Effects Of Gossypol On Production Performance,Gossypol Metabolism,Rumen Fermentation And Microflora Of Dolang Sheep

This experiment was conducted to study the effects of gossypol on production performance,gossypol metabolism,rumen fermentation parameters and microflora of Dolang sheep.Exploring the tolerance of Dolang sheep to high concentration gossypol,providing scientific basis for the rational use of high proportion cotton by-product feed by ruminants.This experiment was divided into three groups:free gossypol group（FG group:free gossypol at 524.12 mg/kg,with free gossypol content in cottonseed meal and cottonseed shell at 1683.53 mg/kg and 442.45 mg/kg,respectively）,gossypol acetate group（AG group:exogenous gossypol acetate added at 524.12mg/kg,calculated based on the FG group’s free gossypol concentration）,and control group（CK group:conventional diet without gossypol）,Six adult Dolang sheep and capricorn sheep were selected as rumen fluid donors for in vitro experiments（fed with a diet without gossypol or cotton by-products）.Gas production,p H,volatile fatty acids（VFA）,ammonia nitrogen（NH₃-N）,and microbial crude protein（MCP）were measured after 24 and 48 hours of in vitro cultivation.Free gossypol concentration in the experimental group was measured at 0,24,and 48 hours,and DM degradation rate was measured by in situ nylon bag method at 24 and 48 hours;18 adult Dolang sheep with permanent fistulas（average weight:37.13±4.78 kg）were selected in the body.Each group had 6 replicates and were raised in a single pen.They were fed twice a day at 9:30 and20:00,with a limit of 1.20 kg/d.The AG group was supplemented with 628.94 mg/d of gossypol acetate（calculated based on the amount of gossypol acetate added to the FG group feeding 1.2kg/d）and mixed with concentrate for 120 days.The pre feeding period of the experiment was 30days,and the sampling period was divided into 31 to 60 days（Phase I）,61 to 90 days（Phase II）,and 91 to 120 days（Phase III）.On the 30th,60th,90th,and 120th days,weights were taken before morning feeding,and venous blood was collected before morning feeding on the 60th,90th,and120th days to measure average daily gain（ADG）and plasma free gossypol.On the 34th to 36th,64 to 66th,and 94 to 96th days,rumen fluid was collected before and after morning feeding at 0,2,4,6,and 8 hours to analyze rumen fermentation parameters The free gossypol indicators in the rumen fluid（as the control group before morning feeding）and the number of protozoa were measured.Digestion tests were conducted on days 40 to 47,70 to 77,and 100 to 107 to determine the apparent digestibility,free gossypol in feces and urine,and other related indicators.On days 54,83,and 113,the rumen fluid was collected for MCP,rumen microbial abundance,and functional prediction.The specific results of the experiment include four parts:Test 1:The effect of gossypol on in vitro fermentation parameters,degradation rate of gossypol and dry matterThe aim of this experiment is to investigate the effect of gossypol on in vitro fermentation parameters of Dolang sheep rumen,and to analyze the degradation rate of gossypol and dry matter（DM）.The results showed that at each time point,the gas production in the FG group was significantly lower than that in the CK group（P<0.01）,while the gas production in the AG group was significantly higher than that in the CK group（P<0.01）;There was no significant difference in p H and TVFA concentration among the groups（P>0.05）;At 24 hours,the NH₃-N concentration in the experimental group was significantly lower than that in the CK group（P<0.01）,and at 48hours,the NH₃-N concentration in the AG group was significantly lower than that in the FG and CK groups（P<0.05）;At 24 hours,the concentration of MCP in the AG group was significantly lower than that in the FG and CK groups（P<0.01）;The free gossypol content in the FG group first increased and then decreased,and degraded to 0.62 mg/L at 48 hours.The free gossypol content in the AG group showed a linear decrease,and degraded to 0.79 mg/L at 48 hours;At 24 and 48hours,the degradation rate of DM in the FG group was higher than that in the AG and CK groups（P<0.01）.The results showed that gossypol had an impact on in vitro gas production,NH₃-N,MCP,and DM degradation rate.The degradation rate of free gossypol at 48 hours was higher in the free gossypol group than in the gossypol acetate group..Test 2:Effect of gossypol on production performance and gossypol metabolism of Dolang sheepThe aim of this experiment is to investigate the effects of gossypol on the production performance and gossypol metabolism of Dolang sheep.The results showed that gossypol had no effect on ADG,water intake,urination,DMI,and fecal output of Dolang sheep（P>0.05）.The digestibility of DM,CP,NDF,ADF,and OM in the experimental group showed a downward trend,while the digestibility of EE and urine p H showed an upward trend.The urinary protein in the stage I FG group was significantly higher than that in the CK group（P<0.05）.The concentration of free gossypol in the rumen fluid and plasma of the experimental group showed an upward trend,while the concentration of free gossypol in the rumen fluid and plasma of the AG group was higher than that of the FG group;The free gossypol concentration in the rumen fluid of the experimental group was the lowest before morning feeding,and after morning feeding,the free gossypol concentration in the rumen fluid of the experimental group showed a trend of first increasing and then decreasing.The FG group had the highest free gossypol concentration at 4hours,while the AG group had the highest free gossypol concentration at 2 hours,and then slowly decreased;The plasma free gossypol concentrations were the highest in the FG group and AG group at stage III,with 0.66 mg/L and 0.89 mg/L,respectively.The concentration of free gossypol in the urine and feces of the FG group showed a decreasing trend,while the concentration of free gossypol in the urine and feces of the Phase III FG group was 0.69 mg/L and 8.78 mg/kg,respectively;The concentration of free gossypol in the urine and feces of the AG group showed an upward trend.The concentration of free gossypol in the urine and feces of the stage III AG group was 1.38 mg/L and 6.68 mg/kg,respectively.The concentration of free gossypol in the urine of the AG group was higher than that of the FG group,which was opposite to the change pattern of free gossypol concentration in feces.The results showed that feeding gossypol reduced the nutrient digestibility of Dolang sheep,and increased urine p H and protein concentration.The concentration of free gossypol in rumen fluid was lower before and after morning feeding,and reached its peak later in the free gossypol group than in the acetic acid gossypol group.The concentration of free gossypol in rumen fluid,urine,and plasma was higher in the gossypol acetate group than in the free gossypol group.However,the pattern of fecal excretion of free gossypol was opposite to the above results,and feces were the main pathway for the excretion of free gossypol.Test 3:Effect of gossypol on rumen fermentation parameters in Dolang sheepThis experiment aims to investigate the effect of gossypol on rumen fermentation parameters in Dolang sheep.The results showed that the rumen p H of each group showed a trend of first decreasing and then increasing.In stage I,the rumen p H decreased to the lowest point at 2 hours,and then slowly increased to the highest point at 8 hours.The p H of the experimental group was higher than that of the CK group at 0 to 2 hours,while it was opposite at 4 to 8 hours;The p H of the Phase II and III experimental groups decreased to the lowest point at 4 hours,while the p H of the CK group decreased to the lowest point at 2 hours,and then slowly increased to the highest point at 8 hours.The p H of the experimental group was higher than that of the CK group,with the FG group having the highest p H,but there was no significant difference in rumen p H among the groups（P>0.05）.The NH₃-N concentration in the rumen showed a trend of first decreasing and then increasing.The NH₃-N concentration in the Phase I and II experimental groups decreased to4 hours and then increased,while the CK group decreased to 6 hours and then increased;The concentration of NH₃-N in Phase III decreased to 6 hours and then increased;The NH₃-N concentration in stage I FG group was significantly lower than that in AG group and CK group at0 and 8 hours（P<0.01）,while the NH₃-N concentration in experimental group was significantly lower than that in CK group at 2-6 hours（P<0.01）.The NH₃-N concentration in FG group was significantly lower than that in AG group（P<0.01）;The NH₃-N levels in the stage II and III FG groups were significantly lower than those in the AG and CK groups（P<0.01）.The concentration of MCP in the experimental group showed a decreasing trend and was significantly lower than that in the CK group（P<0.05）;The concentration of MCP in the Phase I experimental group was significantly higher than that in Phase III（P<0.05）.The concentration of VFA showed a trend of first increasing and then decreasing.The concentration of acetic acid and TVFA in the Phase I experimental group was lower than that in the CK group at 0-2 hours,while the concentration of acetic acid and TVFA was opposite at 4-8 hours,with the FG group having the highest concentration;The concentration of TVFA in the Phase II and III experimental groups was lower than that in the CK group,with the FG group having the lowest concentrations of acetic acid and TVFA.The concentration of propionic acid in the experimental group was higher than that in the CK group,with the FG group having the highest concentration of propionic acid and the CK group having the highest concentration of butyric acid and valeric acid;There was no significant difference in the concentrations of acetic acid,propionic acid,butyric acid,and TVFA among the groups（P>0.05）.The concentration of valeric acid in the 0 hour experimental group of Phase I was significantly lower than that in the CK group（P<0.05）.At 2 hours,the concentration of valeric acid in the FG group was significantly lower than that in the CK group（P<0.05）;At 0 h in phase II,the concentration of valeric acid in the FG group was significantly lower than that in the CK group（P<0.05）.The results showed that feeding gossypol reduced the concentrations of NH₃-N and MCP in the rumen fluid of Dolang sheep,while the p H and propionic acid concentrations in the rumen fluid showed an increasing trend,while the concentrations of acetic acid,butyric acid,valeric acid,and TVFA showed a decreasing trend.Test 4:Effects of gossypol on the diversity,function,and metabolic pathways of rumen microorganisms in Dolang sheepThe aim of this experiment is to investigate the effects of gossypol on the diversity,function,and metabolic pathways of rumen microorganisms in Dolang sheep.The results showed that the number of protozoa fluctuated.The number of protozoa in the stage I FG group at 0 h was significantly lower than that in the AG and CK groups（P<0.05）,and the number of protozoa in the experimental group at 2 to 6 h was significantly lower than that in the CK group（P<0.01）;The number of protozoa in the Phase II experimental group at 0 and 4 hours was significantly lower than that in the CK group（P<0.05）,and the number of protozoa in the experimental group at 2hours was significantly lower than that in the CK group（P<0.01）.The number of protozoa in the FG group at 6 hours was significantly lower than that in the CK group（P<0.05）;There was no significant difference in the number of protozoa among each group in Phase III（P>0.05）.At the phylum level,the bacteria in the rumen fluid of Dolang sheep are mainly composed of Bacteroidetes and Firmicutes（P<0.05）.Compared with the CK group,the relative abundance of Firmicutes and Desulfobacteria in the Phase I experimental group was significantly lower than that in the CK group（P<0.05）,while the FG group significantly increased the relative abundance of Proteobacteria in the gastric juice（P<0.05）;The Phase II FG group significantly reduced the relative abundance of Firmicutes and Desulfobacteria（P<0.05）,but the FG group significantly increased the relative abundance of Proteobacteria（P<0.01）.The relative abundance of Patescibacteria in the experimental group was significantly lower than that in the CK group（P<0.05）;The Phase III FG group significantly increased the relative abundance of Proteobacteria（P<0.05）,but the FG group significantly decreased the relative abundance of Desulfobacteria（P<0.05）.The relative abundance of Fibrobacteres in the experimental group was significantly lower than that in the CK group（P<0.05）.At the genus level,the FG group consists of Prevotella and unclassified＿Succinivibrionaceae is mainly composed of Prevotella and Rikenellaceae＿RC9＿gut＿Group at the AG and CK group genus levels composition.The functional results of the rumen bacteria FAPROTAX in Dolang sheep showed that compared with the CK group,the Phase I experimental group significantly increased animal count＿parasites＿or＿Symbiots function（P<0.01）significantly reduced chemoptosis and fertilization（P<0.05）,and significantly reduced respiration＿of＿sulfur＿Compounds and sulfonate＿respiration（P<0.01）;Phase II FG group significantly reduced xylanolysis and respiration＿of＿sulfur＿Compounds and sulfonate＿res piration（P<0.05）;The FG group in phase III significantly reduced xylolysis（P<0.05）,while the experimental group significantly reduced cellulolysis（P<0.05）and significantly increased intra cellular＿parasites（P<0.05）。At the order level,the methane producing bacteria associated with Dolang sheep rumen fluid are mainly composed of Metanobacteriales,Bacteroidales,and Metalomassilicoccales.Compared with the CK group,the Phase I FG group significantly increased the relative abundance of Enterobacteriales（P<0.05）and significantly decreased Veillonellales＿Relative abundance of Selenomonales（P<0.05）;The Phase II experimental group significantly reduced the relative abundance of Metalomassilicoccales（P<0.05）,extremely significantly reduced the relative abundance of Pedosphaerales（P<0.01）,and the FG group significantly increased the relative abundance of Enterobacterales（P<0.05）;The FG group in phase III significantly reduced the relative abundance of Metalomassilicoccales（P<0.01）and significantly increased the relative abundance of Enterobacterales（P<0.05）;Compared with Phase I,the Phase III FG group significantly reduced the relative abundance of Pirellulales（P<0.01）.At the genus level,the FG group is mainly composed of Metanobrevibater and Prevotella,while the AG and CK groups are mainly composed of Metanobrevibater and unclassified＿Method Ylophilaceae.The functional results of the rumen associated methanogenic bacteria FAPROTAX in Dolang sheep showed that compared with the CK group,the stage III FG group significantly reduced methanogenesis＿by＿reduction＿of＿methyl＿Compounds＿with＿H₂and methylotrophy（P<0.01）;Compared with stage I,the stage III AG group significantly increased chemotherapy（P<0.05）.The KEGG2 level functional prediction results of rumen methane producing bacteria in Dolang sheep showed that compared with the CK group,the Phase I FG group significantly increased the enrichment of the Amino acid metabolism pathway（P<0.01）,significantly increased the enrichment of the Xenobiotics biodegradation and metabolism pathway（P<0.05）,but significantly reduced the enrichment of the replication and repair pathway（P<0.01）,and significantly reduced folding,Enrichment of sorting and degradation and biosynthesis of other secondary metabolites pathways（P<0.05）;The Phase II FG group significantly increased the enrichment of the Amino acid metabolism pathway（P<0.05）and significantly reduced the enrichment of the Biosynthenosis of other secondary metabolites pathway（P<0.05）;The FG group in phase III significantly increased the enrichment of the Amino acid metabolism pathway（P<0.05）,significantly reduced folding,sorting,and degradation（P<0.05）,and significantly reduced the enrichment of the Replication and repair,Biosynthesis of other secondary metabolites pathway（P<0.01）.At the phylum level,the fungi in the rumen fluid of Dolang sheep are mainly composed of Neocollimastigomycota and Ascomycota.Compared with the CK group,there was no significant difference in the levels of rumen fungal phylum between the experimental group and the CK group in different periods（P>0.05）;Compared with Phase I,the Phase II FG group significantly reduced the relative abundance of Ascomycota（P<0.05）,while the Phase II and III AG groups significantly reduced Basidiomycota and unclassified＿Fungi（P<0.05）.At the genus level,the dominant fungi in the FG group are Pyromyces and Pecoramycs;The dominant fungal genera in the AG group are Neocollimastix and Pyromyces;The dominant fungal genera in the CK group are Orpinomyces and Nigrospora.The functional results of rumen fungal FUNGuild showed that compared with stage I,the stage II AG group significantly reduced Pathotroph（P<0.05）.The results showed that gossypol reduced the abundance of rumen microbiota,altered the composition of bacteria,methanogens,and fungal genera,and had an impact on the function and metabolism of some microbial communities.Each stage has an impact on the diversity,function,and metabolic pathways of rumen microorganisms,but the proportion of stage III impact on microorganisms is relatively small.Conclusion:Feeding with gossypol reduced the apparent digestibility of Dolang sheep.The impact of gossypol on the physiological metabolic pathways of Dolang sheep is manifested by increasing urine p H and protein concentration,increasing the concentration of free gossypol in rumen fluid and blood,and feces being the main pathway for the excretion of free gossypol.Tolerance is manifested by an increase in rumen p H,increased utilization of acetic acid,butyric acid,valeric acid,TVFA,NH₃-N,and MCP in the rumen,but decreased abundance of rumen microbiota and altered composition of bacteria,methanogens,and fungal genera.Therefore,feeding gossypol diet affects the fermentation function of rumen microorganisms,which may have a negative impact on the production performance of Dolang sheep.However,long-term feeding of gossypol diet rumen microorganisms can develop a certain tolerance to gossypol.