The Effects Of Glycerol-Enriched Yeast Products On Energy Balance Of Transition Dairy Cows And Its Mechanism

Because of ruminal metabolism characteristic, glucose is directly broken down by ruminal microorganisms but not absorbed across the ruminal wall. Ninety percent of glucose needed for the ruminants depended on the gluconeogenesis. During the perinatal period, especially the high-producing dairy cows, energy demands of fetal growth and lactation are increased, but the dry matter intake (DMI) may decrease. Thus, dairy cows frequently enter a state of negative energy balance, in which energy intake can not meet the energy requirements. To compensate for this energy deficiency, dairy cows increase the mobilization of body fat. However, excessive lipolysis will result in many energy metabolic diseases. Glycerol, an important glucogenic precursor, is closely related with glucose metabolism, and can effectively solve the problems of animal energy metabolism by feeding to ruminants. Recently, glycerol has been excessively produced as a byproduct of biodiesel production, making it a more attractive energy supplement for ruminants. However, this industrial glycerol contains several deleterious impurities, such as methanol and heavy metal, which will affect the health and performance of animals, even affect the health of people. Fermental glycerol is produced when some microorganism metabolize the starch, carbohydrate, or agricultural byproducts under specific environment. Its final broth has many advantages such as free of deleterious impurities, free-extraction and purification of glycerol. It not only contains glycerol, but also contains beneficial microbial strains. It’s a green feed additive without pollution and residue, and will widely used in ruminant husbandry.In this study, glycerol-enriched yeast product (GYP), a microbial feed additive mitigating negative energy balance of ruminants, is successfully prepared by optimization of fermental technology and continuous fed-batch fermentation with a high glycerol-producing yeast strain(Saccharomyces cerevisiae, NAU-ZH-GY1) under hyperosmotic environment. The effects of dietary supplementation of GYP on the performance, blood biochemical indicators, expression of mRNA and protein of key gluconeogenic enzyme and glycerol kinase in liver, rumen fermentation characteristics and the changes of rumen microorganisms of transition dairy cows were investigated in this study to provide the theoretical basis and data support for further application of GYP in ruminants husbandry.Experiment 1. Optimization of fermental technology of GYP and its preparationThe fermental technology was optimized through orthogonal design and continuous fed-batch fermentation methods to maximize the glycerol yield and biomass. The concentration of glycerol and viable count of yeast were determined every 15 d to estimate the stability of GYP. The result showed that the optimal fermentation conditions were as follows:temperature at 30℃, constant pH at 5, and the dissolved oxygen (DO) at 80%. Under these fermentation conditions, glycerol yield, dry cell weight (DCW), and yeast viable count reached 38.7 g/L,13.1 g/L,9.4 1g CFU/ml, respectively. GYP containing 75.8 g/L glycerol and 15.8 g/L biomass was successfully obtained at 72 h through replenishing medium at 24 h and 48 h of fermentation. The glycerol concentration and viable count in GYP did not change when it was stored at room temperature for 3 months. When the GYP was stored for 90 d, its glycerol concentration was 74.5 g/L and yeast viable count was no less than 8 lg CFU/mL, which indicated that the GYP has good stability and can be stored more than three months without bad.Experiment 2. Effects of supplementing GYP on the performance of transition dairy cowA trial was performed to determine the effects of supplementing GYP on the performance of transition dairy cow. Forty-three health multiparous Holstein dairy cows were randomly allocated to four treatments on the basis of weight, parity, time of calving and milk yield of last lactation period:control (C, no additive),150 g/d glycerol (G, food grade,0.998 g/g glycerol),1 L/d yeast culture (Y, containing 31.1 g yeast), and 2 L/d glycerol-enriched yeast products (GYP, containing 151.6 g glycerol and 30.6 g yeast). All additives were topdressed and hand-mixed into the upper one-third of TMR in the morning feeding from d-14 to+28 relative to calving. DMI was measured and recorded daily for each cow throughout the experimental period. Body weight (BW) and body condition scores (BCS) were measured on d-14 of expected calving and d+28 of calving. Calving coefficient (CC) was scored at the time of parturition. Milk yields were recorded daily from parturition to d 28 postpartum and milk samples from each cow were collected and measured weekly from three consecutive milking within a day. The results showed that there were no differences of DMI, change of BW and BCS, and CC among experimental groups (P> 0.05). Dietary supplementation of G, Y and GYP did not affect the milk yield,4% fat-corrected milk (FCM), and levels of milk lactose, milk solids-not-fat and milk urea-N (P> 0.05). Compared with control group, dairy cows in Y and GYP group had higher percentages of milk fat and milk protein (P< 0.05), and higher yield of milk protein (P< 0.05); in addition, cows fed GYP had lower somatic cell counts (SCC) in milk (P< 0.05) than control and G group. These results indicated that dietary supplementation of GYP can significantly improve the milk quality and have preventive and therapeutic effect on the subclinical mammitis of dairy cows.Experiment 3. Effects of supplementing GYP on the metabolic indicators in blood of transition dairy cowA trial was performed to evaluate the effects of supplementing GYP on the energy balance and incidence of ketosis in transition dairy cows. The experiment design was the same as experiment 2. All additives were topdressed and hand-mixed into the upper one-third of TMR in the morning feeding from d -14 to +28 relative to calving. Blood samples from coccygeal vein for examination of blood biochemical indicators were collected approximately 3 h after morning feeding on d -14,-7,-1 of expected calving, and d 7,14,21,28 of actual calving. Subclinical and clinical ketosis was distinguished by the beta-hydroxybutyrate acid (BHBA) levels at 1.0 mmol/L and 2.6 mmo/L, respectively. The results showed that dietary supplementation of G, Y and GYP did not affect the levels of plasma TC, TG, TBiL, DBiL, Crea, and ALT(P> 0.05), however, dietary supplementation of Y and GYP had higher levels of plasma Ca and TP (P< 0.05), a tendency towards higher level of plasma P(P =0.10), and a tendency towards lower levels of plasma Urea and AST (P= 0.07, P= 0.10, respectively). Compared with the control and Y group, dairy cows in the G and GYP group had higher plasma glucose level (P< 0.05), and lower BHBA and nonesterified fatty acids (NEFA) level (P< 0.05). Relative to control group, there was a lower incidence of subclinical and clinical ketosis in GYP group. These results indicated that dietary supplementation of GYP can significantly improve the energy balance and effectively lower the incidence of ketosis of transition dairy cow.Experiment 4. Effects of supplementing GYP on key gluconeogenic enzyme and glycerol kinase in liver of transition dairy cowA trial was performed to reveal the regulation mechanism of supplementing GYP on the gluconeogenesis of transition dairy cows by investigating the activity and expression level of mRNA and protein of pyruvate carboxylase (PC), phosphoenolpyruvate carboxykinase (PEPCK), and glycerol kinase (GK) in liver. The experiment design was the same as experiment 2. All additives were topdressed and hand-mixed into the upper one-third of TMR in the morning feeding from d-14 to+28 relative to calving. Five cows were randomly selected from each group for liver biopsy samples collection by biopsy instruments on d 14 after calving. Liver samples were usded to determine the activity and expression level of mRNA and protein of PC, PEPCK, and GK by spectrophotometry, Real-time PCR, and Western blot, respectively. The results showed that cows in GYP group had higher activity of PEPCK, expression level of mRNA and protein of cytosolic PEPCK (PEPCK-C) (P< 0.05), higher GK activity (P< 0.05), and a tendency towards higher expression level of GK mRNA{P= 0.088) relative to cows in control group, but without difference for PC in the two group (P> 0.05). These results indicated that dietary supplementation of GYP increase the blood glucose level and improve the energy balance of transition dairy cows by up-regulating the PEPCK activity, expression level of mRNA and protein of PEPCK-C, and the activity and expression level of mRNA of GK.Experiment 5. Effects of supplementing GYP on ruminal fermentation performance and microbial flora of transition dairy cowA trial was performed to reveal the regulation mechanism of supplementing GYP on the ruminal fermentation performance and internal environment of transition dairy cows by investigating the change of ruminal fermentation parameters and microbial flora. The experiment design was the same as experiment 2. All additives were topdressed and hand-mixed into the upper one-third of TMR in the morning feeding from d-14 to+28 relative to calving. Ruminal fluid from individual cows for examination of fermentation parameters and microbial flora was collected approximately 4 h after feeding on d-14 and d 14 relative to calving by rumenocentesis. Total genome DNA was extracted from obtained ruminal fluid.16S rDNA was amplified with bacteria universal primer 27F/342R. The amplified products were digested with restriction endonuelease of Hha I and the enzyme-digested products were evaluated by restriction fragment length polymorphism (RFLP).16S rDNA of main bacteria in rumen were amplified respectively, and quantified by real-time PCR. The results showed that dietary supplementation of G, Y, and GYP did not affect the ruminal pH and the molar percentage of acetate and butyrate (P> 0.05) after calving. Cows in the G, Y, and GYP group had higher volatile fatty acid and molar percentage of propionate (P< 0.05), lower ratio of acetate to propionate (P< 0.05), and a tendency towards lower NH3N concentration (P= 0.08) after calving than cows in control group, indicated that dietary supplementation of GYP can significantly improve the ruminal fermentation performance and fermentation pattern of transition dairy cows. Relative to before calving, the bacteria of Phylum Firmicutes were significantly increased in all groups after calving (P< 0.05), and the bacteria of Phylum Proteobacteria were significantly decreased (P< 0.05). Dietary supplementation of G, Y, and GYP had higher bacteria of Phylum Firmicutes after calving than control (P< 0.05), but had no affect on the bacteria of Phylum Proteobacteria, Phylum Bacteroidetes, and Phylum Tenericutes (P> 0.05). Compared with the control, there were higher copies of Streptococcus bovis (formation of lactic acid), and Selenomonas ruminantium and Megasphaera elsdenii (degrading lactate acid to propionate) in GYP group (P< 0.05); Cows in the G and Y group also had higher copies of Selenomonas ruminantium and Megasphaera elsdenii (P < 0.05), but only had a tendency towards higher copies of Streptococcus bovis (P< 0.15). These results indicated that dietary supplementation of GYP can significantly improve the diversity of rumen microbial flora, increase the quantities of propionate-generating bacteria to supply gluconeogenesis with more precursor, following the improvement of energy balance of transition dairy cows.