【作者】 许宝华；

【导师】 曾志将；

【作者基本信息】 江西农业大学 ， 动物遗传育种与繁殖， 2011， 博士

【摘要】 本文结合日本大耳兔生物学特性,运用放射免疫法(RIA)、实时荧光定量PCR(real time-PCR)及同期发情等技术,研究了蜂王浆对日本大耳兔生长及繁殖性能的影响,旨在探索蜂王浆提高动物生长及繁殖性能的作用机制,进而为家畜生产性能的提高、生殖疾病的防治、以及濒临灭绝动物的保种等方面提供新的科学依据和重要借鉴。主要研究结果如下1.蜂王浆对雄性日本大耳兔生长及繁殖性能的影响：分别按体重0.2%(高剂量组)和0.1%(低剂量组)的量,给高、低剂量组的18只2月龄雄性日本大耳兔每天空腹灌胃新鲜蜂王浆,对照组18只雄兔灌胃体重0.1%的生理盐水,每周同一时间称重。运用RIA依次测定灌胃Od、40d、60d和80d的血清性激素六项：促黄体素(LH),促卵泡素(FSH),雌二醇(E2),孕激素(P),睾酮(T)和催乳素(PRL)；70d后活体检测精子密度、活力和畸形率；80d后屠宰并分离主要脏器,计算各自脏器系数；右侧睾丸作成石蜡切片用于组织形态学观察。结果表明：(1)灌胃之前,3个实验组之间的血清性激素六项均差异不显著(P>0.05)。灌胃40d、60d和80d后,高、低剂量组的LH.FSH和T均极显著高于对照组(P<0.01),高、低剂量组之间差异不显著(P>0.05)；而E2、P和PRL在3组之间均差异不显著(P>0.05)。(2)高、低剂量组的精子密度极显著大于对照组(P<0.01),高、低剂量组之间差异不显著(P>0.05)；低剂量组的精子活力极显著高于对照组,而对照组又极显著高于高剂量组(P<0.01)；3组之间的精子畸形率差异不显著(P>0.05)。(3)第0-8周,3组之间的每周龄体重差异不显著(P>0.05)；之后,高、低剂量组极显著高于对照组(P<0.01),高、低剂量组之间差异不显著(P>0.05)。屠宰测定时,高、低剂量组的雄兔睾丸、下丘脑和脾脏脏器系数都极显著大于对照组(P<0.01),高、低剂量组之间无显著差异(P>0.05)；脑、肺和膀胱等其它主要脏器系数均差异不显著(P>0.05)。(4)灌胃80d后,对照组雄兔睾丸曲细精管生精上皮细胞数量明显少于试验组,生精上皮较薄,生精上皮细胞排列疏松,中央管腔更大管腔内大多为初级精母细胞和次级精母细胞；试验组的曲细精管管腔相对缩小,被精子细胞及精子所充实,细胞层数增多,精原细胞附于基膜,初级精母细胞、精子细胞和精子依次紧密排列延伸至管腔内,核染色明显管腔中可见大量的精子。本研究结果说明：蜂王浆能促进雄兔下丘脑、睾丸和脾脏发育；灌胃体重0.1%的蜂王浆能显著提高精子密度和精子活力；蜂王浆灌胃40d后,能显著提高血清中的LH、FSH和T浓度,对E2、P和PRL没有影响。2.蜂王浆对初情期前雌性日本大耳兔生长发育和性激素受体mRNA表达的影响：分别按体重0.2%和0.1%的量,给高、低剂量组12只50日龄的雌性日本大耳兔每天进行空腹灌胃新鲜蜂王浆,对照组12只雌兔灌胃体重0.1%的生理盐水。40d后股动脉放血处死,摘取下丘脑、右侧卵巢和右侧子宫,用于激素受体mRNA表达的检测；摘取心脏、左侧子宫和左侧卵巢等器官,用于脏器系数的测定；采用RIA法测定血清性激素六项。结果表明：(1)灌胃12d,对照组兔子没有换毛现象,高、低剂量组兔子分别出现67%和75%的换毛率。(2)蜂王浆对体重、心脏和卵巢等脏器系数无显著影响(P>0.05)。(3)高、低剂量组血液中的E2浓度极显著高于对照组(P<0.01),高、低剂量组之间差异不显著(P>0.05)；其它性激素在3个实验组之间差异不显著(P>0.05)。(4)高剂量组下丘脑GnRH的mRNA表达量极显著低于对照组和低剂量组(P<0.01),低剂量组与对照组之间差异不显著(P>0.05)；高剂量组卵巢LHR和FSHR的mRNA表达量极显著低于对照组(P<0.01),显著低于低剂量组(P<0.05),低剂量组与对照组之间差异不显著(P>0.05)；高剂量组子宫ERα和卵巢ERβ的mRNA表达量极显著低于对照组(P<0.01),但高、低剂量组之间以及低剂量组与对照组之间均差异不显著(P>0.05)。本研究结果说明：蜂王浆可以促进初情期前雌性日本大耳兔换毛,提高血清中E2水平,抑制下丘脑GnRH、卵巢LHRFSHR、ERβ以及子宫ER。的nRNA表达,对主要脏器系数无显著影响。3.蜂王浆对成年雌性日本大耳兔繁殖性能的影响：首先研究氯前列腺烯醇钠(PG)诱导健康经产母兔同期发情的效果,然后分别按体重0.2%和0.1%的量,给PG处理后的高、低剂量组健康经产母兔每天空腹灌胃新鲜蜂王浆,对照组灌胃体重0.1%的生理盐水,18 d后配种,计算繁殖性能相关指标。结果显示：(1)注射0.25mg／只PG后的健康经产母兔,LH、FSH和E2浓度分别在9h和48h达到最大值和最小值,P浓度则为最小值和最大值；PG处理后每天的LH、FSH、E2均在19：00达最大值,P达最小值：PG处理后第12d的LH、FSH和E2均高于第4d、8d和16d,P则低于第4d、8d和16d,差异达显著水平(P<0.05)。(2)蜂王浆灌胃高、低剂量组的FSH、LH、E2和P浓度均比对照组提前达到最高值和最低值高、低剂量组之间差异不显著(P>0.05),最高值与最低值分别在3个实验组之间差异不显著(P>0.05)。(3)高、低剂量组的窝产仔数和成活率与对照组之间差异不显著(P>0.05)；卵泡数、排卵数和初生个体重、初生窝重泌乳力、断奶个体重、断奶窝重都极显著高于对照组(P<0.01),高、低剂量组之间均差异不显著(P>0.05)。这些结果表明：注射0.25mg/只的PG能使母兔分别在9h和48h达到发情盛期和发情后期,12d后进入下一个发情周期,每天的激素极值出现在19：00。蜂王浆能缩短母兔发情周期,对处于相同生理周期母兔的激素没有影响；蜂王浆能促进母兔卵泡发育和排卵,提高初生窝重、初生个体重、泌乳力、断奶个体重和断奶窝重,对母兔窝产仔数没有影响。更多还原

【Abstract】 A series of techniques including radioimmunoassay (RIA), real-time fluorescence quantitative PCR and estrous synchronization were used in conjunction with the biological characteristics of Japanese white rabbit to detect the effects of Royal Jelly (RJ) on the growth and reproductive performance in Japanese white rabbits, with the aim to give hints for the improvement of animal husbandry production the prevention and therapy of reproductive disease, and the protection of endangered animal species.1. Effects of royal jelly on growth and reproduction performance of male Japanese white rabbits:Fifty four two-month-old male Japanese white rabbits were randomly and equally divided into three treatment groups which received RJ or saline by intragastric administration once a day for 80 days. The high and low dose groups received RJ of 0.2% and 0.1% of body weight, respectively, while the control group was administered with 0.1% body weight of saline. The rabbits were weighted once a week and slaughtered after 80 days. Levels of circulating FSH, LH, E2, P, T and PRL in plasma were determined by RIA on days 0,40,60 and 80 of treatment. Sperm density, vitality and abnormality rate were determined by biopsy prior to slaughter. After slaughter, organs were separated and each organ coefficient was calculated. The right testis was embedded in paraffin for histological examination. The results were as follows.(1) Of the six tested reproductive hormones, no difference was detected among the three groups before treatment. On days 40,60 and 80 of treatment with RJ, the levels of FSH, T and LH in the control group were significantly lower than those in the high or low dose groups (P<0.01). No significant difference was found for P, E2 and PRL levels. All of the six reproductive hormones had no difference between high and low dose groups.(2) The density of sperm was significantly higher in the high dose and low dose treatment groups than in the control group (P<0.01), but there was no significant difference between high and low dose groups. With regard to sperm motility, the low dose group was significantly greater than the control group while the high dose group was significantly lower than the control group (P＜0.01). There was no significant difference in the frequency of abnormal sperm among the three groups.(3) Prior to 8 weeks, body weight among the three groups did not differ significantly. Beginning on day 56, the body weights were significantly higher in the high and low dose groups than in the control group (P<0.01), but high and low dose groups were not significantly different from each other. After slaughter, the organ coefficients of the male rabbit testis, hypothalamus and spleen in the high and low dose group were significantly higher than in the control group (P<0.01), while the organ coefficients of brain, lung and bladder were not significantly different among the three groups. The organ coefficients were not significantly different between the high and low dose groups.(4) Following 80 days of RJ administration, the cell number of the testicular seminiferous tubule epithelium was significantly higher in both treated groups than in the control group. Compared to the histology of the testes in the control group, the following changes were observed in the RJ-treated groups:Characters of the testicular seminiferous tubule appeared to be thin seminiferous epithelium; seminiferous epithelium cells were loosely arranged with larger central lumen; most cells in the lumen were the primary spermatocytes and secondary spermatocytes in the control group; however the seminiferous tube cavity was relatively narrow and was enriched by the sperm cells and sperm. Cell layers were increased. spermatogonia attached base membrane. Primary spermatocytes, spermatids and sperm were packed closely and extended to the lumen, clear nuclear staining. A large number of sperms were observed in the lumen in the RJ given groups.Taken together, these results suggest that RJ can promote the development of hypothalamus, spleen and testis of male rabbits; sperm density and sperm motility were significantly increased in the low dose group, and serum levels of FSH, T and LH (but not of P, E2 and PRL) were significantly increased after 40 d of RJ administration.2. Effects of royal jelly on growth, development and expression of sex hormone receptor mRNA in prepubertal female Japanese white rabbits:Twelve 50-days-old fasting female rabbits were given fresh RJ (0.2% or 0.1% of body weight) or saline (0.1% of body weight) once a day for 40 days. Then the rabbits were sacrificed by femoral artery bleeding and the hypothalamus, the right side of ovary and the uterus were quickly dissected for RT-PCR analysis of hormone receptor mRNA expression. In addition the organs like heart, the left side of the uterus and ovaries were collected for organ coefficient analysis. The serum levels of FSH, LH, E2, P, T and PRL were determined by RIA. The results were as follows.(1) Moulting was observed after 12 d in 67% and 75% of rabbits treated with high and low doses of RJ, respectively, but was not observed in the control group.(2) Royal Jelly had no effect on body weight or the organ coefficients of heart, hypothalamus and ovaries.(3) The levels of serum E2 in high and low dose groups were significantly higher than that in the control group. No significant difference was found in other sex hormones.(4) Uterine ERαand ovary ERp mRNA levels were lower in high and low dose groups than in the control group. There was a significant difference between the high dose and control groups in the level (P<0.01). High and low dose groups, low dose and control group were not significantly different. Hypothalamic GnRH mRNA was lower in the high dose treatment group compared with the control and low dose groups (P<0.01). LHR and FSHR mRNA levels were significantly decreased in the high dose group (P<0.01) and low dose group (P<0.05) relative to the corresponding levels in the control group.Taken together, these results suggest that RJ can increase serum E2 levels in prepubertal female Japanese white rabbits, while suppress the mRNA expression of GnRH in hypothalamus, ERαin the uterus, and ERp, FSHR and LHR in ovary.3. Effects of royal jelly on reproductive performance of mature female Japanese white rabbits:The effect of PG on the homochronous of healthy and multiparous female rabbits was studied at first. After that, the rabbits were injected with PG (0.25mg per rabbit) and divided into three RJ treatment groups as described above. They were then mated after a complete estrous to investigate the effect of RJ on the reproductive performance. The results were as follows.(1) Injection of 0.25mg PG per rabbit caused the levels of LH, FSH and E2 reaching peak and trough at 9h and 48h, respectively; P came into trough and peaked at 9h and 48h, respectively. After PG treatment, the levels of LH, FSH and E2 reached peak at 19:00 each day, and P came into trough; the levels of LH, FSH and E2 on day 12 after PG treatment was higher than those on days 4,8 and 16, P was lower(P<0.05).(2) The peak of LH, FSH, E2 and the trough of P appeared earlier in RJ treatment groups than the control group, no difference was observed between high and low dose RJ teatement groups, neither peak nor trough was different among the three groups.(3) There was no significant effect of RJ on litter size and survival rate. However, the number of ovulation and follicle, individual birth weight, litter weight at birth, lactation ability, litter weight and individual weight at weaning were significantly lower in the control group compared with the high and low dose groups (P<0.01), and high and low dose groups were not significantly different.Taken together, these results suggest that the injection of 0.25mg PG per rabbit can make the female rabbits reach estrus peak at 9h and come into dioestrus at 48h after treatment; it started into the next estrous cycle at 12d; peak and trough of sex hormone mainly appeared at 19:00 each day. RJ can shorten the estrous cycle of female rabbits, but has no significant effect on hormone levels in the same estrous cycle. RJ can promote the development of follicular and ovulating, improve the litter weight at birth, individual birth weight, lactation ability, litter weight and individual weight at weaning, but has no effect on litter size.更多还原