| The purpose of this experiment was to study the effects of different proportions of grape seeds in the diet on the apparent digestibility of nutrients,digestive tract development,intestinal bacteria and fungal flora structure of Duolang sheep.Twenty five three month old healthy Duolang sheep weighing 18.5 ±2.65 kg were randomly divided into five groups,with five sheep in each group and one repetition for each sheep.The experiment lasted 75 days,including pre feeding period of 15 days and positive feeding period of 60 days.10 days before the end of the feeding experiment,the digestion test was carried out with fecal bag to determine the apparent digestibility of nutrients;After the feeding experiment,all the experimental sheep were slaughtered,and the tissue samples of rumen,duodenum,jejunum and ileum were taken for tissue sectioning to observe the changes of digestive tract tissue structure;Two samples of intestinal microflora and one sample of intestinal microflora were collected by sequencing technology to determine the structure of intestinal microflora at the same time.Through the comparative analysis of the changes of apparent digestibility of nutrients,histological structure of digestive tract and the structure of bacterial and fungal flora in duodenum of Duolang sheep,the effects of different proportion of grape seed added diet on the development of digestive tract and intestinal flora of Duolang sheep were explored,so as to provide reference basis for improving the efficient application of grape seed resources in ruminant feed.Experiment 1: effects of different proportion of grape seeds in diet on apparent digestibility of nutrients in Duolang sheep The purpose of this experiment is to verify the appropriate amount of grape seeds in the diet studied by our research group in the early stage.25 healthy Duolang sheep weighing 18.5 ± 2.65 kg at the age of 3 months were selected as experimental animals.They were randomly divided into 5 groups,with 5 sheep in each group and one repetition for each sheep.The experimental sheep were fed in a single pen.The proportion of grape seeds in the diet was 0%(control group,a),4%(b),8%(c),12%(Group D)and 16%(E),respectively.The pre feeding period was 15 days and the trial period was 60 days.Ten days before the end of the feeding experiment,three healthy Duolang sheep with body weight close to the average value were selected from each group,and the feces were collected with fecal bag,pre fed for 5 days and sampled for 5days.The results showed that the apparent digestibility of dry matter in group A was significantly higher than that in Group E(P < 0.05),and there was no significant difference between group A and group B,C and D(P > 0.05);There was no significant difference in apparent digestibility of crude fat Crude fat between groups(P > 0.05);The apparent digestibility of crude protein Crude Protein in group A and group B was significantly higher than that in group E;The apparent digestibility of Neutral Detergent Fiber in groups A and B was significantly higher than that in groups D and E;There was no significant difference in Acid Detergent Fiber apparent digestibility among the groups(P > 0.05).Summary: the results of this experimental study are similar to the previous experimental results of our research group.The appropriate amount of grape seeds in the diet is recommended to be about 8%.More than 12% will significantly inhibit the apparent digestibility of dry matter,crude fat and Neutral Detergent Fiber.Experiment 2: effects of different proportion of grape seeds in diet on digestive tract development of Duolang sheepThe purpose of this experiment was to study the effects of different proportion of grape seeds in diet on the tissue structure of digestive tract of Duolang sheep.The experimental design and the feeding and management of experimental sheep are the same.After the feeding experiment,the experimental sheep were slaughtered and the tissues of rumen,duodenum,jejunum and ileum were taken.The results showed that there was no significant difference in rumen weight and compound stomach weight among the groups(P > 0.05);Compared with group B,group C and group D significantly increased rumen / re stomach weight(P < 0.05);The length of rumen papilla of Duolang sheep in group C was significantly higher than that in group A and group E(P < 0.05),and the length of rumen papilla of Duolang sheep increased first and then decreased with the increase of grape seed in the diet;Compared with group A and group E,the width of rumen papilla in group B and group C was significantly thickened(P < 0.05);The thickness of muscle layer in group C and D was significantly thicker than that in group A(P < 0.05);Compared with group A,the thickness of stratum corneum in groups B,C,D and D increased significantly(P < 0.05);The thickness of lamina propria in Group E was significantly lower than that in the other four groups(P < 0.05);The height of duodenal villi in groups C and D was significantly higher than that in groups a,B and E(P <0.05),the depth of duodenal recess in group A was significantly higher than that in other experimental groups(P < 0.05),and the V / C value in groups C and D was significantly higher than that in groups a,B and E(P < 0.05);The depth of jejunal recess in group A was significantly higher than that in group C(P <0.05),while the V / C value in group C was significantly higher than that in group A;The depth of ileal recess in group C was significantly lower than that in group D and E(P < 0.05),and there was no significant difference between group C and group A and B(P > 0.05);The V / C value of group C was significantly higher than that of the other four groups(P < 0.05).To sum up,when the proportion of grape seeds in the diet was 8% and 12%,it increased the rumen / re stomach weight and promoted the rumen development;At the same time,the rumen papilla height,rumen width,rumen muscle layer thickness and cuticle thickness were increased.When the amount of grape seed was 8%,the villus height / recess depth of duodenum and jejunum was significantly higher than that of the control group,the ileal recess was the smallest and the cell generation rate was the largest.Experiment 3: effects of different proportion of grape seeds in diet on intestinal bacterial flora of Duolang sheepThe purpose of this experiment was to explore the effects of different proportion of grape seeds in diet on the structure and diversity of intestinal bacterial flora of Duolang sheep.The experimental design and feeding management are the same as the experiment.After the duodenal chyme was collected and sequenced by PAC,the intestinal chyme structure was analyzed.The results showed that:(1)the sequencing coverage of samples was 98.22% ~ 98.82%,and there was no significant difference in alpha diversity among groups(P > 0.05);There were 1038 OTUs in 25 samples.Statistical analysis showed that there was no significant difference between the experimental group and the control group(P > 0.05);(2)The dominant bacteria at phylum level are Firmicutes and Actinobacteria.The relative abundance of Bacteroidetes in group A and group B was significantly higher than that in group C,group D and group E(P < 0.05).(3)Aeriscardovia is the first dominant flora of group A,B,C and E,accounting for the highest proportion,while the first dominant flora of group D is Lachnospiraceae_NK3A20_group.To sum up: the dominant bacteria in the intestines of Duolang sheep are Firmicutes and actinomycetes,and the dominant bacteria are aeriscardovia.With the increase of the amount of grape seed,the abundance of Spirillum was significantly increased,the abundance of Bacteroidetes was inhibited,and the abundance of Actinomycetes and Oncococcus was promoted;The addition of different proportion of grape seeds in the diet did not affect the intestinal bacterial diversity and the abundance of some flora of Duolang sheep.Experiment 4: effects of different proportion of grape seeds in diet on intestinal fungal flora of Duolang sheepThe purpose of this experiment was to explore the effects of different proportion of grape seeds in diet on the structure and diversity of intestinal fungal flora of Duolang sheep.The experimental design and feeding management are the same as the experiment.After the feeding experiment,the duodenal chyme was slaughtered,and the structure of intestinal fungal flora was analyzed by pacbio sequence sequencing technology.The results showed that:(1)the sequencing coverage of samples was 98.9% ~ 99.22%,and there was no significant difference in alpha diversity among groups(P > 0.05);There were 896 OTUs in 25 samples.Statistical analysis showed that there was no significant difference between the experimental group and the control group(P > 0.05);(2)The five groups of dominant bacteria are Ascomycota;Basidiomycota in Group E was significantly higher than that in groups A and B(P < 0.05),and Chytridiomycota in group C was significantly higher than that in group B(P < 0.05).(3)At the genus level,Piromyces is the first dominant flora in Group E,Alternaria is the first dominant flora in group C,Neocalimastix is the first dominant flora in groups A and B,and Cladosporium is the first dominant flora in Group D.There was no significant difference(P > 0.05).Conclusion: Ascomycota is the predominant intestinal flora in Duolang sheep;With the increase of grape seed addition,the abundance of Basidiomycetes and Pyricularia was increased,the abundance of Neocallimastix was inhibited,and the abundance of Aspergillus was promoted;The addition of different proportions of grape seeds in the diet did not affect the intestinal fungal diversity of Duolang sheep,but increased the overall abundance.ConclusionIn summary,when the amount of grape seed is 8% ~ 12%,it can promote the absorption of nutrients,improve the apparent digestibility,and promote the development of gastrointestinal tract.With the increase of grape seed addition,it has little effect on the diversity of bacteria and fungal flora,and improves the richness of some flora,which can promote the bacteria involved in some immune effects and the overall abundance of fungal flora. |
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