Animals and management
The experiment was conducted in experimental station of Institute of Dairy Science, Zhejiang University (Hangzhou, China). The study was approved by Animal Care and Use Committee of Zhejiang University. Sixty-four multiparous Holstein cows in early-stage lactation with average DIM of 50 (SD ± 4) and average BW of 726 kg (SD ± 71.3) were selected and assigned to 16 blocks based on parity and milk yield with 4 cows in a block, and then the 4 cows in a block were randomly allocated into 4 groups, and fed basal diets supplemented with different doses of Cr-Met (Availa ® Cr 1000, which supplies 1000 mg Cr/kg from Cr-Met, Zinpro (China) Animal Nutrition Technology Co., Ltd) at the doses of 0, 4, 8, or 16 mg Cr/kg DM, respectively. All cows were housed in a tie stall barn, and fed and milked at 0630, 1400, and 1930 h every day. All cows had free access to drinking water. Feed was given in excess to allow 5% orts during the experiment. The Cr-Met was added once per day at 0630 h by scattering it on the total mixed ration for individual cows. The experiment lasted for 14 weeks, with the first 2 weeks for adaptation. During adaptation stage, Cr-Met was not supplemented to dairy cows.
Sample collection and measurements
Throughout the experimental period, DMI was recorded for 2 consecutive days (d 6 and 7 each wk) and samples of TMR and orts were collected on the same days of every other week. All the samples were analyzed for DM (105°C for 5 h), crude protein (method 988.05; AOAC, 1990) [12], crude ash (method 942.05; AOAC, 1990) [12], and acid detergent fiber (ADF) (method 973.18; AOAC, 1990). Content of neutral detergent fiber (NDF) was analyzed with method described by Van Soest et al [13] with the addition of sodium sulfite and amylase. An ANKOM2000 fiber analyzer (Ankom Technology Corp., Macedon, NY, USA) was used to extract and filter NDF and ADF, respectively. The value for NEL in the experimental diet was estimated based on the Cornell Net Carbohydrate and Protein System (CNCPS) model using the CPM Dairy 3.0 [14]. The ingredient and nutrient composition of the experimental diet are listed in Table 1. The BW was estimated at the beginning and the end of the trail with method described by Yan et al [15].
The milk yield was recorded on d 6 and 7 of each week, and milk samples were collected on d 7 using milk-sampling devices (Waikato Milking Systems NZ Ltd., Waikato, Hamilton, New Zealand). One 50 mL aliquot of the composited milk sample was collected at each milking of the sampling day, and mixed with bronopol tablets (milk preservative, D & F Control Systems, San Ramon, CA). Milk sample was analyzed with a spectrophotometer (Foss-4000; Foss Electric A/S, Hillerod, Denmark) for milk compositions (protein, fat, lactose and milk urea).
Blood samples were collected from the coccygeal vein of each cow 3 h after the morning feeding on d 7 of wk 4, 8 and 12, respectively. The samples were deposited into lithium-heparin-containing vacuum tubes (5 mL, Becton Dickinson, Franklin Lakes, NJ), centrifuged at 3000 × g for 15 min to collect the plasma, and frozen at -20°C for subsequent analysis. Plasma samples were analyzed using an Auto Analyzer 7020 instrument (Hitachi High-technologies Corporation, Tokyo, Japan) with colorimetric commercial kits (Ningbo Medical System Biotechnology Co., Ltd.) to determine total protein, albumin, globulin, BUN, creatinine, glucose, non-esterified fatty acid (NEFA), β-hydroxybutyrate, triglyceride, total bilirubin, alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase, insulin, glucagon, pyruvate, LDH and NADH, according to previously described methods [16]. The superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) were determined using the ELISA method by Jiangsu MeiBiao Biological Technology Co., Ltd, Jiangsu, China [17].
Rumen fluid (50 mL) was collected using an oral stomach tube approximately 3 h after the morning feeding on d 7 of wk 4, 8 and 12, as described by Shen et al [18]. The pH of the rumen fluid was immediately measured using a portable pH meter (FE20-FiveEasy Plus™; Mettler Toledo Instruments Co. Ltd., Shanghai, China). The samples were placed on ice and kept stationary while the supernatant separated, and then, the samples were frozen at −20 °C for future determination of volatile fatty acids (VFAs). Two mL of rumen sample was acidified with 20 μL of 25% orthophosphate acid and then centrifuged at 20,000 × g for 10 min at 4 °C. The supernatant was then subjected to VFAs measurement using a gas chromatograph (GC-2010, Shimadzu, Kyoto, Japan) according to the methods described previously [19].
Statistical analysis
The effect of Cr-Met on DMI, lactation performance, and plasma variables of dairy cows were analyzed using the MIXED procedure in SAS software version 2000 (SAS Institute Inc., Cary, NC), with covariance type AR (1) for repeated measures analysis. A randomized block design with repeated measures was used for the analysis, with wk, treatment, interaction of treatment × wk, and block as the main effects and cow within the diet as a random effect. The linear and quadratic effects of treatment on the variables were evaluated with orthogonal polynomials accounting for unequal spacing of Cr-Met supplement levels. The results were listed as least squares means and were separated using the PDIFF option when the fixed effects were significant. P < 0.05 was defined as statistical significance, and 0.05 ≤ P < 0.10 was considered as tendency of significance.