【作者】 吕建敏；

【导师】 刘建新；

【作者基本信息】 浙江大学 ， 动物营养与饲料科学， 2009， 博士

【摘要】 WHBE兔是由日本大耳白兔突变产生的实验兔新品系,以其白色被毛(White hair)和黑色眼睛(Black eye)的特征而得名,具有能产生高效价抗体的特性。目前对WHBE兔遗传特性的研究较多,而有关营养需要方面的研究几乎空白。由于蛋白质营养与动物的免疫功能密切相关,本文通过饲养试验,氮代谢试验、免疫功能测定等一系列试验,研究了生长期和育成期的WHBE兔对日粮粗蛋白的营养需要特点,并从消化层面(盲肠微生态变化情况)和代谢层面(相关功能基因表达)探讨WHBE兔对蛋白质最佳需要量的产生机理。最后,对WHBE兔日粮中限制性氨基酸的适宜比例作了初步研究。1.日粮粗蛋白水平对生长期WHBE兔生长性能、氮代谢、免疫功能、盲肠发酵参数及肝脏功能基因表达的影响选用40日龄WHBE断奶仔兔60只,进行为期6周的饲养试验。将仔兔随机分为5组(Ⅰ,Ⅱ,Ⅲ,Ⅳ和V组),分别饲以粗蛋白水平为12%,14%,16%,18%和20%的等消化能(10.0MJ／kg)、等粗纤维(12%)日粮,通过测定动物生长性能、血清生化指标和免疫学指标、盲肠发酵参数及相关激素和酶的基因表达峰度,观察日粮粗蛋白水平对兔生长性能和免疫功能影响,确定生长期WHBE兔对粗蛋白的生长需要和免疫需要,并探讨相关机理。另外选取30只WHBE幼兔,并随机分为5组,分别饲喂5种不同粗蛋白水平(12,14,16,18和20%)的等消化能和等粗纤维日粮,进行氮代谢试验,估算生长期WHBE兔的维持氮需要量及净蛋白利用率。试验结果表明：仔兔日增重与粗蛋白水平间存在回归关系(Y=-0.2325X2+7.6371X-40.335,R2=0.6237),经分析,当日粮粗蛋白水平为16.4%时,动物生长性能最佳。日粮粗蛋白水平对动物的采食量无显著影响,但对料重比影响显著。血清碱性磷酸酶的活力以16%粗蛋白组最高,但组间差异不显著；而血清胰岛素样生长因子-Ⅰ(IGF-Ⅰ)的含量以Ⅲ、Ⅳ组为高,与Ⅰ、Ⅱ、Ⅴ组差异显著(P<0.01,P<0.05)。Ⅱ-Ⅳ组的血清总蛋白的含量明显高于Ⅰ组(P<0.05),各组尿素氮含量随蛋白水平提高而显著上升(P<0.01,P<0.05)。在免疫学指标上,随日粮粗蛋白水平提高,各组仔兔的自然杀伤细胞活力、脾脏指数和胸腺指数呈上升趋势,均以V组最高,V组的自然杀伤细胞活力、脾脏指数分别比Ⅰ组提高36%和31%(P<0.05),而V组的胸腺指数比Ⅱ组提高41%(P<0.05)。脂多糖诱导淋巴细胞增殖的刺激指数(LPS SI)以Ⅳ组最高,分别与Ⅰ、Ⅱ组差异显著(P<0.01,P<0.05),而与Ⅲ、Ⅴ组无显著差异。而刀豆素蛋白刺激指数(ConA SI)以Ⅲ组最高,Ⅳ、Ⅴ组次之,Ⅲ-Ⅴ组间无显著差异,但Ⅲ-Ⅴ组分别与Ⅰ、Ⅱ组差异显著(P<0.01,P<0.05)。白介素-1β(IL-1β)的浓度以Ⅳ组最高,显著高于Ⅰ组(P<0.05),免疫球蛋白含量也以Ⅲ、Ⅳ组为高,但各组间差异不显著(P>0.05)。血清NO含量和SOD活性以Ⅲ、Ⅳ组为高,与Ⅰ组差异显著(P<0.01,P<0.05),而MDA含量变化则与SOD活性的变化趋势相反。从盲肠发酵参数看,氨态氮和微生物蛋白的浓度也以16%粗蛋白组最高,且Ⅲ组微生物蛋白浓度显著高于其他4组(P<0.01)。肝脏IGF-1 mRNA的表达峰度以Ⅲ、Ⅳ组最高,与Ⅰ组差异显著。PEPCK mRNA则在16%粗蛋白水平下的表达量最大,并显著高于12%粗蛋白组。从氮代谢试验结果分析可得：生长期WHBE兔平均净蛋白利用率为445g／kg,维持氮需要量为491mg／kg BW0.75。2.日粮粗蛋白水平对育成期WHBE兔生长性能、氮代谢、免疫功能、盲肠微生物环境及肝脏功能基因表达的影响选用90日龄WHBE兔60只,进行为期6周育成期饲养试验。动物分组和采食日粮与生长期试验相同。除测定了生长期饲养试验的相关指标外,育成期试验还进行了抗卵清白蛋白抗体生成实验,以及盲肠微生物区系的DGGE分析。另取30只成年WHBE兔,进行与生长期动物相同的氮代谢试验,随后选用20只16周龄WHBE兔进行无氮日粮代谢试验,即将试兔随机分为5组,分别按55,45,35,25和0g／d饲喂无氮日粮。结果表明：14%-20%粗蛋白水平下,兔子的日增重均显著高于12%粗蛋白水平组(P<0.01),日增重与粗蛋白水平间存在回归关系(y=-0.1147x2+4.1556x-13.154,R2=0.5851),当粗蛋白水平达到18.1%时,动物生长性能最佳。日粮粗蛋白水平对动物的采食量无显著影响。各组兔碱性磷酸酶的活力均低于相应的生长期动物,但IGF-Ⅰ的浓度则高于生长期动物,且IGF-Ⅰ浓度以16%粗蛋白组最高,与12%粗蛋白组差异显著。育成兔的其他各项生化指标的变化趋势基本与生长兔相同。在免疫学指标上,各项免疫学参数随粗蛋白水平的升高均有先上升后下降的趋势,其中,自然杀伤细胞活力、LPS SI、和IL-1β等指标以16%粗蛋白组最高,12%,14%粗蛋白组为低。脾脏指数和胸腺指数都以18%粗蛋白水平组最高,与12%,14%粗蛋白组差异显著(P<0.05),16%和18%粗蛋白组的ConA SI显著高于14%和20%粗蛋白组(P<0.05)。免疫球蛋白含量以16%粗蛋白水平组为高,但各组间差异不显著(P>0.05)。抗卵清白蛋白的抗体生成率也以16%粗蛋白水平组最高,且与12%粗蛋白组差异显著(P<0.05)。血清NO含量和SOD活性的变化趋势与免疫指标的变化一致。从盲肠发酵参数看,18%粗蛋白水平下,氨态氮和微生物蛋白和总挥发性脂肪酸浓度最高,而pH值最低,且盲肠微生物DGGE分析结果显示,在该蛋白水平下,动物盲肠微生物多样性程度最大。WHBE兔在育成期的肝脏IGF-ⅠmRNA和PEPCKmRNA的表达特点与生长期一致。氮代谢试验结果表明,育成期WHBE兔平均净蛋白利用率(NPU)为496g／kg,比生长期增高12%；维持氮需要量则为480 mg／kgBW0.75/d,此结果与通过无氮日粮代谢试验估算结果(486 mg/kgBW0.75/d)基本一致。证实了氮代谢试验结果的准确性。试验结果同时发现,无氮日粮采食量越少,氮排泄量越多,提示当WHBE兔采食氮很低时,可能会通过体蛋白分解代谢来满足机体维持氮的需要。3.成年WHBE兔日粮适宜粗蛋白与限制性氨基酸组合的研究选用100日龄WHBE兔36只,将日粮粗蛋白水平(12%和16%)和限制性氨基酸按满足NRC标准的百分比(80%,100%和120%)不同随机分为6组(12-80、12-100、12-120、16-80、16-100和16-120),分别喂以6种不同粗蛋白和氨基酸组合的日粮,其中前3组日粮粗蛋白水平为12%,赖氨酸含量分别为0.72%,0.9%和1.08%,含硫氨基酸含量分别为0.52%,0.65%和0.78%；后3组日粮粗蛋白水平为16%,赖氨酸与含硫氨基酸组成分别与相应的前3组相同。通过饲养试验、氮代谢试验和免疫功能的测定,观察这6种粗蛋白和氨基酸组合日粮对WHBE兔的氮代谢和免疫功能的影响。试验结果表明,动物的日增重和氮利用率以12-120、16-80组最高,12-80、16-120组最低,12-120、16-80组分别与12-80、16-120差异显著(P<0.01),但12-120组的总氮排泄量明显低于16-80组(P<0.01)。12-120、16-80组动物的脾脏指数和胸腺指数也都高于其他4组,16-80组动物的脾脏指数分别与12-80、12-100、16-100和16-120组差异显著(P<0.01)。抗卵清白蛋白抗体阳性的动物数以12-120组最多,分别与12-100、16-100和16-120组差异显著(P<0.05)。血清总蛋白含量随日粮氨基酸水平提高有上升趋势,血清尿素氮含量以12-100和12-120组最高,与其他各组差异显著(P<0.01,P<0.05)。以上结果表明,12-120组和16-80组的日粮粗蛋白和氨基酸组合比较适宜,考虑到16-80组氮排放量较高,故以12-120组最佳。综上所述,生长期和育成期WHBE兔的维持氮需要量为分别为491和480mg/kg BW0.75 /d,而净蛋白利用率分别为445和496g／kg；生长期WHBE兔适宜生长的日粮粗蛋白水平为16.4%,14%-20%日粮粗蛋白水平均能满足育成期WHBE兔最大生长需要,其最佳点在18.1%。保证生长期和育成期WHBE兔处于最佳免疫状态的日粮粗蛋白水平均在16-18%之间。在这一范围,机体抗氧化能力最强,盲肠微生物数量和种类最多,发酵作用也最强,且肝脏IGF-ⅠmRNA和PEPCKmRNA的最高表达峰度也处于这一适宜水平。此外,适当增加合成氨基酸的比例可降低日粮粗蛋白水平,不仅不影响WHBE兔的生长和免疫,而且可减少氮的排放。更多还原

【Abstract】 A new strain of laboratory rabbit (WHBE rabbit), originated from Japanese White rabbit but characterized by its black eye and higher antibody titer, was used in this study to assess the characteristics of protein requirement. The crude protein requirements for maintenance, growth and maintaing maximal immune function during growing and developing periods were evaluated via feeding trial, nitrogen (N) balance trial and immune function test, and some mechanisms were explained in terms of digestive (ceacal fermentation and bacterial community) and metabolism (functional gene expression)facets. Furthermore, the optimal proportions of limited amino acids in diets were also studied.1. Effects of dietary crude levels on growth performance, N metabolism, immune function, ceacal fermentation, and hepatic functional gene expression in growing WHBE rabbitsSixty weaned WHBE rabbits, at the age of 40 days, were divided into 5 groups (Ⅰ,Ⅱ,Ⅲ,ⅣandⅤ) according to dietary crude protein levels(12%,14%,16%,18% and 20%, respectively). Five diets had similar the digestible energy (10.0MJ/kg) and crude fibre(12%) contents. Feed intake and growth performance were measure during the six-week feeding trial and the blood biochemical indices, immunological parameters, ceacal fermentation, and expression abundant of hepatic IGF-ⅠmRNA and PEPCK-C mRNA were determined when the trial was finished. After feeding trial, another 30 young WHBE rabbits at the age of 10 weeks were used in a N balance trial.The rabbits were divided into five equal groups and fed the same diets as feeding trial, correspondingly. The net protein utilization and the N requirements for maintenance were estimated from the relationship between the N retention and intake.All animals had similar feed intake. Protein levels had significant effects on daily gain (P< 0.05), and the relationship between dairy gain (Y) and dietary protein level (X) was calculated as Y=-0.2325X2+7.6371X-40.335(R2=0.6084), from which the optimal diary gain can be estimated to be at 16.4% CP level.Serum total protein and albumin in 14-20% CP groups were higher than group I. The contents of Urea-N were increased with the increasing dietary protein level, but it was suitable in the third group. The activity of alkaline phosphatase (ALP) was the highest in groupⅢ, but no significant difference was found. At 16 or 18% CP level, The circulation IGF-Ⅰconcentrations were higher than that of at 12,14 and 20%, respectively (P<0.01, P<0.05). The natural killer (NK) cell activity, spleen indices and thymus indices had a trend to increase with the increasing levels of CP, being highest at 20% CP level. The above three indices (NK cell activity, spleen and thymus indices) in group V were significantly increased by 36,31 and 41%, compared with that in groupⅠ,ⅠandⅡ, correspondingly. The lymphocyte proliferation assay showed that the stimulation indices (SI) induced by both T-cell mitogen concanavalin A (ConA) and B-cell mitogen lipopolysaccharides (LPS) were higher in groupⅢ,ⅣandⅤthan that in groupⅠandⅡ, while no significant difference was found among groupⅢ,ⅣandⅤ. The concentration of IL-1βwas highest in groupⅣ, and had significant difference, compared with groupⅠ(P<0.05). The contents of immune globulin A, M and G (IgA, IgM and IgG) had the same trend as the change of IL-1β, but no significant difference was found. The activity of superoxide dismutase (SOD) and concentration of nitric oxide (NO) in groupⅢandⅣwere significantly higher than that of in groupⅠ.However, the change of maleic dialdehyde (MDA) concentration was contrary to that of SOD. The CP levels had significant effects on caecal fermentation, with highest concentrations of ammonia N (NH3-N) and microbial protein in groupⅢ. Expression of hepatic IGF-I was consistent with that of circulating IGF-Ⅰ.Expression abundant of hepatic PEPCK-C mRNA was also highest at 16% CP level.The result of N balance trial showed that average net protein utilization was 445g/kg, and the estimated N requirement for maintenance was 491 mg/kg BW0.75 per day in growing WHBE rabbits.2. Effects of dietary crude levels on growth performance, N metabolism, immune function, ceacal fermentation and bacterial community, and hepatic functional gene expression in developing WHBE rabbitsSixty 90-day WHBE rabbits were carried out in a six-week feeding trial to observe the effects of dietary crude protein levels on performance of developing WHBE rabbits. The experimental design and diets were the same as that of growing rabbits described above. Besides all parameters detected in growing period, antibody response to ovalbumin was tested and the ceacal bacterial community was analized by DGGE technology in this trial.After feeding trial,another 30 adult WHBE rabbit were used in N balance trial as described above. And then, twenty rabbits at the age of 16 weeks were fed a N-free ration to estimate the requirements of N for maintenance. The rabbits were divided into five groups and offered the N-free ration at levels of 55,45,35,25 and 0 g per day, respectively. Daily gain in groupⅡ-Ⅴwere all higher than in group I (P<0.01),the optimal protein level (18.1%) can be obtained from the regression equation (y=-0.1147x2+4.1556x-13.154, R2=0.5851).The changes of some biochemical indices in developing period had the similar trend with that of in growing period. Compared with growing period, the ALP activity in developing period sharply decreased, while the circulation IGF-Ⅰconcentrations increased, with highest IGF-Ⅰconcentrations at 16%CP level in developing period. The NK cell activity, SI induced by LPS, and IL-1βcontents were also highest at 16% CP level, but spleen and thymus indices had a maximam value at 18% CP level, differed significantly with groupⅠorⅡ. SI induced by ConA was higher at 16 and 18% CP level than that at 14 and 20%(P<0.05). The changes of IgA, IgM and IgG were the same as growing period. The positive rate of antibody response to ovalbumin was also highest in groupⅢ, differed significantly from group I (P<0.05). The change with different protein levels in the parameters of SOD, NO and MDA were consistent with that of immune status. The protein levels had strong effects on caecal fermentation and microbial community, with highest concentrations of NH3-N and microbial protein, VFA contents and variety of microbial community in groupⅣ. Expression of hepatic IGF-I and PEPCK-C mRNA were also consistent with that of growing period. Average net protein utilization during developing period was 496g/kg, higher by 12% compared with growing period. The estimated N requirement for maintenance in N balance trial was 480 mg/kg BW0.75 per day, in agreement with the result from the trial in the N-free ration (486 mg/kgBW0.75 per day). The lower intake of N-free ration resulted in more N excretion, suggesting that protein catabolism may occur in the body of rabbit to meet maintenance requirements for N when the dietary N intake was very low.3. Optimal combinations of crude protein and amino acids in developing WHBE rabbitsThe effects of diets with different combinations of crude protein and amino acids on immune function and nitrogen metabolism were studied by nitrogen metabolism trial and immune function test. Thirty six adult WHBE rabbits(100 d old) were randomly divided into 6 groups(12-80, 12-100,12-120,16-80,16-100 and 16-120) according to 6 diets with different combination of CP-AA. The crude protein level in groups 12-80,12-100,12-120 was 12%, while that in group 16-80,16-100 and 16-120 was 16%. From group 12-80 to 12-120 or group 16-80 to 16-120, contents of lysine were 0.72,0.9 and 1.08%, and contents of methionine plus cystine were 0.52, 0.65 and 0.78%, respectively. Both daily weight gain and total N utilization were significant higher in group 12-120 and 16-80 than those in group 12-80 or 16-80 (P<0.01).While compared with the group 16-80, the content of total N excretion in group 12-120 significantly decreased from 2.10 to 1.59 g/d (P< 0.01).Combinations of crude protein and amino acids had significant effects on immune function, with the highest level of antibody response to ovalbumin in group 12-120, which was significant higher than that in group 12-100,16-100 and 16-120 (P<0.05). Both indices of spleen and thymus in group 12-80 and 160-100 were higher than those in the other 4 groups, although there were no significant differences among them except the spleen index in group 16-80.The serum total protein was increased with the increasing amino acids content. The blood urea N was highest in groups 16-100 and 16-120 (5.1mmol/l), which should significant differences from the other 4 groups (P<0.01,P<0.05).The suitable combinations of crude protein and amino acids were the diets in group 12-120 and 16-80, but as the total N excretion concerned, group 12-120 was optimal.In summary, the requirements of N for maintenance are 491 and 480 mg/kg BW0.75 for growing and developing WHBE rabbits, respectively. The net protein utilization during developing period (496g/kg) is higer than that during growing period (445g/kg). The optimal CP levels to assure the maximal growth performance of WHBE rabbit are 16.4 and 18.1% in growing and developing periods, respectively, while the range of 16-18% CP level are suitable to satisfy the need for immune response in either growing or developing periods. The optimal ceacal environment, the maximal antioxidant capacity, and the highest expression of hepatic IGF-ⅠmRNA and PEPCK-C mRNA may be obtained at the suitable CP levels. However, reducing CP level but increasing amino acids contents is effective to enhance immune function and lessen environment pollution in rabbit feeding procedure.更多还原