Effects of Calcium Propionate on Milk Performance and Serum Metabolome of Dairy Cows in Early Lactation

Abstract

Background: Most of the dairy cows in early lactation suffer from a period of negative energy balance (NEB) and hypocalcemia metabolic disorders. Calcium propionate is a source of energy and calcium for alleviating NEB and hypocalcemia. The objective of the study was to investigate the changes of milk compositions and blood metabolites of postpartum dairy cows fed with calcium propionate for 5 weeks.

Methods:

Thirty-two multiparous Holstein cows after calving were randomly allocated to control (CON), low calcium propionate (LCaP), medium calcium propionate (MCaP) and high calcium propionate (HCaP) groups with 8 cows per group balanced with parity, milk yield and body weight. The dairy cows in the four groups were oral drenching with 0, 200, 350, 500 g/d calcium propionate per cow from calving to d 35 in early lactation, respectively.

Results:

The results showed that the milk yield in MCaP group was significantly higher than those in the other groups. At d 35, the somatic cell count (SCC) in MCaP group tended to be lower than those in other groups. Compared with the CON group, serum non-esterified fatty acid (NEFA) in MCaP and HCaP groups tended to decrease. The serum alanine aminotransferase (ALT) concentration in MCaP group was lowest among the four groups. The concentration of aspartate aminotransferase (AST) in HCaP group was significantly higher than the other groups. Feeding calcium propionate had no significant effect on serum calcium, phosphorus and magnesium concentrations. The serum parathyroid hormone (PTH) in HCaP group tended to decrease. The calcium propionate treatments significantly decreased the serum alkaline phosphatase (ALP) level. The MCaP treatment significantly decreased serum total antioxidant capacity (T-AOC), glutathione peroxidase (GSH-Px), superoxide dismutase (SOD) and catalase (CAT) activity, while increased the concentration of malondialdehyde (MDA) when compared with the other groups. The metabolomic results showed that calcium propionate significantly affected the bile acid compositions.

Conclusions: It was concluded that the 350 g/d calcium propionate feeding level could significantly improve milk performance, alleviate body fat mobilization and bone calcium utilization, however decrease antioxidant capacity of dairy cows as well. The effect of calcium propionate on milk performance and serum metabolites in early lactation cows may be regulated through the serum bile acids metabolism.

Introduction

The dairy cows in early lactation suffer a period of negative nutrient balance for the insufficient dietary intake to meet the output nutrients requirements for milk production and maintenance, which is regarded as one of the most challenging elements of the production cycle [1]. The negative nutrient balance makes the dairy cows require a massive mobilization of body reserves, increasing the incidence of such metabolic diseases with hypocalcemia (milk fever), fatty liver and ketosis in early lactation, which may impact animal welfare, productive lifespan and economic outcomes [2]. The hypocalcemia is reported to be a risk factor for limiting milk production and increasing the diseases including of dystocia, uterine prolapse, retained placenta, metritis, displaced abomasum, ketosis, and mastitis [3]. Improving the dietary calcium supplementation is an important method for alleviation hypocalcemia. To avoid negative energy balance (NEB), the dairy cows can regulate lipid mobilization and release non-esterified fatty acid (NEFA) from adipose tissue. The oxidation of fatty acids provides acetyl-CoA, which is then condensed with oxaloacetate and form citrate to enter the tricarboxylic acid (TCA) cycle to produce energy [4]. However, the glucose requirements for milk production are high in early lactation. Oxaloacetate is increasingly used for gluconeogenesis and thus acetyl-CoA cannot be completely oxidized but is converted into ketone bodies, mainly acetone, acetoacetate and β-hydroxybutyrate (BHBA) [2]. Moreover, when the production of NEFA from body fat mobilization exceeds the liver oxidative capacity, it increases the fat accumulation in liver and results in fatty liver. Therefore, the key to solving the metabolic disorder caused by insufficient energy was probably to supple the gluconeogenesis precursor [5] or to make acetyl-CoA enter the TCA cycle effectively, such as providing enough oxaloacetate or which can be converted to oxaloacetate [6]. Propionate is a primary glucose precursor in dairy cows and contributes to the synthesis of oxaloacetate [7, 8]. But the quantity of propionate is often insufficient to satisfy the need for glucose in early lactation [9]. During the postpartum period, a balance of propionate supply to dairy cows could lead to improvements in dry matter intake (DMI), and subsequently, health and production in dairy cows [7].

Calcium propionate, as a source of both calcium and energy can be hydrolyzed into Ca²⁺ and propionic acid in the rumen, has been used for preventing or treating hypocalcemia and ketosis in dairy cows [10, 11]. Goff et al. [12] has reported that oral calcium propionate supplementation to dairy cows after calving was beneficial in reducing subclinical hypocalcemia and decreasing the incidence of milk fever. Pehrson et al. [13] also reported that calcium propionate might be a satisfactory alternative to calcium chloride in preventing milk fever. Liu et al. [14] observed that adding calcium propionate into total mixed ration (TMR) during first 63 days of lactation could improve energy status, as indicated by higher blood glucose, lower blood BHBA and NEFA and lower urine ketones in Holstein cows. Dairy cows fed calcium propionate also improved milk yield during early lactation and the effectiveness of calcium propionate was better than calcium acetate and calcium salts of fatty acids [15]. However, the optimal feeding level and maximum feasible dose of  calcium propionate in cows still need to be investigated [11]. Moreover, few studies have clearly elucidated the metabolites and metabolic pathways associated with the application of calcium propionate in dairy cows.

Therefore, we hypothesize that the milk composition, serum metabolites and metabolic pathways would be changed after feeding the dairy cows in early lactation with different levels of calcium propionate, which might be benefit for alleviating the NEB and hypocalcemia. For the metabolomics-based technologies have been used for biomarker identification, disease diagnosis, and mechanism exploration in ruminants [5], we used the liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) based metabolomics to analyze the mechanisms of calcium propionate in improving the metabolic disorder in early lactation. Thus, this study was aimed to explore the effects of calcium propionate supplement on milk performance, metabolic status and the profile of serum metabolites in the early lactation dairy cows. 

Materials And Methods

All the experimental protocols were approved by the Animal Care Committee of Chinese Academy of Agricultural Sciences (Beijing, China) (Approval Number: IAS2020-93) and were under the academy’s guidelines for animal research.

Animals and experimental design

Thirty-two multiparous Holstein dairy cows (3.29 ± 0.18 of parity; 41.61 ± 1.61 kg/d of milk yield; 779.48 ± 12.51 kg of body weight) were selected for the animal research and divided into four treatments with eight cows per treatment in a randomized block design balanced with party, live weight and previous corrected 305-d milk yield. As the calving occurred randomly, each cow was assigned to the four treatments by the parturition order. The four treatments included control (CON), low calcium propionate (LCaP), medium calcium propionate (MCaP) and high calcium propionate (HCaP) groups with the same TMR diet plus 0, 200, 350 and 500 g/d calcium propionate (Jiangsu Runpu Food Technology Co. LTD., Jiangsu, China) per cow, respectively, from calving to 35 d postpartum. The calcium propionate was oral drenched three times a day in equal amounts after milking. In the experiment, one cow in MCaP group was eliminated due to metritis, and two cows in CON group were eliminated due to left displaced abomasum. The date about the three cows were also discarded accordingly.

The experiment was conducted in China-Israel demonstration dairy farm (Beijing, China) from September to December in 2020. After calving, the cows were fed three times a day at 7:00, 14:30, and 18:00 h with the same TMR after milking. The TMR was formulated to meet or exceed the nutrient requirements of early lactation dairy cows according to the National Research Council [16]. All cows were individually fed and had free access to water. The ingredients and chemical composition of the basal diet in the experiment are shown in Table 1. The orts were collected and weighted before the daily morning feeding. The TMR offered was adjusted every day to make sure the cows were individually fed 110% of their ad libitum consumption. The treatments began at calving and continued until d 35 postpartum. 

Feed samples and chemical analysis

The TMR samples were collected weekly on 2 consecutive days and composited to obtain 1 sample every week. The samples were dried in a forced air oven at 55 ℃ for 48 h and then ground pass through a 1-mm screen for the follow-up nutrient analysis. The TMR samples were analyzed for dry matter (DM) [method 934.01; 17], crude protein (CP) [method 954.01; 17], ether extract (EE) [method 920.39; 17], ash [method 942.05; 17], starch [method 996.11; 17], calcium (Ca) [method 968.08; 17] and phosphorous (P) [method 946.06; 17]. The neutral detergent fiber (NDF) and acid detergent fiber (ADF) of the TMR samples were determined as described by Van Soest et al. [18]. The concentration of net energy of lactation (NE_L) was calculated according to NRC [16].

Milk samples and composition analysis

Milk yield was measured from d 2 to 35 postpartum of each cow. Cows were milked three times a day at 6:00, 14:00 and 22:00 h, and the production was recorded automatically by an AfiMilk MPC milk meter (2 × 14 fishbone milking machine, AfiMilk Ltd. Kibbutz Afikim, Israel). Milk sample of each cow was collected in a plastic bottles (50 mL) containing a preservative (2-bromo-2-nitropropan-1,3-diol) at 3 consecutive milking  at d 35 postpartum and mixed at a proportion of 4∶3∶3. Then the milk samples were stored at 4 ℃ for the analysis of milk composition and somatic cell count (SCC). The milk composition of fat, protein, lactose, urea nitrogen (MUN) and SCC were analyzed using an infrared milk analyzer (Combi Foss 4000, Foss Electric A/S, Hillerød, Denmark) according to the manufacturer’s instructions.

Blood samples and measurement

Six cows from each group were randomly selected for the blood samples collection. The blood sample of each cow was collected using a 10-mL gel vacuum by puncturing the coccygeal vein between 07:00 to 08:00 h on d 35 postpartum. Then the samples were centrifuged at 3 000 × g at 4 ℃ for 15 min to obtain the corresponding serum. The supernatant was harvested in 2 ml centrifuge tubes and stored at -80 ℃ for the later analysis of biochemical parameters and metabolome profiles. 

The concentrations of serum glucose, BHBA, alanine aminotransferase (ALT), aspartate aminotransferase (AST), total calcium (Ca), phosphate (P), magnesium (Mg) and alkaline phosphatase (ALP) were determined following the methods of colorimetric commercial kits (Shanghai Kehua Bio-Engineering CO., LTD, Shanghai, China). The serum insulin, glucagon, NEFA, parathyroid hormone (PTH), calcitonin (CT), and calcitriol (1,25-dihydroxyvitamin D₃) were measured using a commercial ELISA kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The serum activity of total antioxidant capacity (T-AOC), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase (CAT), and concentration of malondialdehyde (MDA) were determined according to the description of Sun et al. [19] by using a commercial assay kit (Nanjing Jian Cheng Bioengineering Institute, Nanjing, China). 

Liquid chromatography-mass spectrometry analysis of serum metabolites

Sample collection and measurement

 The serum samples collected on d 35 postpartum were used for non-targeted metabolomic analysis. A 30 mg serum sample was transferred to a 2 mL tube. After 500 μL of extract solvent (acetonitrile-methanol-water, 2∶2∶1) was added to the tube, the sample was ground under 60 Hz for 2 min, and then incubated at 20 ℃ for one hour. The samples were centrifuged at 4 ° C at 13 000 × g for 15 min. Then 350 μL supernatant was moved to a 1.5 mL tube and dried in vacuum concentrator. The metabolites were redissolved in extract solvent (acetonitrile-water, 1∶1), vortexed for 30 s. After 15 min centrifugation at 13 000 × g at 4 ℃, 50 μL supernatant was harvested into a 2 mL injection flask for further LC/MS analysis.

Identification and quantification of compounds by LC/MS

The ultra-high-performance liquid chromatography (UHPLC) system of an Exion LC™ AC system coupled with a quadrupole-time-of-flight mass spectrometer (QTOF MS; Triple TOF 5600+, SCIEX, Framingham, Massachusetts, USA) system was used to analyze the serum metabolites. The liquid chromatography separation was performed by a Waters ACQUITY UPLC BEH Amide column (1.7 µm × 2.1 mm × 100mm, Waters, Milford, Massachusetts, USA) and the column temperature was maintained at 40 °C. The mobile phase A was 25 mmol/L ammonium acetate and 25 mmol/L ammonium hydroxide in water, and the mobile phase B was 100% acetonitrile. The following gradient program was used: 95% B, 0 - 0.5 min; 95% - 65% B, 0.5 - 7 min; 65% - 40% B, 7 - 8 min; 40% B, 9 min; 40% - 95% B, 9 - 9.1 min; and 95% B, 9.1 - 12 min. The flow rate was set at 0.5 mL/min. The injection volume was 2 µL, and the samples were maintained at 4 °C in the autosampler. The QTOF MS system was performed via an electrospray ionization (ESI) source in both positive and negative-ion mode. The MS conditions were set: ion spray voltage (IonSpray Voltage Floating) at 5 500 V (positive ion mode) and - 4 500 v (negative ion mode); ion gas temperature, 650 °C; curtain gas, 30 psi; ion gas pressure, 60 psi; and the declustering potential at 60 V. The TOF MS data was acquired over the range of 60 - 1200 m/z at 0.15 s/spectra using collision energy 10 eV. The data dependent acquisition scan was performed by selected the 12 most abundant mass peaks in TOF MS. MS/MS data was collected over the range of 25 - 1200 m/z at 0.03 s/spectra using a collision energy 30 eV.

Identification and data analysis of metabolomics

ProteoWizard software was used to convert the MS raw rate files into mzXML format, and then processed use R program package XCMS (V.3.2) to get the three-dimensional data involving the retention time (RT), mass-to-charge ratio (m/z) values, and peak intensity for metabolite identification. If metabolite feature was detected in < 20% of experimental samples or < 50% of quality control (QC) samples (prepared by mixing sample extracts), it would be removed from data analysis. Then the missing values of raw data were filled up by half of the minimum value. In addition, internal standard normalization method was employed in this data analysis. Finally, features with relative standard deviation of QC > 30% should be discarded from the subsequent analysis. The R package CAMERA was used for peak annotation after XCMS data processing. The online HMDB (http://www.hmdb.ca) was used for metabolites identified and validated by aligning the molecular mass data (m/z) [20]. 

Statistical analysis

The completely randomized block design was used in the study. The data of milk yield was averaged to weekly means and analyzed using the repeated measures model. The data of milk composition, rumen fermentation and serum biochemical parameters were analyzed using a completely randomized design with one-way analysis of variance (ANOVA) in SAS 9.4 (SAS Institute Inc., Cary, NC, USA). The differences among the treatments were identified using Duncan’s significant difference tests. Data was shown as mean and standard error of the mean (SEM). Significance difference was considered when P ≤ 0.05, and a trend was accepted when  0.05 < P ≤ 0.10. 

The identified metabolites from positive and negative modes were merged for multivariate statistical analysis [6, 21]. The multiple statistical analysis including principal component analysis (PCA) and orthogonal projections to latent structures-discriminate analysis (OPLS-DA) was conducted using SIMCA 14.1. To refine this analysis, the first principal component of variable importance in the projection (VIP) was obtained. The VIP values summarize the contribution of each variable to the model. The P-value < 0.05 (student's t-test) and the variable importance in the projection (VIP) > 1.0 were considered as significantly changes. The fold-change (FC) value of each metabolite was calculated by comparing the mean value between the CON and the treatment groups, and the log₂FC value was used to indicate the specific variable quantity in the comparison [22]. MetaboAnalyst 4.0 (http://www.metaboanalyst.ca/) was used to search for the enrichment pathway of differential metabolites and hierarchical clustering analysis. In addition, the pathway enrichment analysis was performed by using the commercial databases including KEGG (http://www.genome.jp/kegg/) and PubChem (https://pubchem.ncbi.nlm.nih.gov/) databases. The high impact values and P-values were regarded as the key pathways based on the pathway topology analysis using relative-betweenness centrality in the metabolome map [22].

Results

Lactation performance 

Lactation performance of the dairy cows was presented in Table 2. The MCaP feeding level significantly increased the milk yield during the first 5 weeks of lactation (P = 0.02). The milk yield in LCaP group was also higher than that in the CON group statistically, although there was no significant difference (P > 0.05). The concentrations of milk fat (P = 0.71), protein (P = 0.80), lactose (P = 0.59), MUN (P = 0.90) and the ratio of fat to protein (P = 0.87) were not significantly different among the treatments on d 35. The calcium propionate supplementation tended to reduce the milk SCC compared with the CON group (P = 0.07).

Blood parameters related to NEB and calcium homeostasis

The serum biochemical indices associated with NEB, and calcium homeostasis were shown in the Table 3. Overall, the serum glucose, INS, glucagon, BHBA, NEFA, Ca, P, Mg, PTH, CT, calcitriol did not different among the four groups (P > 0.05), while the NEFA in MCaP tended to be lower compared with the CON group (P = 0.08). The ALT in MCaP group was significantly lower than the other groups (P = 0.05). The AST in HCaP group was significantly higher (P = 0.001), while the PTH in HCaP group tended to be lower (P = 0.06) than those in the other treatments. Feeding calcium propionate significantly decreased the serum ALP (P = 0.003). 

Antioxidant indexes

As shown in Table 4, supplementation with calcium propionate at 350 g/d significantly decreased serum T-AOC (P < 0.001), SOD (P < 0.001), GSH-Px (P = 0.003), and CAT (P < 0.001) activity in comparison with the control group. But the serum MDA of MCaP group was significantly increased when compared with the other treatments (P < 0.001).

LC/MS compound identification and quantification

The ion chromatograms of QC samples in positive or negative ion modes demonstrated the high repeatability and stability of the test results (Figure S1). Metabolomic profiles of the serum contents were generated using the LC/MS, including 6 samples in each group. After data pre-processing of spectra, a total of 42 932 features were obtained in positive ion mode and 41 045 features were obtained in negative ion mode, respectively. Multivariate analysis was conducted to discover the effects of calcium propionate on the serum metabolic profile of the dairy cows in early lactation. After merged the identified metabolites of positive and negative modes for multivariate analysis, 516 metabolites were relatively quantified after rigorous quality control and identification.

Significantly different metabolites in blood 

In order to exhibit the distribution and difference of the serum samples among and within the groups, unsupervised PCA was used by merging the positive and negative ionization modes. The PCA score plots were obtained from the mass spectrometer data (Fig. 1). In order to obtain a higher level of group separation and get a better understanding of variables responsible for classification, supervised OPLS-DA were carried out. According to the OPLS-DA score plots (Fig. 2), there were clear separations among the control and the calcium propionate treated groups, indicating that the different feeding levels of calcium propionate resulted in different potential metabolites in serum metabonomic profile. 

Univariate analysis was used to compare the metabolites in serum between the different calcium propionate treatment groups. The differential metabolites were generated based on the VIP score > 1 obtained from OPLS-DA model and the variables obtained from Student’s t-test with P values < 0.05 among the groups. As shown in Fig. S2 and Table 5, there were 19, 28, 13 and 8 different metabolites between CON vs LCaP, CON vs MCaP, CON vs HCaP, and HCaP vs MCaP, respectively. 

Specifically, 8 different metabolites mainly including L-saccharopine, 3-pyridinemethanol, Leu-Val N, N-diacetylchitobiose, His-Ala, ephedrine choline, and 4-aminobutyric acid were downregulated and 11 different metabolites including glycocholic acid, glycodeoxycholic acid, glycochenodeoxycholate, taurocholate, Phe-Trp, deoxycholic acid, pristanic acid, 1-stearoyl-sn-glycerol-3-phosphocholine, tetracosanoic acid, D-glucuronate, and argininosuccinic acid, were upregulated in LCaP compared with CON group (Fig. 3). Sixteen upregulated metabolites mainly comprised of bilirubin, glycocholic acid, imatinib, L-serine, allopurinol riboside, arachidic acid, (S)-2-aminobutyric acid, glycine, pristanic acid and 12 downregulated metabolites mainly including ubiquinone-10, N-Docosanoyl-4-sphingenyl-1-O-phosphorylcholine, N-Tetracosenoyl-4-sphingenyl-1-O-phosphorylcholine, N-Tetracosanoyl-4-sphingenyl-1-O-phosphorylcholine, (S)-2-hydroxyglutarate, were found in MCaP compared with those in CON group. Gamma-L-glutamyl-L-phenylalanine was reduced and 12 metabolites mainly including (S)-2-aminobutyric acid, N6-(1-iminoethyl)-L-lysine, Phe-Trp, tetracosanoic acid were increased in HCaP group when compared with the CON group. Comparing to the MCaP group, adenylyl (3-5) cytidine, protoporphyrin IX, lathosterol, and trans-cinnamate were significantly increased, and taurochenodeoxycholate, tauroursodeoxycholic acid, taurodeoxycholic acid, and indole-3-pyruvic acid were significantly decreased in HCaP group. The compound classification of different metabolites in serum were processed by the data of HMDB on the comparisons. The major different metabolites were belonging to amino acids, peptides, and analogues; bile acids, alcohols and derivatives; fatty acids and conjugates, respectively, for the top 3 clustered metabolites among the four comparisons. 

The Venn diagram illustrated the individually and mutually different metabolites between the calcium propionate treatment groups and the CON group (Fig. 4). Compared with the CON group, the LCaP and MCaP had the same different metabolites of glycocholic acid and pristanic acid; MCaP and HCaP had the same different metabolites of (S)-2-aminobutyric acid and L-serine; LCaP and HCaP had the same different metabolites of Phe-Trp, tetracosanoic acid and D-glucuronate, respectively. 

Metabolic pathway enrichment of differential metabolites in blood

In order to comprehensively understand the body metabolism changes to the supplement of calcium propionate, metabolic pathway enrichment analysis was performed. The results of the identified significant differential metabolites pathway enrichment analysis of the four comparisons are shown in Fig.5. The significantly different metabolites between the CON and the LCaP groups were mainly involved in cholesterol metabolism (P < 0.001, impact value = 0.3), bile secretion (P < 0.001, impact value = 0.052), primary bile acid biosynthesis (P = 0.003, impact value = 0.039), alanine, aspartate and glutamate metabolism (P = 0.015, impact value = 0.127). The different metabolites between CON and MCaP groups were mainly enriched in biosynthesis of amino acids (P = 0.008, impact value = 0.039), lysine degradation (P = 0.018, impact value = 0.054), mineral absorption (P = 0.035, impact value = 0.069), central carbon metabolism in cancer (P = 0.056, impact value = 0.054), and primary bile acid biosynthesis (P = 0.054, impact value = 0.043). The different metabolites between CON and HCaP groups were mainly enriched in cysteine and methionine metabolism (P = 0.017, impact value = 0.033), sphingolipid signaling pathway (P = 0.051, impact value = 0.067), sphingolipid metabolism (P = 0.084, impact value = 0.04), and mineral absorption (P = 0.097, impact value = 0.034). Between the HCaP and MCaP groups, the significantly different metabolites were mainly involved in cholesterol metabolism (P = 0.035, impact value = 0.10), primary bile acid biosynthesis (P = 0.154, impact value = 0.021), and steroid biosynthesis (P = 0.163, impact value = 0.020). The detailed information about enriched metabolic pathways related with the significantly differential metabolites were shown in the Supplementary Table S1-S4.

Discussion

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.

Conclusions

Calcium propionate effectively alleviated the NEB and mobilization of calcium from bone. The appropriate dietary supplementation of calcium propionate is beneficial to increase milk yield but also increase the oxidative stress for the dairy cows in early lactation. In addition, untargeted metabolomics analysis of serum showed that the mechanism of calcium propionate in improving the dairy cows’ performance mainly through the bile acid-mediated signaling pathways in regulating energy, glucose, lipids, and immunity. The optimum amount of calcium propionate for the dairy cows in early lactation was 350 g/d under the conditions in the current study.

Abbreviations

ADF: acid detergent fiber

ALP: alkaline phosphatase

Apo-B100: Apolipoprotein B100

BHBA: β-hydroxybutyrate

Ca: calcium

CAT: catalase

CON: control

CP: crude protein

CT: calcitonin

DM: dry matter

DMI: dry matter intake

EE: ether extract

FC: fold-change

FXR: farnesoid-X-receptor 

GSH-Px: glutathione peroxidase

HCaP: high calcium propionate

LCaP: low calcium propionate

LC-MS/MS: liquid chromatography coupled to tandem mass spectrometry

L-FABP: liver-type fatty acid binding protein

MCaP: medium calcium propionate

MDA: malondialdehyde

Mg: magnesium

MUN: milk urea nitrogen

NDF: neutral detergent fiber

NEB: negative energy balance

NEFA: non-esterified fatty acid

NE_L: net energy of lactation

OPLS-DA: orthogonal projections to latent structures-discriminate analysis

P: phosphorous

PCA: principal component analysis

PTH: parathyroid hormone

QC: quality control

RT: retention time

SCC: somatic cells

SEM: standard error of the mean

SOD: superoxide dismutase

T-AOC: total antioxidant capacity

TCA: tricarboxylic acid

TGR5: Takeda G-protein-coupled receptor 5

TMR: total mixed ration

UPLC: ultra-high-performance liquid chromatography

VIP: variable importance in the projection

VLDL: very low-density lipoproteins

Declarations

Ethics approval and consent to participate

The experimental protocols were approved by the Animal Care Committee of Chinese Academy of Agricultural Sciences (Beijing, China) (Approval Number: IAS2020-93) and were under the academy’s guidelines for animal research.

Consent for publication

Not applicable.

Availability of data and materials

The datasets used or analyzed during the current study are available from the corresponding author on reasonable request.

Competing interests

The authors state that there is no conflict of interests in this study.

Funding

This work was funded by National Key R&D Programs of China (2017YFD0701604, 2018YFD0500703, 2019YFE0125600), and Beijing Dairy Industry Innovation Team (bjcystx-ny-1).

Authors' contributions

FZ, YG, and BX conceived and designed the experiment. FZ, YZ, and YW involved in the animal experiment and sample collection. FZ analyzed the date and completed the initial manuscript. YZ and HW completed the language editing and revised the manuscript. All authors read and approved the final manuscript.

Corresponding authors

Correspondence to Yuming Guo or Benhai Xiong

Acknowledgments

The authors are grateful for the staff of the China-Israel demonstration dairy farm in Beijing for their help in the animal management. 

Author information

Affiliations

State Key Laboratory of Animal Nutrition, Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing 100193, China

Fan Zhang, Yiguang Zhao, Yue Wang, Hui Wang, and Benhai Xiong

State Key Laboratory of Animal Nutrition, College of Animal Science and Technology, China Agricultural University, Beijing 100193, China

Fan Zhang, and Yuming Guo

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Tables

Table 1 Ingredients and nutrient composition of the basal diet in the early lactation dairy cows

¹ The megalac is a rumen-protected fat supplement produced by Volac Wilmar Feed Ingredients Ltd (Hertfordshire, United Kingdom).

 ² The concentrate for postpartum dairy cows is manufactured by Beijing Sanyuan Seed technology Co., Ltd (Beijing, China). The concentrate ingredients contain: corn, soybean, rapeseed meal, wheat bran, cottonseed meal, CaCO₃, NaCl, CaHPO₄·2H₂O and premix. The chemical compositions of the concentrate: DM, 88.50%; CP, 23.91%; NDF, 13.20%; ADF, 7.40%; Ash, 13.1%; Ca, 1.41%; P, 0.58%; K, 1.20%; Mg, 0.58%; Na, 0.99%; Cu, 46.25 ppm/kg; Fe, 80.30 ppm/kg; Zn, 136.76 ppm/kg; vitamin A, 20.53 kIU/kg; vitamin D, 3 548.50 IU/kg; vitamin E, 116.90 IU/kg.

³ The NE_L is a calculated value according to NRC [16].

Table 2. Effects of dietary supplementation with calcium propionate on milk yield and milk composition of cows in early lactation.

^a,b,c means in the row with different superscripts are significantly different (P ≤ 0.05). 

SEM=Standard error of the mean; SCC = somatic cell count; MUN = milk urea nitrogen.

¹ Treatments: CON = control group, basal diet; LCaP = low calcium propionate, basal diet plus 200 g/d calcium propionate; MCaP = medium calcium propionate, basal diet plus 350 g/d calcium propionate; HCaP = high calcium propionate, basal diet plus 500 g/d calcium propionate.

Table 3. Effects of dietary supplementation with calcium propionate on blood parameters related to NEB and calcium homeostasis of cows in early lactation

^a,b means in the row with different superscripts are significantly different (P ≤ 0.05)

SEM = standard error of the mean; INS = insulin; BHBA = β-hydroxybutyric acid; NEFA = non-esterified fatty acid; ALT= alanine transaminase; AST = aspartate aminotransferase; Ca = calcium; P = phosphorus; Mg = magnesium; PTH = parathyroid hormone; CT = calcitonin; ALP = alkaline phosphatase.

¹ Treatments: CON = control group, basal diet; LCaP = low calcium propionate, basal diet plus 200 g/d calcium propionate; MCaP = medium calcium propionate, basal diet plus 350 g/d calcium propionate; HCaP = high calcium propionate, basal diet plus 500 g/d calcium propionate.

Table 4. Effects of dietary supplementation with calcium propionate on blood antioxidant indexes of cows in early lactation

^a,b,c means in the row with the different superscripts are significantly different (P ≤ 0.05).

SEM = standard error of the mean; T-AOC = total antioxidant capacity, SOD = superoxide dismutase, GSH-Px = glutathione peroxidase, CAT = catalase; MDA = malondialdehyde.

¹ Treatments: CON = control group, basal diet; LCaP = low calcium propionate, basal diet plus 200 g/d calcium propionate; MCaP = medium calcium propionate, basal diet plus 350 g/d calcium propionate; HCaP = high calcium propionate, basal diet plus 500 g/d calcium propionate.

Table 5. Identification of significantly different metabolites in the serum of the calcium propionate supplement groups compared with the control group of dairy cows in early lactation