| With the development of animal husbandry in China,feed production has achieved significant growth but also caused a shortage of feed grains needed to feed livestock and poultry,especially soybean meal,which threatens China’s food security.Cottonseed meal(CSM),an unconventional protein material with abundant sources,high protein content,and a relatively low price,can be used in poultry diets.This research first studied the tolerance of CSM to goslings of different ages and evaluated whether the use of CSM impacted product quality.On this basis,the toxicity mechanism of free gossypol on the intestine of the absorption site and the liver of the accumulation organ of the goslings was investigated by oral administration of gossypol acetate(GA,a form of free gossypol).Four parts are included in this paper.Trial 1:Effects of cottonseed meal on growth performance,serum biochemical parameters,and liver redox status in goslings at 1 to 28 days of ageA total of 300 1-d-old male goslings were used to investigate the effects of CSM on growth performance,serum biochemical parameters,and liver redox status in goslings at 1 to 28 days of age.All birds were randomly allotted into 5 groups(10 goslings/replicate,6 replicates/group)according to a single-factor randomized design and subjected to a 28-d experiment.Five isonitrogenous and isoenergetic diets were formulated such that 0%(a cornsoybean meal basal diet,control),25%(CSM25),50%(CSM50),75%(CSM75),and 100%(CSM100)of protein from soybean meal was replaced by protein from CSM(corresponding to 0,7.08,14.15,21.23,and 28.30%CSM in the feed,respectively).The free gossypol contents in the five diets were 0,56,109,166,and 222 mg/kg,respectively.The results showed as follows:1)Dietary CSM was associated with linear decreases in body weight(BW),average daily feed intake(ADFI),and average daily gain(ADG)and linear increases in the feed-togain ratio(F/G)from 1 to 28 days of age(P<0.05).As the dietary CSM concentration increased,a numerical increase was found in the mortality of goslings,and more deaths occurred at 15 to 28 days rather than at 1 to 14 days.According to a single-slope broken-line model,the breakpoints for the ADG of dietary free gossypol concentration on days 1 to 14,15 to 28,and 1 to 28 occurred at 23.63,14.78,and 18.53 mg/kg,respectively.2)As the dietary CSM concentration increased,serum albumin(ALB)concentrations decreased linearly,and serum uric acid(UA)increased linearly(P<0.05).3)The hydroxyl radical scavenging ability and catalase(CAT)and glutathione peroxidase(GSH-Px)activities of the liver decreased linearly with increasing dietary CSM(P<0.05).4)Dietary CSM did not affect the concentrations of reactive oxygen metabolites,malondialdehyde(MDA),or protein carbonyl in the liver(P>0.05).Overall,the increasing dietary CSM increased the concentration of free gossypol and altered the composition of some amino acids in the diet.A high concentration of CSM reduced the growth performance of goslings aged 1 to 28 days by decreasing feed intake,liver metabolism,and antioxidant capacity.From the primary concern of free gossypol in CSM,the tolerance of goslings to free gossypol from CSM is low(less than 18.53 mg/kg,equivalent to 2.38%CSM in this study),and the toxicity of free gossypol has a cumulative effect over time.Trial 2:Effects of cottonseed meal on performance,meat traits,lipid metabolism,and cecal microbiota in goslings at 29 to 63 days of ageA total of 240 28-d-old male goslings were used to investigate the effects of CSM on performance,meat traits,lipid metabolism,and cecal microbiota.All birds were randomly allotted into 5 groups(8 goslings/replicate,6 replicates/group)according to a single-factor randomized design and subjected to a 35-d experiment.Five isonitrogenous and isoenergetic diets were formulated to produce diets in which 0%(Control),25%(CSM25),50%(CSM50),75%(CSM75),and 100%(CSM100)of protein from soybean meal was replaced by protein from CSM(corresponding to 0.5.66,11.33,16.99,and 22.65%CSM in the feed,respectively).The free gossypol contents in the five diets were 0,44,92,135,and 183 mg/kg,respectively.The results showed as follows:1)Dietary CSM did not affect the growth performance from 29 to 63 d and carcass traits at 63 d(P>0.05).2)Liver gossypol residues were influenced(P<0.05)by dietary CSM and increased linearly(P<0.05)and quadratically(P<0.05)as dietary CSM increased.However,the gossypol was undetectable in the breast muscle of geese in all treatment groups.3)The MDA content of the liver was lower in the CSM100 group than in the other groups(P<0.05).4)There were no effects on the meat quality of breast and thigh muscles,including meat color,pH value,cooking loss,and shear force(P>0.05).5)Dietary CSM did not affect the crude protein in the breast muscle of the goslings(P>0.05).As the dietary CSM concentration increased,the content of threonine,isoleucine,leucine,phenylalanine,total essential amino acids,and essential amino acids/total amino acids in the breast muscle decreased linearly(P<0.05).6)Serum triglyceride and low-density lipoprotein cholesterol(LDL-c)were influenced(P<0.05)by dietary CSM and increased linearly(P<0.05)with increasing dietary CSM.7)Dietary CSM altered(P<0.05)the composition of some fatty acids in the liver and breast muscle.The concentration of linolenic acid and Σn-3 polyunsaturated fatty acid(PUFA)in the liver and breast muscle decreased linearly,but theΣn-6/Σn-3 PUFA ratio increased linearly with increasing dietary CSM(P<0.05).8)As the dietary CSM concentration increased,the hepatic gene expression of fatty acid synthase(FAS)increased linearly(P<0.05)and quadratically(P<0.05),but the hepatic gene expression of acetyl-CoA carboxylase(ACC)and apolipoprotein B(ApoB)increased linearly(P<0.05).9)The CSM diet decreased the relative abundance of the Bacteroidota and Bacteroides(P<0.05),and the CSM50 diet increased the relative abundance of the Firmicutes and Colidextribacter(P<0.05)compared to the control group.Overall,dietary CSM has no adverse effects on the performance and liver function of goslings from 29 to 63 d,and no gossypol residue is detected in breast muscle.However,CSM decreased the contents of essential amino acids and Σn-3 PUFA in breast muscle and altered organismal lipid metabolism.Liver gossypol residues increased with increasing dietary CSM.In addition,goslings adaptively changed the cecal microbiota with CSM.Trial 3:Effects of gossypol acetate on growth,serum biochemical parameters,and intestinal health of goslingsThe aim of this study was to investigate the effects of GA on growth performance,serum biochemical parameters,and intestinal health of goslings.Seventy-two healthy male goslings—7 d old with similar BW were randomly divided into three groups,each of 24 geese.The three groups were the control group and two GA-treated groups(GA25 and GA50),which were orally administered GA(25 and 50 mg/kg BW)daily.The trial period was 14 days.The results showed as follows:1)after 7 days of GA administration,the BW of the goslings in the GA50 group decreased compared with the control group(P<0.05).As the duration of GA administration increased to 14 days,the goslings in the GA25 and GA50 groups had lower BW than those in the control group(P<0.05),and the goslings in the GA50 group had lower BW than those in the GA25 group(P<0.05).2)The residual gossypol in the liver increased with the increase of the dose of GA administration(P<0.05).In addition,as the duration of oral administration of GA increased,there was a significant time effect in the GA50 group(P<0.05).3)After 7 days of GA administration,serum total protein(TP),ALB,globulin(GLOB),total cholesterol(TCH),high-density lipoprotein cholesterol(HDL-c),and LDL-c concentrations of the goslings in the GA50 group were decreased,but serum UA concentration was increased(P<0.05).As the duration of GA administration increased to 14 days,compared with the control group,the serum concentrations of TP,ALB,GLOB,TCH,HDL-c,and LDLc in the GA25 and GA50 groups decreased(P<0.05),and these parameters in the GA50 group were lower than those in the GA25 group(P<0.05).4)After 7 or 14 days of GA administration,goslings in the GA50 group had lower intestinal villus height and villus height/crypt depth than those in the control group(P<0.05).The ileal villus height and villus height/crypt depth in the GA25 group decreased after 7 days of GA administration(P<0.05).After 14 days of GA administration,the intestinal villus height/crypt depth and jejunal villus height of goslings in the GA25 group decreased,but the ileal crypt depth increased(P<0.05)compared with the control group.5)After 7 days of GA administration,goslings in the GA25 or/and GA50 group had higher reactive oxygen species(ROS)levels and MDA content in the intestine(P<0.05)but lower superoxide dismutase(SOD)and CAT activities in the ileum(P<0.05)compared with the control group.As the duration of GA administration increased to 14 days,goslings in the GA25 or/and GA50 groups had higher ROS levels and MDA content in the intestine(P<0.05)but lower GSH-Px,SOD,CAT activities,and glutathione content(duodenum only)(P<0.05)compared with the control group.6)After 7 or 14 days of GA administration,goslings in the GA25 or/and GA50 groups had higher contents of tumor necrosis factor α,interleukin 1β(IL-1β),and IL-6(except in the ileum after 7 days of GA administration)but lower content of IL-10(except in the duodenum and jejunum after 7 days of GA administration)in the intestine compared with the control group(P<0.05).7)The contents of complement 3(C3),immunoglobulin M(IgM),and secretory immunoglobulin A(sIgA)in the ileum of goslings in the GA-treated groups were reduced after 7 days of GA administration(P<0.05).The contents of lysozyme,C3,C4,IgM,and sIgA in the intestine of goslings were decreased after 14 days of GA administration(P<0.05).8)The intestinal gene expression of Occludin decreased(P<0.05),but the intestinal gene expression of ZO-1 was not affected(P>0.05)by the administration of GA for both 7 and 14 days.The intestinal gene expression of Bax and Caspase-3 was up-regulated(P<0.05)in the GA25 or/and GA50 groups,but the intestinal gene expression of Bcl-2(ileum after 7 days of GA administration and duodenum and ileum after 14 days of GA administration)was down-regulated compared with the control group after 7 or 14 days of GA administration(P<0.05).Overall,oral administration of GA(25 and 50 mg/kg BW)inhibited the growth of goslings,damaged the intestinal morphology,caused oxidative stress and inflammation,reduced immune function,and increased intestinal permeability and apoptosis of intestinal cells.GA exhibited apparent toxicity in goslings,with dose and cumulative time effects.Trial 4:Multi-omics techniques reveal the mechanism of gossypol-induced liver damage in goslingsThis study aimed to reveal the damage mechanism of GA on the liver of goslings from morphology,transcriptomics,and metabolomics.The experimental design was the same as that of trial 3.Liver tissue samples were collected after 14 days of oral administration of GA.The results showed as follows:1)Granular degeneration of hepatocytes,hepatocyte enlargement,light staining of nuclei,fine reddish granules in the cytoplasm,and the disappearance of hepatocyte cytoplasm occasionally were observed in the liver of the goslings.A few hepatocytes showed focal necrosis,nucleus breakage,and cytoplasmic disintegration,accompanied by lymphocyte infiltration.Mitochondria in the liver was damaged,with moderate swelling,enlargement in volume,matrix dissolution,and shallowness.The cristae were primarily broken,shortened,reduced,or flocculent and individual membranes were damaged.2)Transcriptome analysis showed that 1137 differentially expressed genes were screened in the liver between the GA50 and control group,among which 702 genes were upregulated,and 435 genes were down-regulated.The enrichment results of the KEGG pathway showed that global and overview maps(carbon metabolism),carbohydrate metabolism(glycolysis/gluconeogenesis,pyruvate metabolism,propanoate metabolism,and TCA cycle),lipid metabolism(fatty acid degradation and primary bile acid biosynthesis),amino acid metabolism(tryptophan metabolism and cysteine and methionine metabolism),cellular community-eukaryotes(focal adhesion)and endocrine system(PPAR signaling pathway)were affected by GA.3)Metabolome analysis showed that 109 differential metabolites were screened between GA50 and the control group,among which 82 were significantly upregulated and 27 were significantly down-regulated.The enrichment results of the KEGG pathway showed that GA greatly affected linoleic acid metabolism,PPAR signaling pathway,arachidonic acid metabolism,cAMP signaling pathway,and serotonergic synapse.Overall,GA administration induced hepatocyte injury,and the prominent damaged organelles in the cells are mitochondria.Transcriptomics and metabolomics further confirmed that GA causes abnormal liver focal adhesion and down-regulates the glycolysis/gluconeogenesis and related pathways,PPAR signaling pathway,and lipid metabolism,resulting in the imbalance of the body’s energy supply.To sum up,CSM is an excellent dietary protein source for goslings,but attention should be paid to the low tolerance of goslings aged 1 to 28 days to dietary free gossypol(less than 18.53 mg/kg,equivalent to 2.38%CSM in this study).The complete replacement of soybean meal with CSM(183 mg free gossypol/kg)has no adverse effects on the performance of goslings from 29 to 63 d,but it reduced the edible value of goose products.Gossypol has a cumulative toxicity to goslings over time,and it will damage intestinal health after entering the intestine.After accumulating in the liver,it can lead to abnormal focal adhesion,damage mitochondria,and down-regulate energy metabolism pathways,causing an imbalance in the body’s energy supply. |
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