锌（Zn）、铜（Cu）、锰（Mn）、铁（Fe）、硒（Se）是保证肉种鸡健康生长及高效生产所必需的微量元素，目前，肉种鸡微量元素添加量缺乏统一的标准，通常导致这些元素添加过量，从而影响肉种鸡生产性能，并造成环境污染等不良影响。复合有机微量元素对肉用种公鸡睾丸发育和精液品质的改善机制仍缺乏大量研究，因此本研究以雪山草鸡和三黄鸡为研究对象，对其饲喂不同形式和浓度的微量元素日粮，通过检测饲粮、血清、睾丸中五种微量元素的含量，不同时间点检测公鸡精液品质，观察各组种公鸡睾丸组织的显微结构，以及睾丸组织和精子的超微结构，血清睾酮浓度以及睾酮分泌相关基因表达测定，并评估复合有机微量元素安全性，研究日粮中复合有机微量元素对种公鸡精液品质的提升作用和对睾丸发育的改善作用，为全面评价日粮中复合有机微量元素对生殖的改善机制提供科学的理论依据。主要研究如下： 1. 复合有机微量元素对种公鸡精液质量和睾丸发育的影响 选用23周龄雪山草鸡种公鸡192只，随机分为2组，即无机微量元素组（Inorg-TM）和有机微量元素组（Org-TM），每组6个重复，每个重复16只公鸡，试验期39周。Inorg-TM组日粮中微量元素（Mn, Zn, Fe, Cu, Se）添加水平按照NRC（1994）肉鸡推荐需要量添加，添加形式为无机微量元素；Org-TM组日粮中微量元素添加水平为NRC（1994）肉鸡推荐需要量的80%，添加形式为60%有机微量元素和40%无机微量元素。饲喂1周后，每组每个重复任选1只，每隔两周采1次精液进行液质量检测；饲喂39周后，12只采精公鸡屠宰，采集血清和睾丸等组织分析。结果显示：Org-TM组饲料中Zn元素含量显著低于Inorg-TM组（P＜0.05），其他四种元素无显著差异。与Inorg-TM组相比，Org-TM组采精量和精子密度在饲喂8周后均极显著提高（P＜0.01），精子活力提高不显著；精子畸形率在饲喂14周后显著提高（P＜0.05）。与Inorg-TM组相比，Org-TM组种公鸡体重、睾丸重和睾丸指数均无显著差异。睾丸组织形态学观察发现，两组种公鸡睾丸精原细胞均存在精原细胞脱落、线粒体空泡、核仁固缩、核内质周缘化等现象，但是Org-TM组相对程度较轻。精子的透射电子显微镜观察发现，两组精子均存在顶体脱落、线粒体膨胀、中段质膜不贴合等现象，但是，Org-TM组较Inorg-TM出现数量相对较少。上述结果表明，在微量元素添加水平为需要量的80%，添加形式为60%有机微量元素和40%无机微量元素条件下，复合有机微量元素能够提高种公鸡精液品质，减缓公鸡睾丸退化状况。 2. 复合有机微量元素对种公鸡睾酮分泌及其相关基因表达的影响 为了进一步阐明复合有机微量元素提高种公鸡精液品质的营养分子机制，本试验选用23周龄的三黄种公鸡240只，随机分为2组，即无机微量元素组（Inorg-TM）和有机微量元素组（Org-TM），每组6个重复，每个重复20只公鸡，试验期为22周。Inorg-TM组日粮微量元素（Mn, Zn, Fe, Cu, Se）的添加水平按照NRC（1994）肉鸡推荐需要量添加，添加形式为无机微量元素；Org-TM组日粮微量元素添加水平是NRC（1994）推荐需要量的70%，全部以有机微量元素形式添加。饲喂一周后，每组每个重复选1只，共12只公鸡，每两周采一次精液进行精液质量检测；分别在饲喂7周、12周和22周后，每组屠宰6只公鸡采集血清和睾丸附睾等组织。结果显示：Org-TM组日粮中Zn和Mn含量显著低于Inorg-TM组日粮（P＜0.05）。与Inorg-TM组相比，Org-TM组采精量在饲喂8周后极显著提高（P＜0.01），精子密度和精子活力在饲喂8周到12周提高显著（P＜0.05），精子畸形率在饲喂2周后提高显著（P＜0.05）。与Inorg-TM组相比，饲喂7周、12周和22周，Org-TM组种公鸡体重、睾丸重和睾丸指数均无显著差异。睾丸组织形态学观察发现，两组公鸡均存在睾丸精原细胞排列不紧密和精原细胞脱落等现象，但是Org-TM组相对程度较轻。Org-TM组公鸡血清睾酮浓度在饲喂12周和22周显著高于Inorg-TM组（P＜0.05）。与Inorg-TM组相比，饲喂7周，Org-TM组公鸡睾丸组织睾酮合成相关基因HSD17B4 mRNA表达量显著性升高（P＜0.05），而CYP19A1 mRNA表达量极显著性降低（P＜0.01），HSD3B2和CYP17A1 mRNA表达量无显著差异；饲喂12周，HSD3B2和CYP17A1 mRNA表达量上升但无显著差异，HSD17B4 mRNA表达量显著上升（P＜0.05），CYP19A1 mRNA 表达量极显著上升（P＜0.01）； 饲喂22周，HSD3B2和CYP17A1 mRNA表达量显著上升（P＜0.05），HSD17B4 mRNA表达量显著下降（P＜0.05），CYP19A1 mRNA 表达量极显著上升（P＜0.01）。上述结果表明，在微量元素添加水平为需要量的70%，添加形式全部为有机微量元素条件下，复合有机微量元素能通过提高睾酮分泌来促进睾丸发育，提高精液质量。 3. 复合有机微量元素提高种公鸡精液品质的安全性评价 为了进一步明确复合微量元素在提升种公鸡精液品质同时，而不影响种公鸡机体健康，本试验采集试验二动物试验的相关样品，通过评估复合有机微量元素在种公鸡体内元素的分布以及血清氧化平衡状态，衡量复合有机微量元素的整体安全性。结果显示：与Inorg-TM组相比，饲喂7周，Org-TM组只有血清Fe含量显著降低（P＜0.01），其他微量元素在血清和睾丸组织均无显著差异；饲喂12周，血清和睾丸组织中五种元素含量均无显著性差异；饲喂22周，Org-TM组睾丸组织Zn含量和血清Mn含量显著高于Inorg-TM组（P＜0.05），其他微量元素在血清和睾丸组织均无差异。与Inorg-TM组相比，饲喂7周，Org-TM组种公鸡睾丸组织GSH水平显著降低（P＜0.05），同时T-SOD活力显著升高（P＜0.05），CAT活力无显著差异；饲喂12周，Org-TM组睾丸组织GSH水平显著升高（P＜0.05），T-SOD和CAT活力无显著差异；饲喂22周，Org-TM组睾丸组织CAT活性显著下降（P＜0.05），T-SOD活性和GSH水平无显著差异。上述结果说明，在微量元素添加水平为需要量的70%，添加形式全部为有机微量元素条件下，复合有机微量元素可通过还原性微量元素对抗氧化应激，降低体内氧化还原水平，缓解氧化应激，其在生产中提高公鸡精液品质的应用是安全的。

Zinc, manganese, copper, iron and selenium serving as trace minerals, were well known to be nutritionally essential for roosters reproduction. In order to obtain the maximum growth rate, dietary inorganic trace minerals were much higher than those recommended by NRC for poultry. However, there was less research on the effect of organic mineral on poultry semen quality and the development of testis. In the present studies, Xue Shan and San Huang roosters were used to detect the effect of different forms and concentrations of complex organic trace minerals on semen quality, the histological structure, the ultrastructure of testis and sperm, serum testosterone concentration and the related-genes mRNA expression of testosterone synthesis in rooster. It could provide a scientific basis for comprehensive assess of the beneficial effects of complex organic trace minerals to improve fertility. 1. Effect of dietary complex organic trace minerals on semen quality and testis in breeder roosters One hundred and ninety-two 23-week-old Xue Shan roosters were randomly divided into two groups: inorganic trace minerals group (Inorg-TM) and organic trace minerals group (Org-TM). According to the NRC (1994) recommended chicken requirements, Inorg-TM were added with 100% level of NRC recommended inorganic trace minerals included Mn, Zn, Fe, Cu, Se. Org-TM were added with 80% level of NRC recommended from the form of 40% inorganic trace minerals plus 60% the organic. Each group repeated six times and every repeat included 16 roosters. One rooster was randomly selected from each replicate for semen collection. The first time of semen collection was taken after 1-week treatment (1W), and the other 19 times was taken by 2-week internal. After 39-weeks treatment, these 12 roosters were slaughtered for collecting serum and testes. The results showed that the concentration of zinc in organic fodder was significantly decreased than the inorganic (P<0.05). Semen quality was improved significantly in Org-TM compared with Inorg-TM. Semen volume was increased in Org-TM from 8W to 22W, compared with Inorg-TM (P<0.01). There was an higher increase in sperm density (P<0.05) than those in Inorg-TM from 8W to 12W. Abnormal morphology was decreased in Org-TM than Inorg-TM from 14W to 22W (P<0.05). There was no significantly differences in body weight and testes’ relative organ weight between two groups. Histological observation showed that complex Org-TM could relieve the shedding of spermatogonium and Leyding cells compared with Inorg-TM. Ultrastructure observation showed that both mitochondria vacuolation in spermatogonium and pyknosis of nucleolus were more in Inorg-TM than those in Org-TM. In Inorg-TM, there were more lost acrosome, condensed mitochondria and damaged plasma membrane than Org-TM. In summary, our results showed that in this supplemented level of 80% level of chicken requirements from the form of 40% inorganic trace minerals plus 60% the organic, it could improve semen quality and promote the testis development. 2. Effect of dietary complex organic trace minerals on testosterone synthesis and the related gene expression in breeder roosters In order to clarify the mechanism underlying the positive effect of organic trace minerals on semen quality in roosters, two hundred and forty 23-weeks-old San Huang roosters were randomly divided into two groups: inorganic trace minerals group (Inorg-TM) and organic trace minerals group (Org-TM). According to the NRC (1994) recommended chicken requirements, Inorg-TM were added with 100% level of NRC recommended inorganic trace minerals included Mn, Zn, Fe, Cu, Se. And then, Org-TM were added with 100% level of NRC recommended organic trace minerals. Every group repeated six times and every repeat includes 20 roosters. The experiment period was 22 weeks. One rooster was randomly selected from each replicate for semen collection. The first time of semen collection was taken after 2-week treatment (2W), and the other 10 times was taken by 2-week internal. Another one rooster was randomly selected from each replicate for slaughter at 3 time points (7W, 12W, 22W).The results showed that the concentration of znic and manganese in organic fodder was decreased significantly than the inorganic (P<0.05). Semen volume was increased in Org-TM from 8W to 22W, compared with Inorg-TM (P<0.01). There was an higher increase in sperm density and motility (P<0.05) than those in Inorg-TM from 8W to 12W. Abnormal morphology was decreased in Org-TM than Inorg-TM from 1W to 22W (P<0.05). There was no significantly differences in body weight and testes’ relative organ weight between two groups. Histological observation showed that for three times slaughter, complex Org-TM could relieve the shedding of spermatogonium compared with Inorg-TM and Leyding cells were closed packed. Serum testosterone concentration was significantly higher than Inorg-TM in Org-TM when 12W and 22W(P<0.05). The relative mRNA expression of HSD3B2 and CYP17A1 increased significantly at 22W than Inorg-TM(P<0.05). The relative mRNA expression of HSD17B4 increased significantly at 7W and 12W than Inorg-TM, and decreased at 22W(P<0.05). The relative mRNA expression of CYP19A1 was significantly lower than Inorg-TM at 7W, but was higher at 12W and 22W(P<0.01). Hence, our results showed that, in this supplemented level of 100% level of chicken requirements in forms of organic trace minerals, it could improve semen quality and the function might be achieved by promoting the synthesis of testosterone. 3. Security evaluation of dietary complex organic trace minerals for promoting semen quality in breeder roosters In order to determine the security of complex organic trace minerals utilized in rooster diet, the tissue trace concentration and serum antioxidative banlance were messured from rooster in the second experiment. The results showed that there was no significantly difference for five trace minerals’ concentrations in serum and testis between two groups and only Fe’s was significantly decreased in serum of Org-TM at 7W(P<0.01). At 12W, there were no significant differences. At 22W, the concentration of Zn in testis and Mn in serum were increased significantly than Inorg-TM(P<0.05). Compared with Inorg-TM, at 7W the GSH level was decreased in testis of Org-TM (P<0.05) and the T-SOD activity was higher(P<0.05). At 12W, the GSH level was increased significantly in Org-TM(P<0.05). At 22W, the CAT activity was decreased in Org-TM(P<0.05). In Inorg-TM, the activity of CAT was decreased for three times. These results suggested that, in this supplemented level of 100% level of chicken requirements in forms of organic trace minerals, complex organic trace minerals could withstand oxidative stress by minerals alleviate the oxidative damage. Alternatively, it was safe to supply the complex organic trace minerals in rooster diet to enhance semen quality.