This experiment was carried out from November 7, 2020 to February 2, 2021 at Nestle Dairy Farm Institute (DFI, Harbin, Heilongjiang, China, E 125°41′, N 45°08′). The average temperature and humidity were −12.7°C and 69.7%, respectively. Treatment of disease followed the standard operating procedures at DFI and sick calves were treated by a veterinarian accordingly.
Calves, Housing, and Diets
A total of twenty-four Holstein male calves were separated from their dams after birth and placed in individual pens (1.8 m × 0.8 m) on straw beddings. Calves were fed 4 L colostrum within 3 h after birth and another 4 L colostrum within 12 h to ensure the successful passive transfer of immunity. Then 4 L of pasteurized whole milk were provided twice daily (1100 h and 1900 h) until 7 d of age. After that, calves were bottled-fed three times a day at 0730 h, 1430 h and 2200 h the whole study with an adjusted step-up/step-down milk feeding protocol as 5, 6, 7, 6, 5, 4 and 3 L at wk 1, 2, 3, 4, 5, 6, 7 and 8, respectively. All calves were weaned at 56 d of age and the experiment was terminated at 70 d of age.
This trial was performed according to randomized complete block design and calves were assigned to 4 blocks based on arrival date. Within each block, calves were randomly allocated to three dietary treatments including 100% milk replacer (MR, Land O' Lakes, Arden Hills, MN), 50% pasteurized waste milk mixed with 50% milk replacer (MM), as well 100% pasteurized waste milk (WM, including milk with antibiotic and transition milk of 2~3 d of postpartum cows at DFI). Milk replacer was mixed with warm water (46 ℃) at a ratio of 1:7 and waste milk was pasteurized by heating up to 72 ℃ for 15 s and cooled down to 38~40 ℃ for bottle feeding. During the experiment, starter and fresh water were fed ad libitum from 8 d of age. Intake of starter was recorded daily, and samples were taken every two weeks, which were kept frozen for subsequent analyses.
Measurements, Sampling, and Analyses
Body weight (BW), height, length, hip width, hip height and heart girth were measured as described by Mirzaei (24) before morning feeding at 8 d, 56 d and 70 d of age. Starter intake were analyzed every two weeks, and average daily gain (ADG), dry matter intake (DMI) and feed efficiency (FE) were calculated from 8 to 56 d (pre-weaned period), 57 to 70 d (post-weaned period) and 8 to 70 d (whole experiment period) of age. Fecal consistency was scored daily before morning feeding according to the guideline by Larson (25): 1 = firm, well-formed (not hard); 2 = soft, pudding like; 3 = runny, pancake batter; 4 = liquid, splatters, pulpy orange juice. Feces were estimated abnormal when the score ≥ 3, and a diarrhea case was defined when fecal score was ≥ 3 for at least 2 days.
Blood samples were obtained from the jugular vein into heparin sodium tubes before morning feeding at 8, 49, and 70 d of age. Following collection, blood samples were separated by centrifuging at 3500 × g for 10 min to obtain plasma, which was divided into several aliquots and stored at −20°C for subsequent analyses. The concentration of IgG, IgM, IgA, IL-2, IL-6, IL-10 and TNF-α were measured using ELISA assays (Enzyme-linked Biotechnology Co., Ltd, Shanghai, China).
Fresh feces were obtained by-hand from the rectum of calves using clean gloves at 49 d and 70 d of age. After collection, part of samples was directly stored at −80 °C for analysis of 16S rRNA gene copies of E. coli and Lactobacillus species. Pour plate method was used for counting bacteria (26) using Maconkey Agar Medium (Aoboxing, Beijing, China) for E. coli and MRS Agar Medium (Aoboxing, Beijing, China) for Lactobacillus species (21, 27). The remaining fecal samples were diluted with water (1:4) for measuring pH using a glass electrode (Sartorius, Göttingen, Germany).
The bacterial community was profiled by sequencing V3–V4 hypervariable region of 16S rRNA genes using 314F (5′-CCTAYGGGRBGCASCAG-3′) and 806R (5′-GGACTACNNGGGTATATAAT-3′) primers (28). The amplicons were sequenced (2 × 250 bp) using the nova PE250 in Novaseq 6000 platform (Novogene, Tianjin, China). Analysis of the sequence data were performed using Quantitative Insights Into Microbial Ecology 2 (29) (QIIME2, version 2021.04). Amplicon sequence variants were generated using DADA2 (30) workflow to remove the barcodes, primers and low-quality sequences. The taxonomic classification was performed using the SILVA database (SILVA Release 138) based on 99% sequence similarity. All sequences were deposited in the NCBI Sequence Read Archive under the project number PRJNA752817.
Growth performance, plasma immune parameters, fecal scores, health-related indices, and copy numbers of E. coli and Lactobacillus) analyses were conducted using PROC MIXED procedure of SAS (version 9.4; SAS Institude Inc., Cary, NC), with treatment and sampling time as fixed effect, and block as random effect. Initial value of body weight, structural measures and blood immune indices were considered as covariates. Treatment, age, and their interactions were included in the model as fixed effects.
Fecal bacterial alpha diversity and relative abundance of bacterial taxa were analyzed using Kruskal-Wallis test in R (version 4.0.2; R Foundation for Statistical Computing, Vienna, Austria), and the P-value was adjusted based on false discovery rate (FDR) using the Benjamini-Hochberg algorithm. Principal coordinate analysis (PCoA) of the fecal microbial profiles was conducted using Bray-Curtis distance and permutational analysis of variance (PERMANOVA) in QIIME2. Statistical significance was declared at P ≤ 0.05 and trends at 0.05 < P ≤ 0.10. One calf became ill and dead at 14 d of age and thus the data for this calf was excluded from the dataset before analysis.