The study was approved by the farm owner and all experimental animals were conducted according to the International Guiding Principles for Biomedical Research. The protocol was approved by the Committee on the Ethics of Animal Experiments of the Heilongjiang Bayi Agricultural University.
Herd and cow selection
In northeastern China (MiShan, Heilongjiang, China), commercial dairy herds from a large dairy farm were selected to participate in the study. Five non-pregnant, non-lactating Holstein–Friesian dairy cows (18 ± 4 months of age) were allocated randomly based on specific condition needs of compliance with the study protocol. All dairy cows were in good health.
This study included ten Holstein cows, five dairy cows were assigned randomly to two groups for the experiment. Group A was treated with a moderate-energy diet (120 % of predicted energy requirements) in the primary stage of the experiment and then they were treated with a controlled-energy diet (30 % of maintenance energy requirements) afterwards. Group B was treated with a controlled-energy diet (30 % of maintenance energy requirements) first and then they were treated with a moderate-energy diet (120 % of predicted energy requirements) in the later stage. Each level of nutrition was achieved by feeding appropriate amounts of a single TMR (Total Mixed Rations). The TMR composition was 1.48 Mcal (Mega-calorie) net energy of lactation (NEL) and 160 g of crude protein per kilogram of dry matter (DM). Feeding duration for both the controlled-energy and moderate-energy diets was 14 days; both groups were given a moderate-energy diet (120 % of predicted energy requirements) for 7 days before the change in diet (Fig. 1).
Blood samples were collected from tail vein in early morning before receving diet every day. After blood collection, tubes without anticoagulant were placed in an icebox and carried to the laboratory within 1 h of collection, after which they were placed at room temperature for 30 min, centrifuged at 3000 g for 15 min, and then stored at −20 °C until analysis.
Blood metabolites and hormones
Concentrations of FGF-21 were measured using a RD Human Fibroblast Growth Factor-21 ELISA kit (R&D Systems Inc. Minneapolis, MN, USA) on an automatic biochemistry analyzer (JianCheng Biological Engineering Research Institute, Nanjing, China). Glucagon (GC) was measured with a RD Bovine Glucagon ELISA kit, growth hormone (GH) was measured with a RD Bovine Growth hormone ELISA kit, and insulin (INS) was measured with a RD Bovine INS ELISA kit. All biochemical parameters were analysed on a Huadong electronic DG5033A microplate reader (Huadong, Nanjing, China). Blood glucose (GLU), serum levels of triglycerides (TG) and nonesterified fatty acid (NEFA) were measured directly by using an automatic biochemistry analyzer.
Data collation and statistical analyses
Data in this study were collated and initially analyzed using Excel 2013 (Microsoft Corp., Redmond, WA, USA). Descriptive and graphical analyses were carried out to verify the data. When appropriate, we used IBM SPSS19.0 software (SPSS Inc. Chicago, IL) to analyze the data. The results are expressed as means ± standard error means (SEM). Changes in FGF-21 levels and hormonal parameters between two groups were evaluated by an Independent Sample T-test. One-way ANOVA was used to compare data from different time periods between the two groups, and multiple testing was corrected using the LSD (Least Significant Difference) method (Equal Variances Assumed) or the Dunnett T3 (Equal Variance not assumed). Pearson correlation tests were used to calculate the relationships between FGF-21 levels and other parameters. Pearson’s correlation analysis and linear regression analysis were used to analyze the correlation of BHBA and serum FGF-21. In data testing, a P value <0.05 was considered statistically significant.