Effects Of Dietary Protein Levels On Growth, Development And Hepatic Gene Expression Of Early Weaned Lambs

This study was conducted to investigate the effects of dietary protein levels on the growth and development and hepatic genes expression in early-weaned Hu twin lambs. Twin lambs were selected as the experimental subject and paired experiment design adopted. Lambs were weaned at 15 d of age, normal protein level and low protein level diets were fed from 15 to 60 d of age, but all lambs consumed the same protein level diets from 61 to 90 days. The growth performance, slaughter performance, development of organs, nutrient digestion variation were determined to evaluate the influence of low protein rations on the growth development, and genes expression in liver by RNA-seq to explore the regulatory mechanism of protein on young lambs growth and development. The outcome of this study was expected to promote the development of young animal cultivation. The present study covered three trails as follows: Experiment 1: Effects of dietary protein levels on growth and development and meat quality of early-weaned male Hu lambsThis study aimed to investigate the effects of dietary protein levels on growth performance, slaughter performance, non-carcass offal development and meat quality of Hu twin lamb. Sixteen pairs of Hu lambs weaned at 15 d after birth were selected and randomly allotted to two treatments. One group was fed a milk replacer and starter with normal protein level(25% and 21%, respectively; NPL), and the other group with equal energy but low protein level(19% and 15%, LPL). The milk replacer was fed from weaning to 60 d of age. LPL group feed the starter with 21% protein from the age of 60 to 90 d. Four paired twins were slaughtered on d 60 and 90 for evaluating carcass quality. The results showed as follows: body weight of LPL group was lower than that in NPL group on d 60 and 90(P<0.05), but no obvious difference was found in feed intake(P>0.05). Feed conversion ratio in the LPL group was significant higher than that of NPL group from d 15 to 60, but was lower than NPL group from d 60 to 90(P<0.05); In addition, average daily gain and feed conversation ratio increased with the age of lambs. Live body weight, empty body weight, carcass weight and GR values were significant higher in NPL group than those in LPL group(P<0.05), while no significant differences were found in dressing percentage(P>0.05) on d 60 or 90; Kidney and spleen weights in LPL group were significant lower than those in NPL group on d 60(P<0.05) and just kidney weight was lower on d 90(P<0.05), but no significant differences were observed in other internal organ indices(P>0.05); Compared with the results in 60 days, all the weight of internal organs were increased, whereas its percentage to the live body weight were remained stable. On d 60, weight of rumen and small intestinal in LPL group were significantly lower in LPL group than those in NPL group(P<0.05), and weight of abomasum and it percentage of total complex stomach weight were significantly lower compared with those in LPL group(P<0.05); however, weight of rumen, reticulum and small intestinal were lower than that in NPL group on d 90(P<0.05). However, the percentage of rumen to complex stomach and large intestine to live body weight were risen from 60 to 90 days, and the percentage of abomasum to complex stomach and small intestine to live body decreased. Drip loss, cooking rate and water holding in LPL group were significantly worse than those in NPL group(P<0.05). However, drip loss and water holding in LPL group were significantly lower than that in NPL group after 30 days of protein recovery(P<0.05). The a* value of meat color, cooking rate and water holding also increased when compared the results of 90 to 60 days, but drip loss decreased. Experiment 2: Effects of dietary protein levels on nutrient digestion and metabolism, rumen fermentation and serum parameters of early-weaned male Hu lambsThis study aimed to investigate the effects of dietary protein level on nutrients apparent digestibility and metabolism, rumen fermentation and serum indices of male Hu lambs. The experiment design and animals were the same as Experiment 1. Four paired twin lambs were selected to detect the nutrient digestion at 50-60 and 80-90 days, blood samples collected from jugular vein at 60 and 90 d of age before the lambs slaughtered, and GLU, BUN, GH, IGF-Ⅰand so on, while rumen fluid sampling after the slaughter to measure NH3-N and volatile fatty acids(VFAs).The results showed as follows: Crude protein apparent digestibility and retain nitrogen in LPL group were significantly lower than that in NPL group(P<0.05), but nitrogen metabolism rate higher than that in NPL group from 50 to 60 d of age(P<0.05). Crude fat apparent digestibility and nitrogen metabolism rate of LPL group were obviously higher than NPL group(P<0.05); Digestibility of NDF and ADF increased from 60 to 90 days, but the nitrogen metabolism decreased sharply. At 60 days, p H, NH3-N, the percentage of acetic, propionic to total volatile fatty acids(TVFA) and acetic/propionic in the NPL group were higher than that in LPL group(P<0.05), but no significant differences were found among the other VFAs between NPL and LPL group(P>0.05). After intaking 30 days of normal protein level ration at 90 days, the ratio of propionic to TVFA in NPL group lower than LPL group(P<0.05), however, the ratio of isobutyric and valeric acid to TVFA and acetic/propionic were significant higher than LPL group(P<0.05). Moreover, no significant difference on p H, NH3-N and acetic were found in the two groups(P>0.05); the concentration of NH3-N went up from 60 to 90 days. At 60 d of age, blood urea nitrogen(BUN) concentration and insulin growth factor-I in LPL group lower than that in NPL group(P<0.05), but the activity of alkaline phosphatase(APL) and glutamic-pyruvictransaminase(ALT) were much higher than NPL group(P<0.05). At 90 days, APL and ALT activity and growth hormone(GH) level higher in LPL group when compared with NPL group(P<0.05). The indices of serum were greatly various with the change of lamb’s age, and BUN increased, but GH and IGF-I have a tendency to decline. Experiment 3: Effects of dietary protein levels on hepatic genes expression in early-weaned male Hu lambsIn this study, liver transcriptional response of protein deficiency and compensation on the early development of Hu lambs was investigated in order to reveal the mechanisms that regulation of protein on young animal. The experiment design and animals were the same as Experiment 1. Liver samples were collected at 60(LPL60, NPL60) and 90(LPL90, NPL90) days to provide comprehensive transcriptional-level data. Analysis of different expression genes(DEGs) of this study revealed that low protein level affected a large number of differentially expressed genes(DEGs: 302, up-regulated: 167, down-regulated 135) from the early-weaned(day 15) lambs to 60 days age. The expression level of the majority of DEGs recovered to a normal levels following protein compensation for 30 days(DEGs: 12, up-regulated: 8, down-regulated 4). With the effects of age-by-protein, 2802 DEGs were found compared LPL90 with LPL60(up-regulated: 1186, down-regulated 1616), while only 8 DEGs between NPL90 and NPL60(up-regulated: 4, down-regulated 4). According to the Gene Ontology(GO) annotation, protein deficiency affected the development of vessels and cells, cell proliferation, and the growth of the animals in the LPL60 and NPL60 groups(P<0.05) but not in the LPL90 and NPL90 groups(P>0.05). The gene expression profiles of the NPL60 and NPL90 groups showed little variation, while the following showed marked changes in the LPL90 and LPL60 groups: cellular protein metabolic and modification processes, protein metabolic and modification processes, intracellular and extracellular regions of protein binding, and cytoplasm and organelles(P<0.05). The analysis of the Kyoto Encyclopedia of Genes and Genomes(KEGG) database suggested that genes which involved in the cytokine pathway and cytochrome P450 and chemical carcinogenesis, were significantly altered in the LPl60 and NPL60 groups(P<0.05). It also pointed to down-regulation of genes governing glutathione, lipid, and amino metabolism pathways(P<0.05). The immune function and anti-infection capacity of the NPL90 group were better than those in the NPL60 group(P<0.05). Compared LPL90 with LPL60, the results also pointed to up-regulation of genes involved in glutathione, lipid, and amino metabolism pathways, in addition to those governing the cytochrome P450 pathway were significantly improved(P<0.05). What’s more, we also found that 18 pathways which related to diseases were regulated by two DEGs(DQA1 and DQA2) between the LPL90 and NPL90 groupsIn conclusion, protein deficiency of pre-weaned Hu lambs depressed the growth and slaughter performance, hindered the development of non-carcass offal, postponed the mature of gastrointestinal tract system and meat quality, affected the rumen fermentation parameters and reduced the nutrient digestion, in consequence caused a negative influence on the early stage growth and development of lamb. What’s more, low protein level exerts large effects on the gene expression profiles of early-weaned lambs, and genes related to growing development and immune function pathways also affected significantly. Though the growth and development of lamb was improved after 30 days of feeding normal protein level ration, it cannot rehabilitate to the state of well-balanced lamb and such compensation does not eliminate the risk of disease in the future.