Transfer Regularity And Metabolomics Study Of Gossypol Enantiomers In Lactating Dairy Cows

Gossypol is one of the main limitations for cottonseed usage in dairy cows.To study the transfer regularity of gossypol isomers in cows and its impact on body metabolism,the following four experiments were conducted.The gossypol intakes by cows on commercial farms were survived and the transfer regularity of gossypol isomers in cows was investigated by designing animal study.Nine cows were blocked and assigned into one of the three treatments: control,500,and 1000 mg/kg DM gossypol for 28 d.Gossypol intakes by cows in two of the three farms survived were upper the limitation by EFSA.Animal study shown that(1)plasma positive gossypol concentrations in 500 and 1000 mg/kg DM gossypol groups reached highest on treatment day 21 and 14(1.63 ?g/mL,2.55 ?g/mL),respectively;(2)plasma negative gossypol contents in both treatment groups were highest on treatment day 21(2.63 ?g/mL and 4.76 ?g/mL,respectively);(3)on withdrawal day 49,plasma positive gossypol contents in both treatment groups were below LOQ,negative gossypol in 500 and 1000 mg/kg DM gossypol groups were 0.19 ?g/mL and 0.29 ?g/mL,respectively.These results indicated that gossypol over feeding is common on dairy farms and the absorptivity is higher and accumulation ability is stronger for negative gossypol in cows,compared with the positive form.The degradation of gossypol isomers by rumen microbes was studied in vitro by supplemented with gossypol at 500 and 1000 ?g/g DM substrate levels.At 6 h,the degradation rates of negative gossypol in the 500 and1000 ?g/g gossypol group was 67.78% and 85.85%,respectively,and increased to 81.73% and 85.22%,respectively,at 48 h.For positive gossypol,65.65% and 86.44% were degraded in the 500 and1000 ?g/g gossypol group,respectively,at 6 h,and that values increased to 84.08% and 87.18%,respectively,at 48 h.These results demonstrated the strong degradation of gossypol by rumen microbes and this degradation is not enantioselective.Twelve dairy cows were randomized block divided into two groups and administrated with 0 and 1000 mg/kg DM gossypol for 28 d to study the impact of high level gossypol on animal health.Milk lactose and milk protein contents were significantly lower in the treatment group than that in the control group.Plasma glucose and lysine contents was decreased,phosphate and HDL-C concentrations,choline esterase,glutathione S-transferase,alanine aminotransferase,alkaline phosphatase,and superoxide dismutase activities were increased,LDL-C and malondialdehyde levels were significantly decreased in the treatment group,compared with that in the control group.Metabolomics analysis showed that 23 metabolites in plasma,8 metabolites in urine,and 64 metabolites in the milk were differened between the control and treatment group animals.These results indicated that administration dairy cows with 1000 mg/kg DM gossypol for 28 d could reduce milk performance through impairing liver function,decreassing gluconeogenesis,amino aicds utilization,and lipids transportation from liver to peripheral.The mechanism of gossypol-induced hemolysis in the above animal studies was explored using metabolomics and proteomics methods.Compared with control group,mean corpuscular hemoglobin and mean corpuscular volume were significantly decreased in the treatment group.Metabolomics and proteomics analysis shown that erythrocytes in the treatment group had(1)lower contents of glucose,glucose-6-phosphate,and fructose-6-phosphate,higher abundance of phosphoglycerate kinase,pyruvate kinase,L-lactate dehydrogenase,unchanged pyruvate and lactate levels;(2)higher abundances of 6-phosphogluconate dehydrogenase and NADPH and increased ribose-5-phoshphate level;(3)up-regulated glutathione S-transferase,superoxide dismutase,catalase,decreased oxidized glutathione and increased the catabolite of glutathione,cysteine levels.Through the global multi-omics analysis,we conclude that glycolysis and pentose phosphate pathways responsible for essential energy metabolism and maintenance of reducing agents,along with the oxidative reduction pathways for antioxidation reactions,were involved in erythrocytes in response to the hemolytic toxicity of gossypol.In conclusion,gossypol over feeding is common on dairy farms in China,and its toxicity is of concern.By comparing the metabolism of gossypol isomers in dairy cows,it's found that the absorptivity is higher and accumulation ability is stronger for negative gossypol in cows,compared with the positive form,and rumen degradation is not responsible for the difference.Animal study found that feeding dairy cows with 1000 mg/kg DM gossypol for 28 d induced hepatotoxicity,decreased anino acids utilization,gluconeogenesis,and transportation of lipid from liver to peripheral,accelerated hemolysis by causing energy depletion and oxidative stress,which all contributed to the lower production performance in the gossypol group,compared with that in the control group.These results may benefit the usage of cottonseeds products in dairy cows and gossypol safety evaluation.