Effects Of Chinese Hebal Feed Additives On Meat Quality, Flavor Of Chinese Merino(Xinjiang Junken Type)

In this paper, three Chinese herb feed additives designed by ourselves were added respectively into basal diet of Chinese Merino(Xinjiang Junken Type).And then the meat moisture,crude protein,intramuscular fat(IMF),ash,muscle fiber diameter,muscle fiberdensity,the contents of IMP and its correlative metabolites, the fatty acid composition and content,the relative expression level of meat quality traits candidate genes,the SNPs at the exon 2 of the H-FABP gene were all detected to analyze the effects of Chinese hebal feed additives on meat quality, flavor of Chinese Merino(Xinjiang Junken Type).According to the results of the above, Chinese herb feed additives Ⅲ was considered the most effective one which could improve meat quality and flavor of Chinese Merino(Xinjiang Junken Type). In a follow-up experiment,we established culture model in vitro of sheep preadipocyte and to investigate the effects of Chinese herb feed additive Ⅲ on the proliferation and differentiation of preadipocyte.In order to investigate the safety of Chinese herb feed additive III for improving the meat quality and flavour of sheep,the acute toxicity test and 30 d feeding test were carried out in mice.The main contents of the experiment and the results are as follows.1. 32 healthy 10-month-old Chinese Merino(Xinjiang Junken Type) were randomly divided into 4groups.The groups 1,2 and 3 were fed basal diets added with 1% Chinese herb feed additiveⅠ,1% Chinese herb feed additiveⅡ and 1% Chinese herb feed additive Ⅲ,respectively.The group 4 was control group which was given basal diet.After 60 days of feeding,the meat quality indexes were determined.The results showed that three Chinese herb feed additives could increase the content of intramuscular fat and the muscle fiber density in various degrees,so as to improve tenderness and juiciness of mutton.The results demonstrated that the Chinese herb feed additive Ⅲ can significantly improve the quality of mutton.2. After 60 days of feeding, the contents of ATP,ADP,AMP,IMP,INO,HYP in mutton were detected by HPLC method.The results showed that ATP was not detected in all of the samples.The contents of ADP and AMP were very low, and there was no significant intergroup difference(P>0.05).Three Chinese herb feed additives all can improved the contents of IMP and its correlative metabolites in different degrees.The contents of IMP、INO and IMPc of meat added with 1% Chinese herb feed additive Ⅲ were extremely significantly higher than control group(P<0.01),and significantly higher than the group ofPrescriptionⅡand PrescriptionⅠ(P<0.05).These results demonstrated that the Chinese herb feed additiveⅢ can significantly improve the flavor of the meat of Chinese Merino(Xinjiang Junken Type).3. After 60 days of feeding, the fatty acids composition and content of the mutton were analyzed by gas chromatography, methods according to compare with relative retention time of the mixed fatty acid methyl ester standard and relative percentage content of each fatty acid by the peak area of each fatty acid,the analogy and quantitative analysis method were adopted for qualitative and quantitative evaluation.The results showed that 14 fatty acids were found, in which there were 4 saturated fatty acids(SFA), 5monounsaturated fatty acids(MUFA), and 5 polyunsaturated fatty acids(PUFA). The major fatty acids were C18:ln9c, C18:0, C16:0 and C17:1, and these four FAs occupied 77.201~ 80.453% of the total FA.Mutton odour and total saturated fatty acid decreased, useful unsaturated fatty acids on the heath component content and proportion increased under treatments. By contrast, improving nutritional value of the Chinese herb feed additives Ⅱ treated is significantly higher than that of other additives and Chinese herb feed additives Ⅲ can not only efficiently weak mutton odour negative impact, but also make more progress on redressing imbalance on the FA side.4. Experimental animals will be slaughtered in the end of the feeding examination(after 60 days),real time fluorescent quantitative PCR was used to quantize the expression abundance of IMF( H-FABP,A-FABP, LPL, HSL, FAS and UCP 3), IMP( ADSL) and tenderness( Calpain I and CAST) candidate genes mRNA in longissimus dorsi muscle tissues. With former research results of meat quality and flavor indexes to analyze the correlation with the relative expression levels of each candidate gene in order to explore the influence of Chinese herb feed additives on them.The results showed that compared with any candidate genes, Chinese herb feed additives had no significant influence on LPL、UCP3 and CAST. The Chinese herb feed additivesⅡcontributes to expression of H-FABP and A-FABP, H-FABP、FAS、ADSL and calpain I are promted by additives Ⅲ while it leads to repression of HSL expression. The expression of H-FABP、LPL and FAS showed positive relation to IMF content, negatively correlated with HSL and UCP3, A-FABP was not related. The expression of ADSL was positively correlated with IMP and IMPc. Calpain I was negatively correlated with muscle fiber diameter but positively correlated with muscle fiber density expression. The muscle fiber diameter was not related with expression of CAST, negatively correlated with muscle fiber density expression. These results suggested that the Chinese herb feed additives used in this study are effective in up-regulating lipogenic,flavor and tenderness candidate genes expression, repressing lipolytic-related gene expression, they are not only increase IMF and umami substances content, but also improve the tenderness of muscle tissue.Among them, additives Ⅲ is the most effective one.5. Intramuscular fat(IMF)content,muscle fiber diameter and muscle fiber density of 150 Chinese Merino(Xinjiang Junken Type) were measured, and polymerase chain reaction-single strand conformation polymorphism(PCR-SSCP) technique was applied to test the SNPs at the exon 2 of the H-FABP gene.The results showed that the exon 2 of H-FABP gene displayed AA、AB and BB genotypes.There were mutation of 939(A/G) and deletion of C both in AA and BB genotypes,and 789(T/C) in BB genotype,which led to valine convert to alanine.The BB genotype was advantageous genotype of IMF and there were significant difference between BB and AB(P<0.05), and extremely significant difference(P<0.01)between BB and AA.The correlation of BB genotype and muscle fiber diameter was negative.These results suggested that the exon 2 of H-FABP gene of Chinese Merino(Xinjiang Junken Type) showsed polymorphism and that H-FABP gene might be a major gene or a major gene-linked gene for meat quality of Chinese Merino(Xinjiang Junken Type).6. The adipose tissue of Chinese Merino(Xinjiang Junken Type) was used to isolate and culture preadipocyte by means of explant technique and monolayer culture method.And then the cultured cells were detected by their morphological changes,growth curve and extracting stained intracytoplasmic lipid with oil red O.The influence of Chinese herb feed additive Ⅲ on proliferation of sheep preadipocytes was detected by MTT method, the effect on lipid accumulation was determined by Oil Red O staining.The results showed that the cells cultured by means of explant technique and monolayer culture method were highly homogeneous and proliferative,and cells obtained from monolayer culture are more than from explant technique in a short period of time.The Chinese herb feed additive Ⅲ could stimulate sheep preadipocyte proliferation and differentiation.7. In acute toxicity test,50 mice were randomly divided into 4 treatment groups and control group,the treatment groups were administered drug by intragastric infusion at single dose of 50,75,100,150mg/(10 g BW)respectively,and then mice poisoning symptoms and death cases were observed and the median lethal dose were calculated for 14 days.In order to estimate the maximum dosage,40 mice were randomly divided into two groups,the Chinese herb feed additive III was administered intragastrically at a maximum concentration(1.0 g/mL)and maximum capacity[0.4 mL/(10 g BW)]with single dose in thetreatment group,0.9% saline was used in control group;After being administered,the mice were observed for 14 days.In 30 days feeding test,160 mice were randomly divided into 3 treatment groups(high,middle and low dose group)and control group,drug was administered orally at does of 30,15 and 7.5 mg/(10 g BW·d)respectively,the mice in the normal control group were oraled with the same volume of saline.After the treatment of 30 days,body weight,blood physiological index,biochemical index and organ coefficient of all groups were measured and compared.The results showed that there was no any poisoning and death case in each group which leaded to cannot calculate LD50.MTD was over 400 m g/(10 g BW)in mice showed that this Chinese traditional medicine prescription was non-toxic.There were no significantly different in routine blood physiological indexes,biochemical indexes and organ coefficient among all groups(P>0.05),and no abnormal alteration in organ observation.Body weight of 3 drug-treated groups were increased significantly and there was significantly different between middle dose group and control group(P<0.05),which suggested that the Chinese herb feed additive III could increased appetite and promoted animal growth.These results suggested that Chinese herb feed additive III was safe, effective and non-toxic side effects in clinical applications.