Animals and experimental design

The experimental protocol was approved by the Animal Care and Use Committee of Shanxi Agriculture University. Forty-eight Chinese Holstein bull calves at 15 days of age and 45.1 ± 0.36 kg body weight (BW) were assigned randomly to one of four groups, 12 calves per group. The treatments were: control (without isovalerate), low-isovalerate (LIV), moderate-isovalerate (MIV) and high-isovalerate (HIV) with 3, 6 and 9 g isovalerate per calf per day, respectively. The supplement of isovalerate (analytical grade, 98.5% of isovalerate) was purchased commercially (Shanghai Aladdin Biological Technology Co., LTD, Shanghai, China) and hand-mixed into milk for pre-weaned calves and into concentrate for post-weaned calves. The study lasted 75 days, including a 15-day adaptation period followed by a 60-day sampling period. Calves were weaned at 60 days of age. During the pre-weaning period, calves were offered whole milk (0.10 of BW) twice daily via nipple feeding at 08.00 and 15.00 h for 25 days. From days 26 to 30, the daily offer of milk was decreased by 50% and calves were fed a commercial concentrate ad libitum. The post-weaning diets consisted of 600 g/kg alfalfa hay and 400 g/kg commercial concentrate based on a DM basis and was offered ad libitum. The chemical composition of the feeds is shown in Table 1. Calves were housed in individual pens (2.5 × 3 m²) and fresh water was available throughout the experimental period.

Table 1. Ingredient and chemical composition of the diet (g/kg dry matter (DM))

^a Contained 8 mg Co, 700 mg Cu, 4000 mg Fe, 2400 mg Mn, 2400 mg Zn, 240 mg I, 120 mg Se, 600 000 IU vitamin A, 1 00 000 IU vitamin D and 3000 mg vitamin E per kg premix.

^b Calculated from NRC (2001).

Data collection, sampling procedures and chemical analysis

Animals were weighed at 15, 30, 60 and 90 days of age. Milk, feed offered and refusals were recorded daily through the experimental period to calculate DM intake. Samples of milk, feed and refusals were collected once weekly for DM determination. Milk samples were preserved with 2-bromo-2-nitropropane-1, 3-diol and pooled for each group per period before chemical analysis. Feeds and refusals were dried in an oven at 55 °C for 48 h and ground to pass through a 1-mm screen with a mill (FZ102, Shanghai Hong Ji Instrument Co., Ltd., Shanghai, China) for chemical analysis. During the experimental period, six calves were chosen from each treatment at random, euthanized (lethal doses of pentobarbital sodium) and slaughtered at 30 and 90 days of age before the morning feeding. The entire digestive tract was removed and placed on ice. The small intestine was separated, laid out on a table and looped around pegs as described by Bauer et al. (Reference Bauer, Harmon, Bohnert, Branco and Huntington2001). After the length of the small intestine was measured, small intestinal chyme and sections were collected from the duodenum (10 cm distal to the pyloric sphincter), proximal jejunum (10 cm distal to ligament of trites), distal jejunum (10 cm distal to ligament of trites) and ileum (10 cm proximal to the ileocecal junction). The chyme in the lumen of each site was collected into 10 ml collection tubes and stored at −80 °C for enzyme activity analysis. Approximately 5-cm intestinal segments were removed, emptied and washed clean with normal saline. For morphometry, a 1 × 1 cm² area of the small intestine was retrieved. Three 1 × 1 cm² samples were collected and stored at −80 °C for RNA isolation and analysis. The entire process from animal stunning through to tissue collection generally took about 20 min. Milk samples were analysed for fat, true protein and lactose using infrared spectrophotometry (Foss 120 Milko-Scan, Foss Electric, Hillerød, Denmark) according to AOAC (1997, method 975.16) procedures. Analytical DM content of oven-dried samples was determined by drying at 135 °C for 3 h (AOAC 1997; method 930.15). Ash content was determined by combustion at 550 °C for 5 h. The content of neutral detergent fibre (NDF) and acid detergent fibre (ADF) were determined using the methods described by Van Soest et al. (Reference Van Soest, Robertson and Lewis1991) with heat stable alpha amylase and sodium sulphite used in the NDF procedure, and expressed inclusive of residual ash. Content of nitrogen (N) in the samples was determined by the Kjeldahl method (AOAC 1997; method 976.05) and multiplied by 6.25 to obtain protein content. Frozen chyme in the lumen was thawed and homogenized, and analysed using an amylase activity assay kit (C016, Nanjing Jiancheng Bioengineering Institute, Nanjing, China), a trypsin activity assay kit (A080-2, Nanjing Jiancheng Bioengineering Institute, Nanjing, China) and a lactase activity assay kit (A082-1, Nanjing Jiancheng Bioengineering Institute, Nanjing, China) by a UV2100 spectrophotometer (Shanghai Wanning Precision Scientific Instruments co., LTD, Shanghai, China). One unit (U) of enzyme activity was defined as the amount of enzyme that hydrolysed 1 µm substrate/min at pH 7.20 and 37 °C for trypsin, amylase and lactase.

Light microscopy and morphometry

Small intestinal tissue (1 × 1 cm² surface) was fixed in 4% formalin solution. After rinsing with water, the small intestinal tissue was dehydrated in a graded series of ethanol (70, 80, 85, 90, 95% and absolute ethanol), cleared twice with xylene, saturated and embedded in paraffin. Serial sections were then cut at 4 µm thicknesses, ten slices of each sample, deparaffinized in xylene, dehydrated, stained with haematoxylin and eosin, and observed under a light microscope. Muscular layer thickness, mucosa layer thickness, villus height and crypt depth of small intestine were determined by the computer operated Image C picture analysis system (Intronic GmbH, Berlin, Germany) and the IMES analysis program (Image-Pro Plus, version 6.0, Media Cybernetics Inc., Bethesda, MD, USA), using a colour video camera (Sony 3 CCD, CCD, Sony Electronics Inc., Tokyo, Japan) and a light microscope (Axiolab, Carl Zeiss Jena, Germany). The mucosa is the innermost layer of intestinal wall and is made up of epithelium, lamina propria and muscularis mucosae. The muscular layer is the third layer of the intestinal wall from the lumen outwards and comprises both longitudinal and circular smooth muscle that also helps with continued peristalsis and the movement of digested material along and out of the gut. In between the two layers of muscle lies the myenteric plexus.

Extraction of RNA and quantitative real-time polymerase chain reaction

Total RNA was extracted from the small intestine by using a Total RNA isolation kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol. The quality and concentration of the isolated RNA were determined by using a NanoDrop ND-1000 Spectrophotometer (NanoDrop Technologies, Rockland, DE, USA). The ratios of absorbance at 260 and 280 nm of all preparations were ~2.0. The integrity of RNA was checked by denaturing agarose gel electrophoresis and ethidium bromide staining. The iScriptTM cDNA Synthesis Kit (Bio-Rad Laboratories GmbH, Munich, Germany) was used to synthesize cDNA from 500 ng total RNA of each sample per 10-μl sample reaction according to the manufacturer's instructions. Reaction conditions were 15 min at 37 °C and 5 min at 85 °C. Negative control reactions without reverse transcriptase were performed on each sample to detect possible contamination of genomic DNA or environmental DNA. The abundance of mRNA for GHR, IGF-1R, PEPT1 and SGLT1 was quantified by quantitative real-time polymerase chain reaction (qRT–PCR) using the iCycler and the iQ-SYBR green detection (Bio-Rad Laboratories, Hercules, CA, USA). The primer sets used for real-time PCR were forward primer (5′–3′) CAG ACA AAT CAC TCC ACC AA, reverse primer (5′–3′) GAA GGG CAC CAC CAG GAG T for 18s rRNA as reference gene, forward primer (5′–3′) AAA TTC ACC AAG TGC CGT TC, reverse primer (5′–3′) GGG GCA TTC TTT CCA TTC TT for GHR (GenBank No. AF044258, 141 bp), forward primer (5′–3′) CCT CAT TAT TCC TGC TAA CCA A, reverse primer (5′–3′) AGA TAG AAG AGA TGC GAG GAG GAT for IGF-1R (GenBank No. JN204287, 159 bp), forward primer (5′–3′) TGG CTG GGG AAG TTC AAG AC, reverse primer (5′–3′) TCC TGG CCC TCT TCA AA for PEPT1 (GenBank No. NM001099378, 239 bp), forward primer (5′–3′) TTT CTG GGG CCA TAT TCA TC, reverse primer (5′–3′) CGT CTG CAA GGT GTC TGT GT for SGLT1 (GenBank No. AF508807, 137 bp). Subsequent qPCR was performed on a MxPro-Mx3000P multiplex quantitative PCR systems (Stratagene, La Jolla, CA, USA) at a minimum in triplicate. A reaction mixture (20 µl) consisted of 2 µl cDNA, 10 µl SYBR Premix Taq™ II (TaKaRa Biotechnology Co., Ltd., Dalian, China), 0.8 µl PCR Forward Primer, 0.8 µl PCR Reverse Primer, 0.4 µl ROX Reference Dye II and 6.0 µl dH₂O. The PCR was performed under the following cycle conditions: 1 cycle of 95 °C for 20 s, 45 cycles of 95 °C for 20 s, and annealing temperature for 30 s and 62 °C for 20 s, followed by a melting curve analysis (Denman & McSweeney Reference Denman and McSweeney2006). Fluorescence detection was performed at the end of each denaturation and extension step. The relative quantity of mRNA for GHR, IGF-1R, PEPT1 and SGLT1 were done as a proportion of 18S rRNA according to the equation:

$${\rm {Relative \; quantification}} = {\rm 2}^{{ -} \left( {\rm {Ct \; target \; genes - Ct \; total \; reference \; genes}} \right)}$$

where C_t represents threshold cycle.

Statistical analysis

Data were analysed using the mixed model procedure of SAS (Proc Mixed; SAS 2002) with a 2 (age) × 4 (isovalerate supplementation) factorial arrangement of treatments. The treatment with different levels of isovalerate supplementation and age was considered as a fixed effect, while animals within treatment were considered as random effects. In addition, linear and quadratic orthogonal contrasts were tested using the CONTRAST statement of SAS with coefficients estimated based on the isovalerate application rates. Effects of the factors were declared significant at P < 0.05.