Protein Requirement And Approach To The Related Mechanisms In A New Strain Of Laboratory Rabbit

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.