| This study was conducted to study the effect of feeding Bupleurum extract (BE) on production performance, serum hormone, material metabolism, antioxidate status and immune function of cows under heat stress and the effect of Saikosaponin a (SSa) and d (SSd) on antioxidant activity and the expression of heat shock protein gene level of heat-induced bovine mammary epithelial cells. The object of this study was to approach the mechanism that BE may possibly reduce heat stress of cows, find out the active ingredient and provide theoretical basis and reference for the application of heat stress dairy cows. The research is mainly including the following aspects:1. Effects of BE on lactation performance and blood metabolism. Forty lactating Holstein cows were randomly divided into one of four groups (n=10) according to milk yield (37.5±1.8 kg/d), days in milk (75±15 d) and parity (1.7±0.4). One of four treatment diets, assigned randomly to one of four groups, consisted of BE supplementation at 0,0.25, 0.5 or 1.0 g/kg based on dry matter (DM). The experiment lasted for 10 wks in hot summer. Cows were housed in a tie-stall barn and were individually fed the treatment diets. During the experiment, daily average temperature-humidity index (THI) was overall more than 72, which indicated that the cows were subjected to heat stress model during 10 wks experiment. The results revealed that average respiration rates (RR) were decreased for cows fed BE supplementation, especially significantly for cows fed at 0.5 g/kg(P< 0.05). Moreover, average rectal temperatures (RT) were decreased regardless of levels at any time, especially at the hot time of a day (P< 0.05). Additionally, cows supplemented with BE increased DMI and milk production compared with control (P< 0.05), of which cows fed BE at 0.25 and 0.5 g/kg significantly improved milk yield.Supplemented with BE decreased COR level (P< 0.05), but increased T3, PRL, GH and LEP content (P< 0.05). BE supplementation has the tendence to reduce the GLU content (P< 0.1), but has no effect on the level of T4, INS, NPY, IGF-I and HSP70 (P> 0.05). Cows supplemented with BE increased the level of TP(P< 0.05), but had no effect on the concentrations of GLU, NEFA, TG, TC, LDL-C and HDL-C (P> 0.05). Cows fed BE at 0.25 g/kg significantly improved ALB content (P< 0.05), and 0.25 and 0.5 g/kg significantly lowed BUN content(P< 0.05), BE supplementation improved K+,but had no effect on Mg2+ content (P> 0.05). Cows fed BE at 0.5 and 1.0 g/kg significantly improved Na+, and Ca2+ concentrations and 0.5 g/kg significantly improved Cl" concentration (P< 0.05). Cows fed BE had lower P level(P< 0.05).No difference was observed in WBC, MC V, MCHC, PLT and MPV contents (P> 0.05) among dietary groups. RBC, HGB, HCT and RDW contents was improved in 0.5 g/kg BE group (P< 0.05). MCH content was increased (P< 0.05) especially with 1.0 g/kg BE compared with the control. BE supplementation improved SOD and GSH-Px activity (P< 0.05)and had tendence to increase T-AOC activity and decreased MDA content, especially at 0.25 g/kg level (P< 0.05). IgA, IgG, IL-2, IL-4 and IL-6 concentrations in BE supplementation treatments were higher than in control especially at 0.25 and 0.5 g/kg levels (P< 0.05). No difference was observed in IgM, TNF-a, the percentage of CD4+, CD8+and CD4+/CD8+ ratio (P> 0.05) among dietary groups (P> 0.05).2. Effects of Saikosaponin a (SSa) and SSd on antioxidant enzymes activity and the expression of heat shock protein. Put the cells at 38℃ as the control group, the medium added 0,0.1,1.0 μmol/L SSa and 0,0.01,0.1 μmol/L SSd as treatment group, the cells induced by 42℃ high temperture for 1,4,8,12, and 24 h to detect the activity of SOD, GSH-Px and CAT and the expression of heat shock protein gene in cells and MDA content in culture medium. The results indicated that:the activity of SOD, GSH-Px and CAT were significantly decreased and MDA was increased in 42℃ treatments for different time compared with the control (P< 0.05). Supplementation with SSa and SSd increased (P< 0.05) SOD, GSH-Px, and CAT activity and decreased (P< 0.05) MDA content compared with 42℃ positive control group, of which cell enzyme activity increased significantly in SSa and SSd treatments within 4h before heat treatment (P< 0.05), and MDA content was significantly decreased at 0.01 and 0.1μmol/L SSd treatment for 12h heat treatment (P< 0.05). The expression of HSF-1, HSP27, and HSP70 mRNA up-regulated within 4h before heat treatment (P< 0.05), and HSP90 mRNA up-regulated significantly at 8h (P< 0.05). HSP70 and HSP90 mRNA level higher HSP70 and HSP90 mRNA level significantly higher at SSa and SSd groups with 4h before heat treatment (P< 0.05). The Hsp27mRNA lever in SSa and SSd group were higher in the 1h heat treatment (P< 0.05), while the HSF-1mRNA lever in SSa and SSd group were higher than that in 38℃ and 42℃ treatment. Therefore, SSa and SSd improved the SOD, GSH-Px and CAT activity, reduced the MDA content, and improved the gene expression abundance of heat shock protein family, which suggeste that SSa and SSd have good protection effect on the oxidative stress and cell damage caused by thermal stress.Overall, BE improved feed intake, milk production, metabolism, antioxidant ability, and immune function in heat-stressed cows. Therefore, BE may alleviate side effects of high tempture on lactating cows, which showed a dose-response effect and the optimal dose was 0.25～0.5 g/kg. SSa and SSd improved antioxidant enzymes activity and increased the expression of heat shock protein family genes of dairy cow mammary epithelial cells, which may be the active ingredient in BE and which indicated that there existed some other active ingredients still to further study. |
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