterine involution and resumption of ovarian activity became progressively shorter as the number of parity increased in Chinese Holstein cows2.The effects of immunization against a novel somatostatin DNA vaccine—pVGS/2SS-asd on fertility and lactation in female rats.In order to investigate the effects of immunization against a novel somatostatin DNA vaccine—pVGS/2SS-asd on fertility and lactation in female rats, and analyze the effects of immunization doses and delivery routes on immune effect of this DNA vaccine,216 female Sprague-Dawley rats were randomly divided into 9 groups,24 per group. Test groups:rats of T1-T3 groups were primarily intramuscular immunized against pVGS/2SS-asd in high (2×109 CFU/200μL), middle (2×108 CFU/200μL) or low (2×107 CFU/200μL) dose; rats of T4-T6 groups in the same dosage as T1-T3 groups were primarily subcutaneous immunized against pVGS/2SS-asd s. Control groups:rats of CO group (half intramuscular injection and half subcutaneous injection) were immunized against 10% aluminum hydroxide gel saline (200μL); rats of C1 group were intramuscular immunized against C500 (2×109 CFU/200μL); rats of C2 group were subcutaneous immunized against C500 (2×109 CFU/200μL). Booster immunizations in the same dosage as the primary immunization were performed twice at 4-week intervals. The results showed that this novel pVGS/2SS-asd somatostatin DNA vaccine had good immunogenicity, induced a high level of immune response, and maintained a longer time (approximately 11-13 ws) by booster immunization in female rats. In the delivery routes, intramuscular immunity in the antibody absolute value (OD= 0.823±0.075) and persistent immune response (approximately 13 ws) was more advantages than subcutaneous immunity. In dose effect, there was no dose-dependent among all tests, and middle dose groups of two delivery routes were obvious advantages than other dose groups. Whether intramuscular or subcutaneous, the reproductive performance of all rats immunized with different doses was not affected. However, pVGS/2SS-asd somatostatin DNA vaccine in postpartum lactating rats contributes to the growth of offspring, and improves the lactation performance of female rats indirectly. Weaning weight gain (215.8±18.8 g) of offspring of the middle dose intramuscular group was significantly higher than those of other groups, with a significant difference between the middle dose intramuscular group and the control group, and followed by the middle dose subcutaneous group (212.2±13.6 g). 3.Safety evaluation of somatostatin DNA vaccine in chromosomal integration.At the end of immunization against pVGS/2SS-asd somatostatin DNA vaccine in rats with intramuscular or subcutaneous injections, multiple tissues such as heart, liver, spleen, lung, kidney, ovary and muscle were removed from each female rat of test groups, respectively. Moreover, tissue samples of the pups, such as heart, liver, spleen, lung and kidney were also collected after weaning, respectively. The genomic DNA was extracted, and then the genomic DNA samples were detected by sensitive PCR method. The results showed that the plasmid was not integrated into maternal and offspring genomic DNA, and the result revealed that the somatostatin DNA vaccine in chromosomal integration was safe to rats.4. Effects of Somatostatin gene immunization on the reproduction and lactation performance of dairy cows.The purpose of this study was to explore the effects of the new somatostatin DNA vaccine—pVGS/2SS-asd on the reproduction and lactation performance of postpartum dairy cows.40 postpartum cows were randomly divided into 4 groups,10 per group. At 7d after calving,3 test group cows were primarily intramuscular immunized against pVGS/2SS-asd in high (1×1011 CFU/1mL), the middle (1×1010CFU/1mL), and low (1×109 CFU/1mL) dose; the control group cows were intramuscular immunized against 10% aluminum hydroxide gel saline(1mL). Booster immunizations in the same dosage as the primary immunization (injection day as 0 d) were performed twice at 4-week intervals. The results showed that the novel pVGS/2SS-asd somatostatin DNA vaccine for cows had good immunogenicity and could induce a certain intensity of immune response. High dose group in the induction of the maximum antibody peak value (OD= 0.490±0.171) and the persistent high proportion of antibody positive cattle (100.0% for 7 w) had obvious advantages than other dose groups. After immunization, there was no significant difference among all the groups in the gravid uterine horn and uterine cervical involution, intervals of first ovulation and ovulation follicle diameter, this somatostatin DNA vaccine did not adversely affect the reproductive performance of dairy cows. Moreover it also confirmed that in the similar nutritional conditions, this vaccine improved the lactation performance of cows. The middle dose group had more advantages than other dose groups. Milk yields of the middle dose group were significantly higher than other groups for 5 weeks (2w to 6w) after primary immunization.更多还原