Metabolic disorders and immunosuppression exist in peripartal cows,and glutamine(GLN)has the potential efficacy to improve the negative energy balance(NEB)and the function of neutrophil(polymorphonuclear granulocyte,PMN)in peripartal cows.The aim of the present study was to investigate the effects of supplementation with rumen-protected GLN on the metabo-lism and the phagocytosis function of PMN in peripartal cows.Twenty Chinese Holstein cows with similar age,parity,body condition score and expected date of delivery were selected and divided into CON(n=10,fed basal diet)and GLN groups(n=10,supplemented with 180 g/d of rumen-protected GLN).During the test period of 42 d,blood samples were collected at-21,-14,-7,0,7,14 and 21 d.Milk production was recorded at 7,14 and 21 d,respectively.The results showed that:serum GLN concentrations in peripartal cows continued to decrease until they increased after 7 d postpartum,suggesting that peripartal cows may be in a state of GLN deficiency.The serum GLN content of the cows increased significantly(P＜0.05)from-7 to 21 d after GLN supple-mentation,indicating that supplementation was effective.The blood glucose concentration of peri-partal cows increased on the day of parturition but decreased postpartum,and the concentrations of non-esterified fatty acid(NEFA)and β-hydroxybutyrate(BHBA)in the blood increased postpar-tum,which indicated that peripartal cows had a physiological NEB.Blood glucose concentration in-creased significantly(P＜0.01)at-7,14 and 21 d after GLN supplementation,and blood concen-trations of NEFA and BHBA decreased significantly(P＜0.01)postpartum,suggesting that peri-partal cows had a NEB alleviated by GLN supplementation.Serum malondialdehyde(MDA)con-centration of periparturient cow peaked on the day of parturition but declined postpartum,and the concentration of MDA reached a peak on the day of delivery but declined postpartum,and the total antioxidant capacity(T-AOC)level continued to decline until it increased on the day of parturi-tion,indicating that peripartal cows were in a state of oxidative stress.Serum MDA concentration significantly decreased(P＜0.05)and serum T-AOC level significantly increased(P＜0.05)after GLN supplementation,indicating that antioxidant capacity of peripartal cows was improved by GLN supplementation.The concentration of serum pro-inflammatory factor IL-β peaked on the day of parturition and then decreased,the concentration of TNF-α peaked on the day of parturition and then decreased,and the concentration of anti-inflammatory factor IL-10 continued to decrease,which indicated that there was an inflammatory reaction in the cows during the periparturient peri-od.However,the levels of IL-1β on the day of delivery and postpartum decreased significantly(P＜0.01),the levels of TNF-α decreased(P＜0.05),and the levels of IL-10 increased significantly(P＜0.01)throughout the peripartum period,which indicated that GLN supplementation im-proved the anti-inflammatory ability of periparturient dairy cows.The phagocytosis function of PMN in peripartal cows was significantly reduced from-14 d to the day of parturition(P＜0.01),and the phagocytosis function of neutrophils was significantly enhanced after GLN supple-mentation at-14(P＜0.05),14 and 21 d(P＜0.01),which indicated that intrinsic immune func-tion of peripartal cows was enhanced by supplementation of GLN.GLN supplementation did not significantly affect milk production of cows at three weeks postpartum.In conclusion,supplemental rumen-protected GLN alleviated NEB,alleviated systemic oxidative stress and inflammatory re-sponse,and enhanced phagocytosis of PMN in peripartal cows,which has some potential for clini-cal application.

Metabolic disorders and immunosuppression exist in peripartal cows,and glutamine(GLN)has the potential efficacy to improve the negative energy balance(NEB)and the function of neutrophil(polymorphonuclear granulocyte,PMN)in peripartal cows.The aim of the present study was to investigate the effects of supplementation with rumen-protected GLN on the metabo-lism and the phagocytosis function of PMN in peripartal cows.Twenty Chinese Holstein cows with similar age,parity,body condition score and expected date of delivery were selected and divided into CON(n=10,fed basal diet)and GLN groups(n=10,supplemented with 180 g/d of rumen-protected GLN).During the test period of 42 d,blood samples were collected at-21,-14,-7,0,7,14 and 21 d.Milk production was recorded at 7,14 and 21 d,respectively.The results showed that:serum GLN concentrations in peripartal cows continued to decrease until they increased after 7 d postpartum,suggesting that peripartal cows may be in a state of GLN deficiency.The serum GLN content of the cows increased significantly(P＜0.05)from-7 to 21 d after GLN supple-mentation,indicating that supplementation was effective.The blood glucose concentration of peri-partal cows increased on the day of parturition but decreased postpartum,and the concentrations of non-esterified fatty acid(NEFA)and β-hydroxybutyrate(BHBA)in the blood increased postpar-tum,which indicated that peripartal cows had a physiological NEB.Blood glucose concentration in-creased significantly(P＜0.01)at-7,14 and 21 d after GLN supplementation,and blood concen-trations of NEFA and BHBA decreased significantly(P＜0.01)postpartum,suggesting that peri-partal cows had a NEB alleviated by GLN supplementation.Serum malondialdehyde(MDA)con-centration of periparturient cow peaked on the day of parturition but declined postpartum,and the concentration of MDA reached a peak on the day of delivery but declined postpartum,and the total antioxidant capacity(T-AOC)level continued to decline until it increased on the day of parturi-tion,indicating that peripartal cows were in a state of oxidative stress.Serum MDA concentration significantly decreased(P＜0.05)and serum T-AOC level significantly increased(P＜0.05)after GLN supplementation,indicating that antioxidant capacity of peripartal cows was improved by GLN supplementation.The concentration of serum pro-inflammatory factor IL-β peaked on the day of parturition and then decreased,the concentration of TNF-α peaked on the day of parturition and then decreased,and the concentration of anti-inflammatory factor IL-10 continued to decrease,which indicated that there was an inflammatory reaction in the cows during the periparturient peri-od.However,the levels of IL-1β on the day of delivery and postpartum decreased significantly(P＜0.01),the levels of TNF-α decreased(P＜0.05),and the levels of IL-10 increased significantly(P＜0.01)throughout the peripartum period,which indicated that GLN supplementation im-proved the anti-inflammatory ability of periparturient dairy cows.The phagocytosis function of PMN in peripartal cows was significantly reduced from-14 d to the day of parturition(P＜0.01),and the phagocytosis function of neutrophils was significantly enhanced after GLN supple-mentation at-14(P＜0.05),14 and 21 d(P＜0.01),which indicated that intrinsic immune func-tion of peripartal cows was enhanced by supplementation of GLN.GLN supplementation did not significantly affect milk production of cows at three weeks postpartum.In conclusion,supplemental rumen-protected GLN alleviated NEB,alleviated systemic oxidative stress and inflammatory re-sponse,and enhanced phagocytosis of PMN in peripartal cows,which has some potential for clini-cal application.

Rotundic acid(RA),an ursane-type pentacyclic triterpene acid isolated from the dried barks of Ilex rotunda Thunb.(Aquifoliaceae),possesses diverse bioactivities.To further study its pharmacokinetics,a simple and sensitive liquid chromatography with triple quadrupole mass spectrometry(LC-QqQ-MS/MS)method was developed and validated to quantify RA concentration in rat plasma and tissue using etofesalamide as an internal standard(IS).Plasma and tissue samples were subjected to one-step protein precipitation.Chromatographic separation was achieved on a ZORBAX Eclipse XDB-C18 col-umn(4.6 mm×50 mm,5 μm)under gradient conditions with eluents of methanol:acetonitrile(1∶1,V/V)and 5mM ammonium formate:methanol(9∶1,V/V)at 0.5mL/min.Multiple reaction monitoring transitions were performed at m/z 487.30 → 437.30 for RA and m/z 256.10 → 227.10 for IS in the negative mode.The developed LC-QqQ-MS/MS method exhibited good linearity(2-500 ng/mL)and was fully validated in accordance with U.S.Food and Drug Administration bioanalytical guidelines.Dose proportionality and bioavailability in rats were determined by comparing pharmacokinetic data after single oral(10,20,and 40 mg/kg)and intravenous(10 mg/kg)administration of RA.Tissue distribution was studied following oral administration at 20 mg/kg.The results showed that the absolute bioavailability of RA after administration at different doses ranged from 16.1％to 19.4％.RA showed good dose proportionality over a dose range of 10-40 mg/kg.RA was rapidly absorbed in a dose-dependent manner and highly distributed in the liver.In conclusion,this study is the first to systematically elucidate the absorption and distribution characteristics of RA in rats,which can provide additional information for further development and evaluation of RA in drug metabolism and pharmacokinetic studies.