The transition period from late gestation to early lactation is a critical period for the dairy cows to challenge many metabolic and infectious diseases. Generally, high-production dairy cows have more serious NEB and hypocalcemia during the transition period, which also considerably decrease the full productivity potential of dairy cows. Thus, these metabolic disorders should be prevented or treated immediately [10]. Propionate supplementation provides better energy supply by being converted to glucose in liver and support lactose synthesis in the mammary gland [15]. Calcium propionate, as a source of both calcium and energy, has been administered for dairy cows for prevention or treatment of hypocalcemia and ketosis [10].
Lactation performance
In the present study, the MCaP group had more than 3 kg/d milk yield than the CON group, which indicated the effectiveness of calcium propionate in alleviating NEB in early lactation dairy cows. Martins et al. [15] also found that the Holstein cows fed calcium propionate had greater milk yield which is in line with our results. After calving, large amounts of glucose are required for the synthesis and secretion of steeply increasing milk in dairy cows [23]. Propionate produced from ruminal fermentation is quantitatively the greatest contributor (60%-74%) to gluconeogenesis during the periparturient period [24]. Propionate was absorbed through the rumen wall and transported to the liver for gluconeogenesis. Glucose is the precursor for the synthesis of lactose, which is the primary osmoregulatory of milk synthesis. The available glucose is important to improve dairy cows milk yield and immunity during the transition period. The supplementation of propionate not only decreased the lipolysis and the formation of ketone bodies, also promoted the milk synthesis. However, Liu et al. [14] observed that feeding different amounts of calcium propionate (100 g/d, 200 g/d, and 300 g/d) to the Holstein cows during the first 63 days of lactation didn’t affect the DMI, milk production and composition. Peralta et al. [25] reported calcium propionate-propylene glycol drenching had no significant effect on milk production and milk component, but increased the plasma calcium concentration. The different results of the studies may be related to the amount of supplementation, basal diet, method of feeding, and the conditions of the experiment’s animals. Hypophagic effect of propionate [26] and the excess dietary calcium concentration [16] in the early lactation explain that the milk yield of the cows offered 500 g/d calcium propionate was lower than that of 350 g/d.
In this study, the supplement with calcium propionate did not significantly change the milk compositions of milk fat, milk protein, milk lactose and MUN. But the increasing available glucose and calcium by the feeding of calcium propionate could possibly support postpartum immune function, as the SCC of the calcium propionate treatment groups decreased. McNamara and Valdez [27] reported that calcium propionate (125 g/d) tended to decrease milk fat percentage, milk lactose, milk protein and milk SCC. The ratio of milk fat to protein beyond the values between 1.35 to 1.50 was considered to be at higher risk of energy deficiency [28]. The ratios of milk fat to protein in the present study were all in the threshold on d 35 of lactation.
Therefore, the higher milk yield and the decreased SCC in MCaP group suggested the optimal calcium propionate supplementation in early lactation dairy cows was 350 g/d. And, calcium propionate is an effective additive in improving milk performance and immunity.
Blood parameters related to NEB and calcium homeostasis
In the early lactation, the nutrient intake could not meet the high energy requirements for the milk synthesis, leading to NEB. Consequently, the body will increase the production of endogenous glucose to maintain glucose homeostasis [23]. The serum NEFA and BHBA concentrations were negatively correlated with the energy balance and were used as the indicators of energy balance and body fat mobilization. In this study, the lower concentration of NEFA in the MCaP and HCaP groups suggested that the decreasing body fat mobilization in early lactation. But there were no significant differences in serum glucose, BHBA and insulin in the present study. The results of Liu et al. [14] showed that the calcium propionate supplementation was beneficial to improve energy status for the higher blood glucose, lower blood BHBA and NEFA, and lower concentration of urine ketones. Oba and Allen [29] reported that propionate infusion linearly increased plasma insulin and glucose concentrations for early lactation. But the results of Rivas et al. [30] did not observe the supplement of gluconeogenic precursors (containing propylene glycol, glycerol, and calcium propionate, et al) during the transition changed the plasma glucose, BHBA, NEFA, and insulin. We also did not observe significantly difference in serum glucose, BHBA and insulin may be attribute to the effect of calcium propionate in stimulating milk yield, which improve the requirement of energy.
The healthy liver metabolic function plays a key role to maintain the healthy and normal production performance for the dairy cows in early lactation. The serum ALT and AST are important indicators in amino acid metabolism and gluconeogenesis in the liver [31]. The serum ALT and AST concentrations will increase when the hepatocytes are damaged and the liver function decreases. In our study, the significantly decreased ALT concentration in MCaP group and the significantly increased AST in HCaP group indicated that 350 g/d calcium propionate improved liver function, however 500 g/d calcium propionate impaired liver metabolic function.
During the early lactation, for the dietary calcium absorption, bone calcium mobilization and renal calcium resorption cannot meet the sudden increasing calcium demand for the production of colostrum and milk, therefore, most dairy cows have some degree of hypocalcemia [32, 33]. Calcium chloride is widely used to prevent hypocalcemia by suppling a soluble form of calcium and acidifies, but it could cause a severe acidosis and may irritate the oral and ruminal mucosa [34]. Feeding calcium propionate at calving and 12 h after calving has been proved to reduce the number of cows suffered subclinical hypocalcemia [12]. In this study, the increasing in the blood glucose and calcium concentrations was expected with the calcium propionate supplement, because it would increase energy and calcium intake. However, the numerically increases were not significant. In the multiparous cows, blood calcium concentration was decreasing 1 to 2 d before parturition and reached its nadir between 24 and 48 h after calving [3]. Then the blood calcium concentration did not vary greatly and were within a physiological range (2.1 and 2.5 mmol/L) [35]. The dairy cows can homeostatic control blood glucose and calcium in a relative stable status by mobilizing the body fat for energy and body bone for calcium in early lactation. Blood Mg and P play important roles in the milk fever. In this study, the serum Mg and P concentrations also had no difference among the treatments. This may be the dairy cow can also maintain the serum Mg and P in a relatively stable state by mobilizing body reserves in the period. Under normal physiological conditions, serum calcium is tightly regulated by PTH, calcitriol and CT [36]. The increase PTH levels stimulate osteoclast proliferation and contribute to calcium transferred from bones, which increases the size of the lacunar area. In this study, the HCaP group tended to have lower serum PTH concentration than the CON group suggested that lower calcium was released from bones. Osteoporosis is defined as a skeletal disorder of compromised bone strength predisposing those affected to an elevated risk of fracture [37]. When a large amount of bone calcium loses through milk production, the bone density decreases and is more susceptible to fracture. The serum ALP is a main biomarker for the diagnosis of metabolic bone disease. The higher blood ALP activity is associated with bone metabolism in response to increased calcium demands during early postpartum [38]. The lower bone mineral density is associated with the higher serum ALP levels [39]. In this study, the decreasing ALP concentrations in the calcium propionate treatments suggested the decreasing of calcium mobilization from bones, which could prevent the risk of osteoporosis. The supplementation of calcium propionate may have increased the amount of calcium obtained from the intestines for the dairy cows in early lactation, thereby reducing calcium transferred from bones.
Therefore, suppling appropriate calcium propionate has the advantage of alleviating NEB, improving liver function and decreasing calcium mobilization from bones for dairy cows in early lactation, especially at the dose of 350 g/d.
Serum antioxidant indices
T-AOC is a relatively independent index that describes the dynamic balance between oxidation and antioxidant activity in the blood circulation [40]. The serum SOD and GSH-Px as the key enzymes of antioxidant system can scavenge free radicals generated from oxidant stress, reduce oxidative damage, and maintain cell structure [41]. The CAT is an antioxidant enzyme that catalyzes the conversion of H2O2 into H2O and O2. MDA, a product of lipid peroxidation, is another biomarker of oxidative damage. The dairy cows in transition period were particularly vulnerable to oxidative stress for the imbalance between oxidation and the antioxidant systems. The present study showed that calcium propionate supplementation decreased serum T-AOC, SOD, GSH-Px, and CAT activity and increased the serum MDA concentration (especially the MCaP group), which suggested that calcium propionate decreased the antioxidant capacity. The metabolic level increased with the increasing of milk yield. The reactive oxygen species (ROS) generation were also increased due to the increasing cellular metabolism. Higher-producing animals have higher metabolic activity rates and greater loss of antioxidants in the milk [42]. Therefore, in the present study, the MCaP group had the highest blood MDA concentration and the lowest activity of serum T-AOC, SOD, GSH-Px, and CAT.
Serum metabolomics profiling
In dairy cows, the blood metabolites are easily affected and can be directly used to evaluated the metabolic pathway changed by the dietary treatment [43]. To better understand the physiological and biochemical status of the different treatments, metabolomics was used in the present study. For the high sensitivity and high resolving power, LC/MS based metabolomics is now widely used to screen the changes in the biological systems [44]. When the feeding level of calcium propionate increased from 350 g/d to 500 g/d, the milk yield was decreased. Therefore, we not only compared the difference metabolites between the treatments and the CON, but also compared the differences between the HCaP and MCaP groups.
In this study, the bile acids constituents related metabolites in the groups of LCaP (glycocholic acid, glycodeoxycholic acid, glycochenodeoxycholate, taurocholate, deoxycholic acid) and MCaP (bilirubin, glycocholic acid, glycine) were significantly increased compared with CON group. When compared with the MCaP group, the bile related metabolites of taurodeoxycholic acid, tauroursodeoxycholic acid, taurochenodeoxycholate in HCaP group were significant decreased. Considering the milk yields in the LCaP and MCaP groups were increased compared with the CON group, and that in the HCaP group was decreased compared with MCaP group, we speculate that the appropriate level of calcium propionate on milk yield and other performance was related to the generating of bile acids. Bile acids, a large group of cholanic acid skeleton synthesized from cholesterol in the liver, are divided into unconjugated and conjugated bile acids which are the corresponding glycine or taurine conjugates [45]. The primary bile acids including cholic acid and chenodeoxycholic acid were biosynthesized in liver. Then the water solubility bile aids were conjugated with either taurine or glycine to be glycocholic acid, taurocholic acid, glycinodeoxycholic acid and taurodeoxycholic acid, respectably. The conjugated bile acids were released by the gallbladder into the duodenum after the meal and were deconjugated and dehydroxylated to secondary bile acids such as deoxycholic acid and lithocholic acid by the gut microbiota in small and large intestine [45]. The secondary bile acids are reconjugated with taurine or glycine to be conjugated bile acids. Bile acids not only are essential for the excretion, absorption, and transport of fats in the liver and intestine [43], but also play a key role as signaling molecules in modulating epithelial cell proliferation, gene expression, and lipid and glucose metabolism in the liver, intestine, muscle and brown adipose tissue [45].The bile acids can help to regulate energy, glucose, lipids, lipoprotein metabolism, intestinal integrity and immunity [46, 47].
Bile acids can promote dietary lipids and fat-soluble vitamins absorption in mammals by acting as “intestinal soaps” [47], which is benefit for alleviating NEB in early lactation. Increasing secretion of bile acids [48] or supplementation of bile acids in diets [49] were proved to enhance the fat digestion and absorption in high fat containing dietary. The molecules of bile acids have both hydrophilic and lipophilic properties. The amphoteric structure makes it a kind of surface-active emulsifier, which can effectively emulsify lipids and accelerate the absorption and digestion of lipid nutrients. In the postprandial state, the gallbladder contracts and releases bile acids (in the form of mixed micelles containing bile acids, cholesterol and phospholipids) into the intestinal lumen, thus facilitating the emulsification and absorption of lipids in the small intestine [50]. Improving fat level is a widely used strategy to increase dietary energy density in early lactation, resulting in increased energy intake, reduced body fat mobilization, and improved energy balance of dairy cows [51, 52]. The increase of dietary fat digestion and absorption plays a key role in improving energy efficiency and minimizing NEB, especially in high fat supplementation diet in early lactation. In addition, the increasing fat digestion also benefits the other nutrient digestion, since the unhydrolyzed fat particles would make the digestive enzymes unavailable to the rest feed ingredients. Therefore, the supplement of calcium propionate at 200 and 350 g/d improved the synthesis of bile acids and contributed to alleviating NEB for the dairy cows in early lactation. Xu et al. [53] found a high intake of calcium in veal calves would reduce apparent fat digestibility by 5.6% and increase bile acid excretion in feces by 90%, because the high calcium dietary increased the amount of insoluble calcium, magnesium, and phosphate complexes in the intestinal lumen. The insoluble complexes interrupted the enterohepatic cycle of bile acids and thus increased bile acid excretion in feces, which would decrease the availability of bile acids for the process of fat digestion [53]. In this study, the bile acids in HCaP group were decreased compared with MCaP group may be because the high calcium feeding level inhibited reabsorption of bile acids and then reduced the fat digestibility.
Bile acids play a key role in preventing fatty liver in early lactation. Fatty liver is a common metabolic disorder disease in modern high-yielding dairy cows, occurring from hepatic uptake of lipids exceeding the oxidation and secretion of lipids by the liver [54]. Bile acids are endocrine signaling molecules that affect host physiology via activation of bile acid receptors such as the Takeda G-protein-coupled receptor 5 (TGR5) and nuclear hormone receptors such as farnesoid-X-receptor (FXR) [55]. The activation of hepatic FXR can induce upregulation of the expression of enzymes related to β-oxidation of fatty acids, and subsequently reduce lipid accumulation [56]. Watanabe et al. [57] demonstrated that administration of bile acids to mice increased energy expenditure in brown adipose tissue by activating TGR5 receptor, which could prevent obesity and decrease resistance to insulin. The decreasing of insulin resistance is benefit for decreasing body fat mobilization. Lai et al. [49] reported that the bile acids decreased the activity of hormone sensitive lipase, which will decrease the mobilization of fatty acids from adipose tissue. Hepatic very low-density lipoproteins (VLDL) secretion rate is controlled by both hepatic fat content and Apolipoprotein B100 (Apo-B100) availability [58]. Yin et al. [59] found supplementation of bile acid (chenodeoxycholic acid) in high-fat diet increased Apo-B100 and decreased lipid accumulation in liver of juvenile largemouth bass. Prakash and Srinivasan [48] found that the increasing of bile acids secretion also prevented the accumulation of triglyceride in high-fat fed rats’ liver. Therefore, the bile acids can inhibit degradation of adipose tissue and accumulation of fat in liver. While some results showed the serum bile acid concentration was significantly increased in cows with moderate and severe fatty liver [60]. The bile acids have different compositions and the abnormally high levels of concentrations of bile acids impairs liver function. Conjugated bile acids are more hydrophilic with less cytotoxic effect compared with the unconjugated forms [61]. In this study, the different bile acids among the treatments were mainly in conjugated forms, which were beneficial for the health of the dairy cows. But the complex mechanism of bile acids on liver health and lipid metabolism in dairy cows still needs further study.
In this study, the decreasing of milk SCC in the LCaP and MCaP group may be related to the increase of bile acids which improved intestinal health and immunity. The study of Li et al. [62] found dietary bile acids supplementation was an effective way to improve the intestinal health status by upregulating the relative expression of intestinal mucosal barrier-related genes and reducing the abundance of potential pathogenic bacteria. The activation of TGR5 and FXR counter-regulates macrophages effector functions and shifts the macrophage polarization toward an anti-inflammatory phenotype [63]. In this study, the bile acid concentration in HCaP group was decreased compared with the MCaP group with the increasing concentrations of serum ALT and AST. Therefore, 500 g/d calcium propionate may impair the liver function through the bile acid metabolism.
When compared with the CON group, the LCaP and MCaP groups had similar serum metabolites such as glycocholic acid and pristanic acid. Moreover, the milk performance of these two groups were improved. Glycocholic acid is one of the conjugated bile acids mentioned above. Pristanic acid is an activator of the peroxisome proliferator activated receptor α (PPAR) which in liver cells regulates expression of genes encoding peroxisomal and mitochondrial β-oxidative enzymes as well as cytosolic / nuclear liver-type fatty acid binding protein (L-FABP) [64]. It was reported that pristanic acid significantly increased MDA levels and reduced GSH levels of young rats [65]. This agreed with the results in the LCaP and MCaP groups, where the antioxidant ability was significantly decreased with the supplementation of calcium propionate. The increasing of pristanic acid improved the liver β-oxidation, regulated lipid metabolism and thus increased oxidative stress.
The serum metabolites of imatinib, L-serine, and glycine were significant higher in MCaP group compared with the CON group. The imatinib could prevent injury-induced neointimal hyperplasia, improve insulin resistance and glucose tolerance, and decrease visceral fat accumulation in high fat diet-fed mice [66]. Therefore, the increasing of imatinib in MCaP group benefit for the healthy and performance of the dairy cow in the group. L-serine, a non-essential amino acid, plays important roles in boosting immune function, formatting phospholipids and acting as neuroprotective for brain function. L-serine is also a precursor of glycine. The increasing of L-serine promoted the production of glycine. Ubiquinone-10 is an important lipid-soluble antioxidant. The decrease of ubiquinone-10 in MCaP group may be related to the reduced antioxidant capacity in the group. The increasing of D-Glucuronate in both LCaP and HCaP groups indicated the gluconeogenesis is enhanced with the addition of calcium propionate. The effects of other differential metabolites such as (S)-2-aminobutyric acid, tetracosanoic acid, N6-(1-Iminoethyl)-L-lysine are rarely reported and the functions need further study.