【作者】 张敏；

【导师】 邹晓庭；

【作者基本信息】 浙江大学 ， 动物营养与饲料， 2011， 博士

【摘要】 以美国王鸽为试验对象,研究不同生理阶段蛋鸽生殖激素变化及就巢相关基因表达特性,以探讨不同时期生理变化规律和就巢的内分泌机制。在此基础上,研究功能性氨基酸对蛋鸽生产性能的影响,以探讨通过营养调控手段缓解蛋鸽就巢习性、缩短产蛋间隔的效果及机理。主要研究结果如下：1.蛋鸽血清生化指标和输卵管形态结构差异（1）产蛋期、就巢期与产蛋期相比,就巢期蛋鸽雌激素水平下降41.29%（P<0.05）,催乳素（PRL）水平上升110.28%（P<0.05）。产蛋后蛋鸽血清PRL水平呈现先升高后下降的趋势,雌激素变化趋势相反。输卵管切片观察结果显示产蛋期输卵管腔较就巢期狭窄,管壁褶皱排列较紧密,褶皱上绒毛密集,腺体组织较明显。（2）不同阶段青年鸽（150±10日龄）、初产（180±10日龄）、产蛋2次（210±10日龄）、产蛋5次（270±10日龄）、停产（5-6岁）蛋鸽血清指标显示：PRL、FSH水平随产蛋次数增加升高,停产时最高（P<0.05）；血清白蛋白水平随产蛋次数增加显著下降,停产时达到最低（P<0.05）。停产时血清孕激素水平显著升高（P<0.05）,血清钙水平急剧下降（P<0.05）。光镜观察输卵管形态显示：停产蛋鸽输卵管管腔变大,褶皱排列较为疏松且不规则,呈现萎缩迹象。提示高水平的PRL是维持就巢的关键因素,PRL、FSH、孕激素水平升高与钙流失可能是卵巢衰老的原因之一。2.就巢相关基因克隆及分析通过比较相近鸟类保守序列设计简并引物,采用巢式PCR成功克隆得到鸽VIP、PRL和FSH片段长度分别为322bp、658bp和354bp。将核苷酸序列登录到Genbank,获得登录号分别为FJ913875、GU968532和FJ913876。推断的氨基酸片断相似性比对结果显示：美国王鸽VIP与北京鸭DQ023227.1、鸡CAA56867、鹅AAY59005、爪哇禾雀BAF96737、火鸡P45644、白喉雀ADK26700、斑胸草雀XP002187804大部分氨基酸序列高度相似,同源性较高,一致性分别为95.9%、95.3%、95.5%、95.3%、95.3%、97.2%和95.3%；美国王鸽PRL基因与火鸡AAB60604.1、鹦鹉BAF81527.1、鸡NP₉90797、瘤鸭CAJ55836、鸭BAD14942.1、家鹅AAY59658、珠鸡BAG68294.1、蓝孔雀BAG68293、爪哇禾雀BAF96736.1、鸵鸟BAF81528.1、鹌鹑BAD10927、环颈雉BAG68292.1氨基酸序列一致性分别为90.4%、93.2%、91.3%、94.5%、95.0%、92.7%、92.2%、89.0%、91.3%、91.8%、和88.6%,与鸭、鹅同源性较高；FSH基因与日本鹌鹑BAC01164、鸭ABB60052.1、朱鹮BAC07314、鹅ABX39193、鸡NP 989588、斑胸草雀XP 002194767和鸵鸟P80663基因一致性分别为94.1%、97.5%、99.2%、94.3%、94.9%、93.2%和92.5%,与朱鹮同源性最高。3. VIP. FSH. PRL和PRLR mRNA基因的表达特点采用荧光定量PCR方法检测VIP、FSH、PRL和PRLR mRNA基因在蛋鸽不同时期、不同组织的表达量。（1）不同组织mRNA表达量结果显示：VIP表达量由多到少的顺序为脑、输卵管、脾脏、卵巢、肝脏、肌肉；FSH表达量顺序为卵巢、输卵管、脾脏、脑、肝脏、肌肉；PRLR表达量顺序为输卵管、卵巢、脑、肝脏、脾脏、肌肉；PRL表达量顺序为垂体、脾脏、输卵管、肝脏、心脏、卵巢、胰腺、肾脏。结果表明,VIP、PRL、FSH和PRLR基因在脑、垂体和性腺均有较高表达量。（2）产蛋期、就巢期mRNA表达量结果显示：就巢期卵巢PRL表达量显著高于产蛋期（P<0.05）。（3）不同产蛋次数就巢相关基因mRNA表达量结果显示：垂体VIP在停产和初产期相对表达量较高（P<0.05）。卵巢PRLR基因随产蛋次数增加显著下降,到停产时最低（P<0.05）)。卵巢FSH、垂体PRL基因表达量随产蛋次数增加而增加,到停产时最高（P<0.05）。推测就巢期PRL水平升高与卵巢PRL基因mRNA表达量增加有关,蛋鸽卵巢功能退化与VIP、PRL基因mRNA表达量增多和PRLR基因mRNA表达量减少有关。4.Y-氨基丁酸（GABA）和谷氨酸（Glu）对蛋鸽生产性能的影响选取360日龄美国王鸽120对,随机分为5组,每组3个重复,每重复8对,分别在日粮中添加0（对照组）、20（试验Ⅰ组）、40（试验Ⅱ组）、80mg/kg GABA（试验Ⅲ组）和40 mg/kg Glu（试验Ⅳ组）,研究日粮中添加γ-氨基丁酸（GABA）和谷氨酸（Glu）对蛋鸽产蛋性能的影响,并探讨其作用机理。结果显示：与对照组相比,80mg/kg GABA显著提高产蛋间隔期蛋鸽血清PRL水平,缩短产蛋间隔（P<0.05）；降低产蛋期血清胆固醇水平（P<0.05）。40mg/kg Glu显著提高产蛋期、产蛋间隔期蛋鸽血清PRL水平及产蛋期FSH水平；显著提高产蛋间隔期PRL、FSH、VIP基因mRNA表达量（P<0.05）,降低PRLR基因mRNA表达量（P<0.05）。提示GABA可能通过调节PRL水平及血清中营养成分的途径缩短产蛋间隔。Glu具有促进激素分泌的作用,但具有加速卵巢衰老的风险。更多还原

【Abstract】 The studies were conducted to investigate the incubation and reproduction mechanism of American King Pigeon by analyzing the physical parameters and incubation related gene expression characters in different phases. Furthermore, investigated the nutrimental regulation of incubation behavior and laying performances in laying pigeon.The main contents and results are as follows:1. Change of biochemical parameterns and difference of viduct configration in different phases.The mechanism of incubation arising and ovary fouctional degradation was investigated by analyzing biochemistry parameters and observing the histological section of oviduct in different physiological stages of American king pigeon. （1） As compared to the laying period, the level of PRL increased whereas E2 decreased significantly in incubation （P<0.05）. And the oviduct lumen became cabined, glandular organ became more obvious and the villi arranged more trimly and close in laying. （2） The level of prolctin（PRL） in serum had a tendence to increase first and then decriease, whereas the level of estradiol （E2） have a tendence to decriease first and then increase （P<0.05）. （3） The results of different laying experience pigeon showed that, the level of PRL, E2 and FSH increased with laying experience. While albumin decreased with the laying experiences, which reach to the minimum at spent laying （P<0.05）. Serum calcium decreased numerically from the primiparous to second laying, while increased from the second laying and reached to the maxmum at the fifth laying （P<0.05）, and then decreased significantly at spent laying （P<0.05）. There were higher level of progestogen observed in the primiparous and spent pigeon （P<0.05）. According to the electronic microscope photograph of oviduct, the oviduct lumen became cabined, glandular organ became more obvious and the villi arranged more trimly and close in laying. Oviduct lumen from the primiparous, second and 5th laying was more combined, the villi was more trim and close, and better arranged than pullets. The lumen of oviduct from spent laying became loose and atrophied, and the villi was arranged ruleless. It’s indicated that the physical condition of laying and incubation pigeon was stable, and the pigeon recovered well from nutrient loss during laying. And the relative high level of PLR in serum was crucial for incubation maintainance. Speculated that the ovary senescence have relation to the increasing of PRL, FSH and progestin, while decreasing of calcium in serum.2. Cloning and analyzing of incubation related genes.In order to understand the phylogenetic diversity of incubation related gene, the cDNA of PRL, vasoactive intestinal peptide （VIP） and folliclestimulating hormone （FSH） was isolated and cloned from female American silver king pigeon （Columbia livia） pituitary gland and ovary by a degenerate primer and nested reverse transcription polymerase chain reaction （RT-PCR） based strategy. The results showed that the cloned cDNA contains 658bp,322bp and 354bp nucleotides, respectively, and submitted to Genbank with accession number GU968532, FJ913875, and FJ913876,corresponding sequences of turkey （AAB60604.1）, budgerigar （BAF81527.1）, chicken （Genbank accession no. NP₉90797）, comb duck （CAJ55836）, duck（B AD 14942.1）, domestic goose （AAY59658）, helmeted guineafowl （BAG68294.1）, Indian peafowl （BAG68293）, Java sparrow （BAF96736.1）, ostrich （BAF81528.1）, quail （BAD10927）, and Ring-necked pheasant （BAG68292.1） were 90.4%,93.2%,91.3%,95.0%,92.7%,92.2%,89.0%,91.3%,91.8%, and 88.6%, respectively. Phylogenetic analysis indicates that pigeon PRL is more closely related to the duck and goose orthologs compared to that of other species. The identities of VIP deduced amino acid sequence with the corresponding sequences of Beijing duck （DQ023227.1）, chicken （CAA56867）, goose （AAY59005）, Java sparrow （BAF96737）, turkey （P45644）, white-throated sparrow （ADK26700）, and zebra finch （XP002187804） were 95.9%,95.3%,95.5%,95.3%,97.2%, and 95.3%, respectively. And the identities of FSH deduced amino acid sequence with the corresponding sequnence of Japanese quail （BAC01164）, duck （ABB60052.1）, crested ibis （BAC07314）, goose （ABX39193）, chicken （NP₉89588）, zebra finch （XP₀02194767）, and ostrich （P80663） were 94.1%,97.5%,99.2%,94.3%,94.9%, 93.2%, and 92.5%, respectively.3. Tissue distribution and expression character in different physical phases of VIP, FSH, PRL and PRLR gene.This study was conducted to investigate incubation related gene tissue distribution and expression characters in different physical phases by detecting the mRNA expression in different tissues and phsaes through real-time quantitative PCR （FQ-PCR）. （1） The results showed that, all the gene was widely expressed in tissues analyzed. Highest level of VIP mRNA expression was observed in the brain, and then oviduct, spleen, liver, and muscle （P< 0.05）. PRL mRNA expressed highest in pituitary, and then spleen, oviduct, liver, heart, ovary, pancrease, and kidney （P< 0.05）. The expression order of FSH was ovary, oviduct, spleen, brain, liver, and mucle （P< 0.05）. And the PRLR expressed highest in oviduct, ovary, brain, liver, spleen, and mucle （P< 0.05）. The high expression of aboved gene in brain and gonad indicated the modulation role in pigeon edocrine and reproductive behavior. （2） The ovary PRL mRNA expression increased significantly in incubation, compared to laying （P< 0.05）, while the pituitary PRL and VIP mRNA increased numerically. The amount of VIP mRNA expression reached maxmum at spent laying （P< 0.05）, whereas PRLR reached maxmum at pullets （P< 0.05）. Interstingly, FSH and PRL expression increased with laying experience, and reached maxmum at spent laying （P < 0.05）. Speculated that the increased plasma PRL in incubation pigeon have relation to the elevation of ovary mRNA expression. And the reproduction senescence might be related to the increasing of VIP and PRL mRNA expression and decreasing of PRLR.4. The effect of dietary y-aminobutyric acid （GABA） and glutamine （Glu） on laying performance of American King Pigeon.The study was conducted to evaluate the effect of nutrients on laying performance and incubation related gene expression on American King Pigeon by administration of GABA and Glu. Furthermore, investigated nutritional modulation on laying performance and approached to shorthen the laying interval. The results showed that, compared to the control group, dietary GABA significantly increased the level of plasma PRL and triglyceride, and the activity of plasma GTP in pigeon at laying interval period, and shorthened the laying interval （P<0.05）. While dietary Glu significantly increased the level of plasma PRL in pigeon at laying interval period, and FSH in laying （P<0.05）. And increased the PRL, VIP, and FSH mRNA expression whereas decreased the PRLR mRNA expression in pigeon at laying interval period （P<0.05）. Suggested that dietary GABA shorten the laying interval by modulating the PRL level and nutrents content in laying and incubation. Glu stimulated the PRL, VIP, and FSH expression, while potentially accelerated the reproduction senescence in laying pigeon.更多还原