Effects Of Dietary Marigold Meal On Performance,Serum Indexes,Rumen Fermentation And Microflora Of Sheep

This experiment was conducted to study the effects of different proportions of marigold meal on rumen microflora structure,performance,serum indexes and rumen fermentation parameters of sheep,aiming to provide theoretical support for efficient utilization and appropriate dosage of marigold meal.The experiment was designed as a feeding experiment,This experiment includes the following four parts:Test1:Effect of different proportions of marigold meal on sheep performance.The purpose of this experiment was to study the effects of different proportions of marigold meal added to the diet on sheep production performance,and to screen the appropriate range of addition.A total of 50 crossbred F1 male lambs of Dorper sheep and Hu sheep with a weight of（28.30±3.95）kg were selected from the sheep farm of Fengtai Agriculture and Animal Husbandry Technology Co,Ltd.in Bachu,Kashgar Prefecture,Xinjiang.After drug deworming,a single factor completely randomized trial design was used based on their weight,and they were divided into a control group（CK）,a 5%group（G5）,a 15%group（G15）,a 25%group（G25）,and a 35%group（G35）.Each group had 5 replicates,2 for each replicate.The experimental sheep were kept in the same environment in a feeding pen for 40 days,with a pre feeding period of 10 days and a regular feeding period of 30 days.They were fed twice daily at 09:00 and 19:30,free choice feeding and drinking,and each group maintained a 15%residual feed amount every day.Samples of marigold meal were taken and used for the assay of active ingredients.During the test period,the intake and residual amount were recorded daily.At the end of the pre feeding period and before morning feeding on the 31th day,the experimental animals were weighed on an empty stomach to measure average daily gain（ADG）and calculate the feed weight ratio.The results showed that there were1242 active substances detected in marigold meal,which were divided into 46 different types;Among the top 5 different types of active substances at the peak,there are 170 lipids,221 phenolic acids,98 alkaloids,118 amino acids and derivatives,and 263 flavonoids.Dietary supplementation with marigold meal can improve dry matter intake（DMI）in sheep.DMI in group G15 and group G25 is significantly higher than that in group CK and group G35（P<0.01）,while group G5 is significantly higher than that in group CK（P<0.01）.There is no significant difference between other groups（P>0.05）.ADG in group G15 was significantly higher than that in group CK（P<0.05）.Group G15 had the lowest Feed/Gain（F/G）and had no significant difference compared to group G5（P>0.05）,but had significant differences compared to other groups（P<0.01）.During the trial,the diarrhea rate of sheep in the group CK,group G5,and group G15 was 0,while the diarrhea rate of sheep in group G25 was 8.33%,and the diarrhea rate of sheep in group G35 was 25.00%.In summary,when the marigold meal addition level reached 15%in this experiment,DMI significantly increased,reducing the feed weight ratio.Test2:Effects of Different Proportion of Marigold Meal on Serum Indexes in Sheep.Experimental animals and groups are the same as those in Experiment 1.Blood was collected through the jugular vein before morning feeding on the 30th day of the feeding period.Centrifuge at 4℃and 3 000 r/min for 15 minutes,take the supernatant and sub pack it into a 1.5 m L centrifuge tube,and store it at-20℃for the determination of serum biochemical indicators,serum antioxidant indicators,and heat shock 70 protein.At the same time,record the temperature and humidity inside the sheep shed daily.The results showed that in the serum biochemical indicators of energy metabolism,the content of glucose（GLU）in group G5 was significantly higher than that in group G25（P<0.05）,while there was no significant difference among other groups（P>0.05）;The activity of creatine kinase（CK）in group G15 was significantly higher than that in group G25（P<0.01）,and significantly higher than that in other groups（P<0.05）;There was no significant difference in the content of cortisol（COR）among the groups（P>0.05）.In protein metabolism,the content of total protein（TP）in group G35 was significantly higher than that in group G5（P<0.01）,while there was no significant difference between the other groups（P>0.05）;The content of albumin（ALB）in group G25 was significantly higher than that in group CK and group G5（P<0.05）,while that in group G35was significantly higher than that in group G5（P<0.05）;The content of globulin（GLOB）in group G35 was significantly higher than that in group G5（P<0.01）,and there was no significant difference in the activities of alanine aminotransferase（ALT）and aspartate aminotransferase（AST）among the groups（P>0.05）.In lipid metabolism,there was no significant difference in alkaline phosphatase（ALP）among the groups（P>0.05）;The content of triglyceride（TG）in group G35 was significantly lower than that in group CK（P<0.01）,and significantly lower than that in group G15（P<0.05）;The content of high-density lipoprotein（HDL）in group G35 was significantly higher than that in group G5（P<0.05）;The content of low density lipoprotein（LDL）in group G35 was significantly higher than that in group CK and group G5（P<0.05）;The content of very low density lipoprotein（VLDL）in group G35 was significantly higher than that in group G5（P<0.05）;The content of total cholesterol（TC）in group G35 was significantly higher than that in group CK and group G5（P<0.01）.Serum antioxidant indicators:There was no significant difference in the content of Malondialdehyde（MDA）among the groups（P>0.05）,but there was a downward trend with the increase in the amount of marigold meal added（P=0.086）.There was no significant difference in the contents of Total Antioxidant Capacity（T-AOC）,Superoxide Dismutase（SOD）,Glutathione Peroxidase（GSH-Px）and Catalase（CAT）among all groups（P>0.05）.With the increase in the proportion of marigold meal added,the heat shock protein 70（HSP70）in sheep serum significantly decreased,with group G35 significantly lower than group CK（P<0.01）,group G25 significantly lower than group CK（P<0.05）,and there was no significant difference between the other groups（P>0.05）.Test3:Effects of different proportions of marigold meal on rumen fermentation in sheep.Experimental animals and groups are the same as those in Experiment 1.Before the morning feeding on the 30th day of the feeding period,the rumen fluid was collected from the mouth using a pedal type rumen fluid collection device.After being filtered with four layers of gauze,the rumen fluid was used for p H measurement,the remaining rumen fluid was subpackaged,and liquid nitrogen was frozen and transported to the laboratory.The concentration of volatile fatty acids（VFA）and ammonia nitrogen（NH₃-N）were measured.The results showed that there was no significant difference in the p H of rumen fluid among the groups（P>0.05）.There was no significant difference in the influence of NH₃-N concentration among groups（P>0.05）.In VFA,the concentration of acetate in group G25 was significantly higher than that in group G35（P<0.05）.The concentration of butyrate increased with the increase of marigold meal addition level,and the concentration of butyrate in group G35 was significantly higher than that in group CK（P<0.05）,while the differences in other groups were not significant（P<0.05）.There were no significant differences in the concentrations of propionic,isobutyrate,isovalerate,pentanoate,total volatile fatty acid（TVFA）,and acetate/propionic（A/P）among the groups（P>0.05）.Test4:Effects of different proportions of marigold meal on rumen microbial structure in sheep.Experimental animals and groups are the same as those in Experiment 1,and the collection of rumen fluid is the same as that of Experiment 3.This experiment analyzed the effects of different proportions of marigold meal on the number and structure of rumen microorganisms in sheep through macrogenomic sequencing.The results show that:The ACE index of group G35 was significantly lower than that of group CK（P<0.01）,while that of group G25 was significantly lower than that of group CK（P<0.05）.The Chao1 index in group G35 was significantly lower than that in group CK（P<0.01）,while that in group G25 was significantly lower than that in the group CK（P<0.05）.The Shannon index of the G35 group was significantly lower than that of the CK group（P<0.01）.The relative abundance of Bacteroidota in group G35 was significantly higher than that in group CK（P<0.01）,and the relative abundance of Bacteroides in group G25 was significantly higher than that in group CK（P<0.05）.The relative abundance of Proteobacteria in group G25 and group G35 were significantly lower group CK（P<0.01）.Compared to the CK group,the groups G15,G25,and G35 showed a significantly lower relative abundance of Verrucomicrobiota（P<0.01）.The group G25 and group G35 also demonstrated a significantly lower relative abundance of Cyanobacteria compared to the CK group（P<0.01）.The relative abundance of Planctomycetota in group G25 and group G35 were significantly higher than that in the group CK（P<0.01）.At the genus level,the dominant bacteria were Prevotella,Bacteroides,Succiniclasticum and Quinella.The relative abundance of Prevotella in group G35 was significantly higher than that in group CK（P<0.01）.The abundance of Succiniclasticum in group G35 was significantly lower than that in group CK（P<0.01）,group G15 and group G25 were significantly lower than that in group CK（P<0.05）.The relative abundance of Succinivibrio in group G25 and group G35 were significantly lower than that in group CK（P<0.05）.The relative abundance of Anaerovibrio in group G35 was significantly lower than that in group CK（P<0.01）.To sum up,adding 5%-15%marigold meal in sheep diet will not affect the richness and diversity of sheep rumen flora,while adding 25%-35%marigold meal will reduce the richness and diversity of sheep rumen flora,change the flora structure of sheep,and increase the relative abundance of Bacteroides and Prevotella.Conclusion:In summary,the appropriate proportion of Marigold meal can improve the performance of sheep,affect serum protein metabolism indexes and serum lipid metabolism indexes,did not significantly affect the serum antioxidant indexes,and can reduce the content of heat shock protein70 under heat stress.And 35%Marigold meal to the rumen increased the content of Bacteroides and Prevotella on the rumen and decreased the content of Proteobacteria,25%of Marigold meal increased the content of acetate in the rumen,and the diarrhea rate of 35%group was much higher than other groups,comprehensive consideration of the addition of marigold meal should not exceed25%,the most suitable addition ratio in this trial conditions is 15%.