Serum NEFA Composition In Ketotic Cows

Introduction: Periparturient period is a period when dramatic changes in the endocrine,energy metabolism and immune system of dairy cows take place,and it is also a period of high incidence of nutritional metabolic and infectious diseases.Ketosis is one of the perinatal diseases with the highest morbidity and the greatest losses,characterized by high blood non-esterified fatty acids(NEFA)and β-hydroxybutyric acid(BHBA)in dairy cows.The pathogenesis of ketosis has made great progress,but its warning and treatment are still difficult.At present,most studies focus on the lipotoxicity of high NEFA,but there are very limited reports on serum NEFA composition(i.e.,the mass ratio of each fatty acid component to total fatty acids).Therefore,the purpose of this study was to compare the serum NEFA composition of clinical ketosis,sub-clinical ketosis and healthy cows in order to provide a basis for screening early warning indicators of ketosis and lay a theoretical foundation for further revealing the pathogenesis of ketosis.Methods: Holstein cows with similar parities and lactation days were selected for clinical examination and laboratory diagnosis in the same dairy farm.Blood samples were collected to measure serum BHBA,glucose(GLU),and NEFA concentrations using commercial kits.They were further divided into different groups according to the blood BHBA concentration: those with serum BHBA concentration > 3 mM were the clinical ketosis group(CK,n = 15);those with 1.2 mM ≤ BHBA concentration ≤ 3 mM were the sub-clinical ketosis group(SCK,n = 15);and those with BHBA concentration < 1.2 mM and no clinical symptoms were divided into the healthy group(CON,n = 12).Serum samples were subjected to lipid extraction by the methanol-n-hexane method.Lipid purification was performed by solid phase extraction(SPE),and NEFA were washed out by anhydrous diethyl ether and glacial acetic acid solution(95 : 5,V : V).The purified NEFA were further methylated by incubation with 5% sulfuric acid/methanol solution.Then n-hexane and saturated sodium chloride solution were added to collect the supernatants.Fatty acids were analyzed by gas chromatography-mass spectrometry(GC-MSD,7890A/GC-5975 C,Agilent).Calculation formula: sample concentration = mixed standard-mix concentration ×(sample peak area / mixed standard-mix peak area)/ sample concentration factor;sample mass =sample concentration × volume of extractant added after methyl esterification ×(volume of extractant / post-column volume)× conversion factor.Results: There were significant differences in serum BHBA,GLU and NEFA concentrations among CK,SCK and CON group(P < 0.05).The BHBA concentration of the CK group(3.81 ± 0.62 mM)was the highest,followed by the SCK group(1.81 ± 0.45 mM)and the CON group(0.64 ± 0.21 mM).Similarly,the NEFA concentration was highest in the CK group(1.12 ± 0.26 mM),followed by the SCK group(0.73 ± 0.13 mM)and the CON group(0.49 ± 0.09 mM).Inversely,the GLU concentration in the CK group(1.86 ± 0.30 mM)was the lowest,followed by the SCK group(2.48 ± 0.41 mM),and the highest in the CON group(2.97 ± 0.26 mM).There was a negative correlation between BHBA and GLU(r=-0.8),also GLU and NEFA(r =-0.76);There was an positive correlation between BHBA and NEFA(r = 0.9).A total of 16 FAs were detected in cow serum,of which the highest concentrations were C16:0,C18:0 and C18:1n9.Seven fatty acids were statistically different in composition among the CK,SCK and CON groups(P < 0.05).C10:0%,C12:0%,C16:0%,C18:0% and C22:1n9% in CON group were significantly higher than those in SCK and CK group(P <0.05),but C17:0% and C18:1n9% were significantly lower than those in SCK group(P <0.05).The monounsaturated fatty acid percentage(MUFA%)and unsaturated fatty acid percentage(UFA%)in the CK group were higher than those in the SCK and CON groups(P< 0.05),but the saturated fat percentage(SFA%)and saturated fatty acid / unsaturated fatty acid ratio(SFA/UFA)were the lowest.The ratios of C18:1n9/C12:0 and C18:1n9/C22:1n9in the CK and SCK groups were higher than those in the CON group(P < 0.05).There was a correlation among C18:1n9 and C17:0(r = 0.93),C18:0 and C20:0(r = 0.88),also C18:3n3and C18:2n6(r = 0.88).Conclusion: The serum NEFA composition of ketosis cows and healthy cows is different.Serum C18:1n9/C12:0 and C18:1n9/C22:1n9 of ketosis cows have the potential as warning indicators for ketosis.