Digestion And Metabolism Of Nutrients In Duolang Sheep By Adding Grape Seed In The Diet And The Influence Of Rumen Flora

In order to alleviate the current situation of shortage of forage resources in Xinjiang and make more reasonable use of grape seed resources,this study analyzed the effects of grape seed on rumen fermentation indexes and nutrient degradation rate of Duolang sheep in vitro experiments.On the basis of in vitro experiments,animal experiments were conducted to analyze the nutrient digestion and metabolism,rumen fermentation parameters,digestive enzyme activity concentration,blood indexes and the influence of rumen flora structure of different proportions of grape seed added to Duolang sheep diet.The study is to explore the suitable range of grape seed added to the diet,and providing a theoretical basis for the rational use of grape seed resources.Test ?: Different supplemental ratios of grape seed to basal diet were applied as 0%,4%,8%,12%,16%,20%,24% and 28%,resulting in 8 experimental substrates.In vitro gas production monitoring system was used to continuously record the gas production(GP)for 48 h,and collect the culture fluids at different incubation time to measure the p H,the concentration of ammonia nitrogen(NH3-N)and bacterial protein(MCP).Meantime,nylon bags containing different experimental substrates were used to measure in vitro degradability,and the corresponding culture fluid was collected to measure the p H of ruminal and the concentration of NH3-N.As a result,with the increase of grape seed inclusion level,p H in culture fluid increased.Potential GP in 16% ratio group was higher than that of 0% adding group(P<0.05).The concentration of MCP in 0%,4% and 8% adding groups were higher than other adding groups(P<0.05).The concentration of NH3-N in 16% group was lower than that in 0% group(P<0.05).Test ?: The test selected 15 healthy Duolang sheep emasculated of 1.5-year-old and body weight41.0±1.0 kg,with ruminal cannulas.The sheep were randomly divided into 5 groups and fed with different proportions of 0%(D1),4%(D2),8%(D3),12%(D4)and 16%(D5)total mixed diet.The nutrient apparent digestibility(DM,OM,N,EE,NDF and ADF)and energy metabolic parameters of Duolang sheep were calculated by collecting total fecal and urine.The results showed that: The apparent digestibility of DM and OM in D5 group was significantly lower than D1 group(P < 0.05);the apparent digestibility ofNDF in D5 group was significantly lower than D1 group(P<0.05);the nitrogen retention rate in D5 group was significantly lower than D3 and D4 group(P<0.05);the ADF and EE apparent digestibility of D4 group was the largest,but there was no significant difference among the five groups(P>0.05).The change of total energy digestibility and total energy metabolic were the same(D2>D1>D4>D3>D5),and the rate of digestive energy metabolic was D1>D4>D2>D5>D3,but there was no significant difference(P>0.05).The addition of 16% grape seed in the diet is not conducive to the digestion of nutrients in Duolang sheep.Test ?: On the basis of the diet of trial 2,the p H,NH3-N,volatile fatty acids(VFA)and digestive enzyme activity of rumen fluid were measured by time,and venous blood was collected before morning feeding to determine the blood index.The results showed that:(1)The dietary level had no effect on the NH3-N concentration at each time point after feeding(P>0.05),but it could significantly increase rumen fluid p H(P<0.05).The relative content of total volatile fatty acids(TVFA)in rumen fluid decreased with the increase of grape seed ratio in diet.The relative content of TVFA in low rumen fluid in D4,D5 group(P<0.05).The proportion of acetic acid,propionic acid,butyric acid,valerate and isovalerate in the TVFA in rumen solution was changed,and the value of acetic acid/propionic acid in D4,D5 group was significantly less than D1,D2,D3(P<0.05).(2)the Diet in D3 and D4 groups increased rumen fluid ?-amylase and proteolytic enzyme activities(P<0.05).(3)16% of grape seed increased the number of white blood cells and neutrophils in animal blood(P<0.05).The serum content of grape seed creatinine was significantly higher than that of 0%~8%(P<0.05),and the antioxidant capacity of blood increased with the addition of grape seed in diet.The addition of 16% grape seed significantly increased the superoxide dismutase,malondialdehyde and total antioxidant capacity(P<0.05).Test ?:On the basis of the diet of trial 2,the rumen chyme was collected before morning feeding,and high throughput sequencing was carried out by illuminam miseq sequencing platform.The results showed that:(1)There was no significant effect on the abundance of rumen bacteria by adding grape seed to the diet.16% of grape seed increased the abundance of rumen fungus(P<0.05).(2)The absolute dominant genera at the level of the bacterial phyla were Bacteroides and Firmicutes,abundances of the two phyla were 89.56%~92.85%.The absolute dominant genus at the bacterial genus level is prevoella-1.The abundance of rumenella-1 and christensenella R-7 decreased with the increase of grape seed ratio in diet(P>0.05).(3)The absolute dominant phyla of rumen fungus in Duolang sheep were the Neocallimastigomycota and the Ascomycota.A total abundance of two phyla in rumen fungus was90.18%~96.13%,and the abundance of Neocallimastigomycota and Ascomycota in the D2 group was13.92%,higher than that of the D1 group(P<0.05)and 33.69%(P<0.05).The absolute dominant bacteria at the fungal genus level were Anaeromyces.The lowest abundance of Anaeromyces in group D5 was15.93% and 68.29% smaller than that in group D1.The genus Piromyces decreased first with the addition of grape seed in the diet,then increases.The D3 group was lowest 9.68%,79.68% lower than that of control group.In D3 group unclassified_c__Sordariomycetes,unclassified_p_Ascomycota and Latorua abundance was significantly higher than in the other 4 groups(P<0.05).(Results)Different proportions of grape seed were added to the diet to alter the flora structure of rumen bacteria and fungus but could affect the abundance of some bacteria.In conclusion,(1)In vitro: GP was increased in complete diet with the adding ratio of grape seed from 8% to 16%,but the in vitro degradability was decreased and ruminal p H increased when the adding ratio of grape seed was more than 16%.The suitable adding ratios of grape seed to diet should be no more than 8%~12%based on the current study.(2)Animal experiments: The addition of 16% grape seed in the diet is not conducive to the digestion of nutrients in Duolang sheep;the addition of 4%~8% grape seed in the diet can improve the digestion of starch and protein in the rumen;the addition of more than 16% grape seed in the diet may not be conducive to animals Good health;the addition of more than 8% grape seed in the diet can improve the antioxidant capacity of animals;the proportion of grape seed added does not change the bacterial flora of rumen bacteria and fungi.Above all,the proportion of grape seed added to the diet should be below 12%,and it is suggested that the proportion of grape seed added should be 4%~8%.