Glycerol-Producing Yeast Culture Alleviates Heat Stress In Dairy Cows And Its Mechanism

Heat stress (HS) is considered a primary factor negatively affecting performance, reproductive ability and immunity of dairy cows during the summer, especially in the south area where shows continually high temperature and humidity and cause huge economic loss to dairy industry. Thus, it becomes one of the research focuses in husbandry to study on the mechanism of HS and search for nutritional strategies to alleviate HS during the recent years. It has been demonstrated that dietary supplementation of yeast culture (Saccharomyces cerevisiae) could improve milk yield, milk protein, milk lactose and solids-not-fat in HS dairy cows. Forthermore, foreign researchers have indicated that HS cows have an extra need for glucose and that any dietary component which could increase propionate (a primary glucogenic precursor) production, without reducing ruminal pH, would probably increase milk yield. On the basis of the above rationales, we hypothesize that glycerol as another glucogenic precursor would also probably increase milk yield of HS cows. However, considering glycerol used currently in husbandry is mainly produced from chemical industry, and has negative effects on cows’production and health, even human health, we have isolated Saccharomyces cerevisiae, and used it to prepare a glycerol-producing yeast culture (GY) by fermentation under a hyperosmotic environment, which contained glycerol, yeast, amino acids and vitamin, as well as metabolic products of yeast. This study aimed to evaluate the effects of supplemental GY on physiological indicators, performance, plasma metabolites, antioxidant abilities, hormonal level, immune function, and heat shock protein and apoptosis-related genes expressions in HS lactating cows in order to search for a new pathway to alleviate HS.Experiment 1. Effects of supplemental glycerol-producing yeast culture on physiological indicators and performance in lactating Holstein cows under heat stressThis study aimed to evaluate the effects of supplemental glycerol-producing yeast culture in diet on physiological indicators and performance in lactating Holstein cows under heat stress. It was conducted as a randomized complete block design comprising a continuous period of 60 d (from June 9,2012 to August 8,2012), during which time all 30 Holstein cows received the same basal diet. Based on parity, previous milk yield, and DIM, these cows were randomly allocated to 3 treatments (10 cows per treatment): control (C, no additive),1 L/d of common yeast culture (Y,33.1 g yeast) per cow, and 2 L/d of glycerol-producing yeast culture (GY,153.2 g glycerol and 31.6 g yeast) per cow. All supplements were individually hand mixed into the TMR offered at the morning feeding. During the 60-d experiment, values of air temperature and relative humidity inside the barn were recorded hourly every 3 d to calculate temperature-humidity index (THI). Weekly rectal temperatures (RT) and respiration rates (RR) and daily DMI and milk yield were recorded for all cows. Milk samples were taken twice monthly, and BW and BCS were obtained on d 0 and 60. In this experiment, THI values indicated cows experienced a moderate heat stress. The RT of control tended to be greater compared with Y and GY at 1400 h (P< 0.15) but was similar to these 2 treatments at other times. Dietary treatments had no effect on RR at any time. Cows supplemented with Y and GY had greater yields of milk,4% fat-corrected milk and milk fat, and milk fat percent than control (P< 0.05). The loss in BW tended to be lower for GY compared with control and Y (P< 0.15). No dietary treatments effects were observed on RR, DMI and BCS change. The results showed that dietary glycerol-producing yeast culture improved the lactation performance of heat stressed cows.Experiment 2. Effects of supplemental glycerol-producing yeast culture on blood biochemical indicators, antioxidant status, and hormonal level in lactating Holstein cows under heat stressThis study aimed to evaluate the effects of supplemental glycerol-producing yeast culture on blood biochemical indicators, antioxidant status, and hormonal level in lactating Holstein cows under heat stress. Experiment design was the same as the experiment 1. Blood samples from all cows were obtained after the morning milking and before morning feeding on d 0,15,30,45, and 60. Plasma was collected for the analysis of glucose, urea nitrogen (UN), triglyceride (TG), total cholesterol (TC), total protein (TP), albumin (ALB), globulin (GLO), Na+ and K+ concentrations, alkaline phosphatase (ALP), alanine aminotransferase (ALT), aspartate transaminase (AST), superoxide dismutase (SOD) activities, malonaldehyde (MDA) and total antioxidant capability (T-AOC); erythrocyte lysates were obtained for GSH analysis; serum was analyzed for triiodothyronine (T3), thyroxine (T4), cortisol (Cor) and insulin (INS). The results showed that cows supplemented with Y and GY had higher concentrations of plasma TP and ALB (P< 0.05) and tendencies towards lower plasma UN and higher GSH concentrations (P< 0.15) compared with control. Supplementing GY also increased plasma glucose and decreased NEFA concentrations compared with control (P< 0.05). Lower plasma NEFA concentration (P< 0.05) and a tendency towards greater plasma glucose concentration (P< 0.15) were observed in GY relative to Y. Treatments had no effects on other parameters.Experiment 3. Effects of supplemental glycerol-producing yeast culture on the immune function in lactating Holstein cows under heat stressThis study aimed to evaluate the effects of supplemental glycerol-producing yeast culture on the cellular and humoral immunity in lactating Holstein cows under heat stress. Experiment design was the same as the experiment 1. Blood samples from all cows were obtained after the morning milking and before morning feeding on d 0,15,30, 45, and 60. Serum was analyzed for the levels of IL-2, IFN-y, TNF-a and IgG. Lymphocytes were separated from the anticoagulated blood and cultured for the analysis of lymphocyte transformation rate. The results showed that supplementing Y and GY enhanced the T cell transformation induced by ConA (P< 0.05), and the stimulation index was significantly higher than control (P< 0.05). Cows supplemented with Y and GY had greater IL-2 level in lymphocytes on d 30 and 45 compared with control (P< 0.05). A tendency towards higher IL-2 level was observed in GY on d 60 compared with control (P< 0.15). Both dietary Y and GY improved IgG level on d 45, being higher in GY compared with control (P< 0.05) and Y(P< 0.15). There were no dietary treatment effects on levels of IFN-y and TNF-a.Experiment 4. Effects of supplemental glycerol-producing yeast culture on heat shock and apoptosis-related genes expressions in peripheral blood lymphocytes of lactating Holstein cows under heat stressThis study aimed to evaluate the effects of supplemental glycerol-producing yeast culture on heat shock protein and apoptosis-related genes expressions in peripheral blood lymphocytes of lactating Holstein cows under heat stress. Experiment design was the same as the experiment 1. Blood samples from all cows were obtained after the morning milking and before morning feeding on d 0,15,30,45, and 60. Expressions of HSP70, HSF1, Bcl-2 and Bax-a mRNA in peripheral blood lymphocytes were quantitatively determined by real-time PCR. The results indicated that supplementing Y and GY reduced expressions of HSP70 mRNA on d 30,45 and 60 (P< 0.05), HSF1 mRNA on d 45 and 60, as well as Bax-a mRNA on d 45 (P< 0.05). Cows supplemented with GY had lower Bax-a mRNA expression on d 30 than control (P< 0.05) and reduced HSP70 mRNA expression on d 45 compared with Y (P< 0.05). Furthermore, supplementing Y and GY significantly reduced mean expressions of total HSP70, HSF1 and Bax-a mRNA in peripheral blood lymphocytes (P< 0.05) and increased the rate of Bcl-2 mRNA/Bax-a mRNA expression (P< 0.05); Lower total HSP70 expression (P< 0.05), tendencies towards lower HSF1 and Bax-a mRNA expression and higher rate of Bcl-2 mRNA/Bax-a mRNA expression (P< 0.15) were observed in GY compared with Y. It was concluded that supplementing Y and GY reduced the susceptibility of cows to heat stress and enhanced thermotolerance, being better in GY compared with Y.Experiment 5. Effects of different concentrations of zymosan on peripheral blood lymphocytes in vitro under heat stressPeripheral blood lymphocytes from non-heat stressed cows were incubated with 0 (control),10,30,60 and 90 μg/L zymosan, in or without the presence of PHA, at 37℃ (non heat stress, NHS) or 45℃ (heat stress, HS). The result showed that lower proliferation rate of lymphocytes, GSH concentration, SOD activities, IL-2 production and Bcl-2/Bax-a mRNA rate but higher MDA concentration and HSP70 mRNA expression were observed in HS compared with NHS (P< 0.05). Compared with control, zymosan increased SOD activities and Bcl-2/Bax-a mRNA rate but decreased MDA concentration and HSP70 mRNA expression in HS (P< 0.05), and improved PHA-induced proliferation of lymphocytes and IL-2 production in NHS and HS (P< 0.05). It was concluded that zymosan could improve redox state of lymphocytes stimulated by PHA, reduced heat stress-induced oxidative damage, regulated apoptosis-related genes expressions, and enhanced the adaptation of to lymphocytes heat stress conditions.