【作者】 林燕；

【导师】 吴德；

【作者基本信息】 四川农业大学 ， 动物营养与饲料科学， 2009， 硕士

【摘要】 苏氨酸是免疫球蛋白中含量最丰富的一种必需氨基酸,它在精液或精细胞中的含量很丰富。免疫应激条件下免疫球蛋白的大量合成很容易引起日粮苏氨酸的缺乏和体蛋白的动员,进而影响动物的繁殖性能。因此,本研究设计了两个试验,以感染伪狂犬病毒雄鼠为模型,以雄鼠的精子质量、雄鼠的细胞及体液免疫参数,以及感染伪狂犬病毒雄鼠配种的雌鼠繁殖性能为评价指标,探讨苏氨酸对免疫应激雄鼠繁殖性能的影响及其可能的机制。试验一苏氨酸对对感染伪狂犬病毒雄鼠免疫性能和精液品质的影响试验采用2×4因子试验设计,选用168只昆明一级雄鼠(24.5±2.3g),按体重随机分配到8个组。4个日粮苏氨酸水平组分别为80%(0.7%组),100%(0.88%组),120%(1.1%组)和140%的(1.3%组)的GB(2001)的小鼠生长苏氨酸推荐量;每个水平组下再分为攻毒组和对照组(分别注射PRV和PBS)。雄鼠饲喂试验日粮5周后分别腹腔注射相同体积的PRV和PBS,并于注射后第9天收集样品,计算雄鼠死亡率、脏器指数(胸腺指数、脾脏指数、睾丸指数)、精子密度和精子畸形率;利用透射免疫比浊法测定血清免疫球蛋白(IgG、IgA、IgM)的浓度;利用ELISA法测定血清中的伪狂犬gB抗体、睾丸组织中的细胞因子(IL-1β、TNF-α)的浓度、睾丸组织中的睾酮的浓度,利用RT-PCR方法检测目的基因Toll样受体2(Toll-like receptor2,TLR2)、9 mRNA在睾丸中的相对表达量。试验二:苏氨酸对感染为狂犬病毒雄鼠繁殖能力的影响本试验选用单因子试验设计,选用80只昆明一级雄鼠(22.7±0.6g),按体重随机分到四个组。4个苏氨酸水平组分别为80%(0.7%组),100%(0.88%组),120%(1.1%组)和140%的(1.3%组)的GB(2001)的小鼠生长苏氨酸推荐量。雄鼠饲喂试验日粮5周后腹腔注射伪狂犬病毒,在攻毒后9天,与雌鼠1:1合笼,考察雄鼠的交配率,雌鼠的妊娠率和雌鼠的产仔数。试验结果表明:攻毒组的睾丸指数高于对照组,但只有0.7%达到显著性差异(P＜0.05)。与对照组相比,小鼠感染PRV可降低雄鼠精子密度,其中在0.7%组、0.88%组、1.1%组差异显著(P＜0.05),但在1.3%组却无差异(P＞0.05)。攻毒组第9天,精子密度随苏氨酸水平的升高而升高,0.88%组、1.1%组、1.3%组显著高于0.7%组(P＜0.05),1.1%组最高(P＜0.05)。与对照组相比,小鼠感染PRV可提高雄鼠精子畸形率,1.3%组攻毒处理后精子畸形率上升,但与对照组相比不显著(P＞0.05)。攻毒组第9天,精子畸形率随苏氨酸水平的升高而降低,1.3%组显著低于0.7%组(P＜0.05)。与对照组相比,0.7%组、0.88%组感染PRV的雄鼠睾丸组织中的睾酮的浓度显著降低(P＜0.05),攻毒雄鼠中,随着苏氨酸水平的提高,睾酮的浓度升高,其中1.1%组、1.3%组显著高于0.7%组、0.88%组(P＜0.05),但1.1%组、1.3%组间无差异(P＞0.05)。随着苏氨酸水平的提高,雄鼠的交配率(P=0.264)和雌鼠的妊娠率均有提高的趋势(P=0.139),但雌鼠的产仔数无差异(P＞0.05)攻毒组雄鼠的死亡率随日粮苏氨酸水平的升高而降低,但差异不显著。攻毒组的胸腺指数和脾脏指数高于对照组,其中在0.7%组、0.88%组、1.1%组攻毒后的胸腺指数均显著高于PBS对照组(P＜0.05),但是在1.3%组攻毒后的胸腺指数与对照组相比却没有差异(P＞0.05)。在攻毒处理中,胸腺指数随着苏氨酸水平升高而升高,在1.1%组达到最高(P＜0.05)。攻毒后,1.1%组的脾脏指数显著高于PBS对照组(P＜0.05)。在攻毒处理中,1.1%组的脾脏系数显著高于0.7%组(P＜0.05)。与对照组相比,感染PRV显著增加雄鼠血清IgG的浓度(P＜0.05),但对IgA、IgM浓度的影响不显著(P＞0.05)。攻毒后第9天血清中IgG浓度随着苏氨酸水平的升高而升高,在1.3%组达到最高(P＜0.05)。与对照组相比,感染PRV能增加雄鼠血清gB抗体滴度,但只有1.1%组、1.3%组达到显著性差异(P＜0.05)。攻毒后第9天,gB抗体滴度随苏氨酸水平升高而升高,其中1.1%组gB抗体滴度与0.7%组相比有显著性差异(P＜0.05)。这说明随着苏氨酸水平的提高,雄鼠的免疫性能增强。与对照组相比,感染PRV提高睾丸组织中的IL-1β的浓度,其中0.7%组、1.1%组、1.3%组显著高于对照组(P＜0.05)。攻毒雄鼠中,0.7%组的IL-1β显著高于其他组(P＜0.05),随着苏氨酸水平的提高,IL-1β的浓度逐渐下降,但都未达到显著水平(P＞0.05)。与对照组相比,感染PRV提高睾丸组织中的TNF-α的浓度(P＜0.05),攻毒雄鼠中,随着苏氨酸水平的提高,TNF-α的浓度下降,其中0.88%组、1.1%组、1.3%组显著低于0.7%组(P＜0.05),但1.1%组、1.3%组间未达到显著水平(P＞0.05)。攻毒后,0.7%组睾丸TLR2相对表达量显著高于1.1%组(P＜0.05)和1.3%组(P＜0.05)组,其余各组之间差异不显著(P＞0.05)。攻毒雄鼠中,0.7%组睾丸TLR9相对表达量显著高于其余三组(P＜0.05),0.88%组显著高于1.1%组、1.3%组(P＜0.05),1.1%组、1.3%组之间差异不显著(P＞0.05)。这说明,随着苏氨酸水平的提高睾丸TLR2、TLR9的相对表达量降低。各组雄鼠中,攻毒处理后睾丸TLR2、TLR9相对表达量显著高于PBS组(P＜0.05)。注射PBS条件下,各处理组之间睾丸TLR2、TLR9相对表达量差异不显著(P＞0.05)。根据以上结果,可得到结论:①攻毒提高雄死亡率和精子畸形率,降低精子密度,添加苏氨酸可降低攻毒雄鼠的死亡率和精子畸形率,提高精子密度,攻毒雄鼠精子密度达最大值时,雄鼠对苏氨酸的需要量达正常需要量的1.26倍。②苏氨酸通过提高攻毒雄鼠的免疫力,降低睾丸组织的炎症因子等提高的攻毒雄鼠的繁殖性能。③病毒感染情况下,提高饲料中的苏氨酸含量有利于缓解病毒对雄鼠繁殖性能造成的损害。更多还原

【Abstract】 Threonine is one of the most abundant amino acids constituting immunoglobins and is rich in semen and sperm cells.A bulk of synthesis of immunoglobulin against immunological stress could result in dietary threonine deficiency and the mobilization of body protein,which could exert profound negative effects on the reproductive performance.Therefore,the experiment was based on a PRV-infected mice model,to investigate the effects and possible mechanisms of different dietary thrconine levels on sperm quality,cellular and humoral immunity parameters,and the reproductive performance of female mice mated by PRV-infected male mice.Experiment 1 effects of threonine on immune and reproductive performance in male mice challenged with Pseudorabies VirusAccording to a 2×4 experimental design,168 first class Kunming male mice with similar body weight(24.5±2.3g) were allocated to 4 dietary thrconinc levels of 0.7%, 0.88%,1.1%and 1.3%,which were 80%,100%,120%and 140%of the recommended threonine requirement by GB(2001),and after 5 weeks’ adaptation to their respective diets,male mice were injected by PRV or PBS.On 9 days post-infection,mice were sacrificed to collect the data on mortality,organ index (thymus index,spleen index and testis index),sperm density and sperm deformity rate. Serum immunoglobulins(IgG,IgA and IgM) were determined by radioimmunoassay. Serum gB antibody,testis cytokines(IL-1βand TNF-α),and testis testosterone concentrations were determined by enzyme linked immunosorbent assay.Testis toll-like receptor 2 and 9 mRNA expressions were detected by RT-PCR.Experiment 2 Effect of threonine on Reproductive capacity of male mice challenged with PRV80 first class Kunming male mice with similar body weight(22.7±0.6g) were allocated to 4 dietary threonine levels of 0.7%,0.88%,1.1%and 1.3%,which were 80%,100%,120%and 140%of the recommended threonine requirement by GB(2001),and after 5 weeks’ adaptation to their respective diets,male mice were injected by PRV.on 9 d post PRV challenge,the male mice were copulate with adult female mice in a ratio of(1:1).The pregnancy rate and average number per litter in the copulated female mice were observed and the male mouse copulation rate in each group were calculated.Results indicated that PRV infection in resulted in a higher testis index compared with PBS injected mice,particularly in PRV infected mice in 0.7%Thr level(P＜0.05). Compared with PBS injection,PRV injection decreased sperm density in 0.7%Thr, 0.88%Thr,1.1%Thr mice(P＜0.05),but it did not affect that in 1.3%Thr mice (P＞0.05).On 9 days post-infection,however,sperm density increased as the increasing Thr levels,it was higher in the 0.88%Thr,1.1%Thr and 1.3%Thr mice than the 0.7%Thr mice(P＜0.05),and it was maximal in 1.1%Thr mice(P＜0.05).PRV infection mice had a higher sperm deformity rate(P＜0.05),but it was lower as the increasing dietary Thr level,and 1.3%Thr mice had a significantly lower sperm deformity rate than the 0.7%Thr mice(P＜0.05).PRV-injected mice in 0.7%and 0.88%Thr mice had a lower testosterone compared with PBS-injected counterparts.But Thr supplements rescued the testosterone decrease,and it was higher in 1.1%and 1.3%Thr than the 0.7%and 0.88%Thr mice(P＜0.05).copulation rate,conception rate were numerially higher as the increasing Thr level(P=0.139),but litter size was not affected(P＞0.05).PRV infection resulted in a higher mortality,but it was attenuated by Thr supplement(P＞0.05).PRV infected mice had a higher organ(thymus and spleen) index than the PBS injected mice(P＜0.05).The thymus index was not affected in 1.3%Thr mice between PRV and PBS infection(P＞0.05).Higher Thr level increased the thymus index in the PRV injected mice,and the organ index was higher in 1.1% Thr mice than the others(P＜0.05).Spleen index of PRV injected mice in 1.1%Thr was significantly higher than their PBS counterparts(P＜0.05),and was also higher than the PRV injected mice in 0.7%Thr level(P＜0.05).Testis TLR2 and 9 mRNA expression was significantly higher in response to PRV infection compared with PBS control(P＜0.05),and they were not affected by Thr supplemental level in PBS injected mice.TLR2 expression in testis of PRV infected mice was regulated by Thr, it was higher in 0.7%Thr than the 1.1%Thr and 1.3%Thr mice(P＜0.05).TLR9 expression was also down-regulated by Thr supplement in dose-dependent manner, and it was significantly higher in 0.7%Thr level than the other three Thr levels, providing evidence that the immune status was enhanced as the higher Thr levels.The mice in 0.7%,1.1%and 1.3%Thr mice had a higher IL-1βconcentration in response to PRV injection compared with PBS injection(P＜0.05).IL-1βconcentration was significantly higher in 0.7%Thr than the other Thr levels(P＜0.05),but as the higher Thr supplemental levels,IL-1βconcentration tent to decrease(P＞0.05).PRV infection also resulted in a higher TNF-αconcentration compared with PBS control mice(P＜0.05),but its secretion was decreased as the higher Thr supplemental level, 0.88%,1.1%and 1.3%Thr had a lower TNF-αconcentration than the 0.7%Thr mice(P＜0.05),but no difference were observed between 1.1%and 1.3%mice (P＞0.05).PRV infection resulted in a higher mortality,but it was attenuated by Thr supplement(P＞0.05).PRV infected mice had a higher organ(thymus and spleen) index than the PBS injected mice(P＜0.05).The thymus index was not affected in 1.3%Thr mice between PRV and PBS infection(P＞0.05).Higher Thr level increased the thymus index in the PRV injected mice,and the organ index was higher in 1.1% Thr mice than the others(P＜0.05).Spleen index of PRV injected mice in 1.1%Thr was significantly higher than their PBS counterparts(P＜0.05),and was also higher than the PRV injected mice in 0.7%Thr level(P＜0.05).Testis TLR2 and 9 mRNA expression was significantly higher in response to PRV infection compared with PBS control(P＜0.05),but they were not affected in response to different Thr supplemental levels in PBS injected mice.TLR2 expression in testis of PRV infected mice was regulated by higher Thr levels,it was higher in 0.7%Thr than the 1.1%Thr and 1.3%Thr mice(P＜0.05).TLR9 expression was also down-regulated by Thr supplement in dose-dependent manner,and it was significantly higher in 0.7%Thr level than the other three Thr level.Collectively,these results demonstrated that:Thr supplement could rescue the deleterious effects of the PRV invading on mortality,sperm deformity rate and sperm density,Thr requirement of male mice with highest sperm density was 1.26 times that of mice with normal sperm density.Thr could enhance the immunity of the male mice to decrease the pro-inflammatory response to the male reproductive tract.Upon pathogen invading,higher Thr than normal requirement could attenuate the deleterious effects on the reproductive performance.更多还原