The effects of milk replacer allowance and weaning age on the performance, nutrients digestibility, and ruminal microbiota communities of lambs

Animal Feed Science and Technology 257 (2019) 114263

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The effects of milk replacer allowance and weaning age on the performance, nutrients digestibility, and ruminal microbiota communities of lambs Qian Zhanga,b, Chong Lia,c, Xiaolin Niua, Zhian Zhanga, Fadi Lia, Fei Lia,

T

⁎

a State Key Laboratory of Grassland Agro-ecosystems; Key Laboratory of Grassland Livestock Industry Innovation, Ministry of Agriculture and Rural Affairs; Engineering Research Center of Grassland Industry, Ministry of Education; College of Pastoral Agriculture Science and Technology, Lanzhou University; Lanzhou 730000, China b Institute of Grassland Research of CAAS, Chinese Academy of Agricultural Sciences, Hohhot, China c College of Animal Science and Technology, Gansu Agricultural University, Lanzhou, China

A R T IC LE I N F O

ABS TRA CT

Keywords: Lambs Performance Digestion Morphological Fermentation Rumen microbiota

There has been a recent shift in raising lambs towards early weaning and intensive feeding, with the aim of improving growth rates and feed utilization efficiency of the lamb. This change has prompted studies on the effects on rumen development, which is influenced by a complex interaction between the host, solid feed, ruminal microbiota and fermentation. In this study, 30 male Hu lambs were divided randomly into four treatment groups in a 2 × 2 factorial-design with two different weaning ages (21 or 35 d) and two different milk replacer (MR) feeding allowance (2% or 4% average body weight). This study aimed to examine the effects of weaning age and MR levels on: 1) ruminal morphology, fermentation and microbial composition; and 2) feed intake, growth performance and nutrient digestibility from pre-weaning to post-weaning in lambs. Results showed that weaning age and MR levels had limited effect on apparent digestibility of nutrients, ruminal microbiota and fermentation at 50 d. The digestibilities of dry matter (DM), crude protein (CP), fat and acid detergent fiber (ADF) declined progressively from 8 d to 49 d. There was an interaction between weaning age and MR treatment on the abundance of Firmicutes (P = 0.020), Ruminococcaceae UCG 014 (P = 0.033) and Roseburia (P = 0.032). The 21 d weaning groups had a significantly higher (P = 0.035) relative abundance of Ruminococcus 1 than the 35 d weaning groups. Lambs weaned at 35 d had greater starch intake (P = 0.004), fat intake (P < 0.001), total average daily gain (ADG) (P = 0.001), final body weight (BW) (P = 0.013) and carcass weight (P = 0.008) than 21 d weaned lambs, and lambs given 4% levels of MR had greater fat (P < 0.001), CP intake (P = 0.019), total ADG (P = 0.013) and carcass weight (P = 0.036) than lambs consuming 2% levels of MR. In conclusion, weaning age and MR levels had limited influence on apparent digestibility of nutrients, ruminal microbiota and fermentation at 50 d. Feeding a high level of MR and later weaning age provided a large amount of

Abbreviations: ADF, acid detergent fiber; ADG, average daily gain; BW, body weight; CP, crude protein; DM, dry matter; NDF, neutral detergent fiber; LSD, least significant difference; MR, milk replacer; NMDS, non-metric multi-dimensional scaling; OTUs, operational taxonomic units; PCA, principal component analysis; SEM, standard errors of the mean; VFA, volatile fatty acid ⁎ Corresponding author. E-mail addresses: [email protected] (Q. Zhang), [email protected] (C. Li), [email protected] (X. Niu), [email protected] (Z. Zhang), [email protected] (F. Li), [email protected] (F. Li). https://doi.org/10.1016/j.anifeedsci.2019.114263 Received 24 November 2018; Received in revised form 21 August 2019; Accepted 22 August 2019 0377-8401/ © 2019 Elsevier B.V. All rights reserved.

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easily absorbed nutrients which led to greater ADG and BW, and had a sustained positive effect on lambs’ body weight gain.

1. Introduction Before weaning at 8–10 weeks, lambs consume a changing balance of milk and pasture and dried feed (Langlands, 1973). This feeding program limits the growth rate of lambs and increases the lambing interval of ewes, which can lead to economic and animal welfare losses. As an alternative, a pre-weaning intensive MR feeding program can support accelerated growth and production, and also has long-term benefits for sheep health and performance (Khan et al., 2011; Bach, 2012). Many studies have reported that robust milk or MR feeding program could increase pre-weaning ADG and weaned BW of young ruminants (Cowles et al., 2006; Chapman et al., 2016). In dairy calves, ad libitum milk or MR feeding programs during the early pre-weaning period resulted in greater nutrient intake and higher feed utilization efficiency, allowing higher ADG and BW to be achieved than in a restricted milk feeding system (Maccari et al., 2015). However, a higher MR intake could compromise post-weaning starter intake and ADG compared to conventional quantities of MR (Chapman et al., 2016; Hill et al., 2016). Several studies also found that feeding high quantities of MR could reduce post-weaning starter digestibility, especially for NDF and ADF, and attributed this to late initiation of starter intake (Terré et al., 2006, 2007; Hill et al., 2010; Chapman et al., 2016; Hill et al., 2016). However, few studies have shown a dynamic change of nutrient digestibility in an intensive MR feeding program from pre-weaning to post-weaning. Therefore, there is an increasing interest in the impact of weaning age and MR feeding programs on feed intake patterns, growth performance and nutrient digestibility. Previous studies have reported that age, weaning and the introduction of solid feed all affected the composition of rumen microbiota (Malmuthuge et al., 2014; Meale et al., 2016; Dias et al., 2017). An adequate starter intake can stimulate the development of the rumen, which is necessary to establish rumen microbiota (Khan et al., 2016). A complex and diverse microbial ecosystem can play an important role in the ruminal function and nutrient digestion (Henderson et al., 2015). Previous studies reported that low digestibility in young ruminants fed large quantities of MR was likely to be associated with sub-optimal development of the rumen (Chapman et al., 2016; Hill et al., 2016). However, only a few studies have been done on lambs to assess the effects of intensive MR feeding and weaning age on ruminal fermentation and microbiota, which is crucial for ensuring that early weaning is properly carried out and may also explain the low digestibility during weaning. This study aimed to examine the effects of weaning age and different MR feeding allowance from pre-weaning to post-weaning on: 1) ruminal morphology, fermentation and microbial composition; and 2) feed intake, growth performance and dynamic change of nutrient digestibility.

Table 1 Ingredient and chemical composition of the diet. Starter1

Item Ingredient (g/kg DM) Alfalfa hay Corn Extruded corn Soybean meal Extruded soybean Corn gluten meal Bran Limestone Premix2 Salt Chemical composition (g/kg DM) Dry matter Crude protein Fat Starch Neutral detergent fiber Acid detergent fiber

Milk replacer

180.5 210.0 220.3 210.5 40.0 50.0 60.0 3.0 1.0 4.0 938.6 195.0 13.3 331.0 189.4 86.0

1

969.1 232.2 132.0 0.0 0.0 0.0

Starter was pelleted. Premix composition (per kg feed): 25 mg Fe as FeSO4 × H2O; 40 mg Zn as ZnSO4 × H2O; 8 mg Cu as CuSO4×5H2O; 40 mg Mn as MnSO4 × H2O; 0.3 mg I as KI; 0.2 mg Se as Na2SeO3; 0.1 mg Co as CoCl2; 940 IU vitamin A; 111 IU vitamin D; 20 IU vitamin E, and; 0.02 mg vitamin B12. 2

2

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2. Materials and methods This study followed the recommendations of the Biological Studies Animal Care and Use Committee of Gansu Province, China (2005–12). The experiment was approved by Lanzhou University (2017YFD0500502) and was conducted according to their established guidelines. All efforts were made to minimize animal suffering. 2.1. Animal management Thirty-two male Hu lambs (from Minqin Zhongtian Sheep Industry Co. Ltd., Minqin, China) of the same age and similar birth weight (mean ± SD: 3.29 ± 0.68 kg) were chosen. Two lambs were removed because of serious diarrhea. The remaining 30 healthy lambs were divided randomly into four treatment groups in a 2 × 2 factorial-design with two different weaning (weaned from MR abruptly) ages and two different MR feeding allowance. One group was received MR at 2% BW (n = 15) and one group at 4% BW (n = 15) and within each group, half the lambs weaned at 21 d and half at 35 days (n = 8 for 21 d; n = 7 for 35 d for each). The 2% MR (standard level) group follows the feeding guidelines of the Feed Research Institute Chinese Academy of Agricultural Sciences, China (Yue et al., 2011). The lambs were housed indoors with their ewes from birth to 6 d to ensure sufficient intake of colostrum. At 5 d, the lambs were trained to use the nipple bottle containing MR (Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing, China). At 7 d, the lambs were separated from their ewes and placed in individual pens (0.65 m wide × 1.10 m long; 0.715 m2). All lambs were weighed at birth and then every 7 d to calculate average body weight and adjust MR. No MR fed was rejected. Starter (formulated to meet the requirements of the feeding standard of meat-producing sheep [NYT816-2004]) and water were supplied ad libitum from 7 d to 49 d. The nutrient content of MR and starter are presented in Table 1. 2.2. Measurements and sample collection Individual starter intake was recorded daily from 8 to 49 d. Starter, ort and feces were collected daily at 11:00 h from 8 to 49 d, and slatted floors and gauze were used to separate feces from urine. Feces collected during a 24 h period were weighed and mixed uniformly, then preserved immediately with sulfuric acid. At 50 d, the lambs were slaughtered at the experimental station of Lanzhou University in accordance to the Biological Studies Animal Care and Use Committee of Gansu Province, China (2005–12). Before morning feeding, lambs were euthanized by penetrative captive bolt followed by exsanguination from the jugular vein. After slaughter, the abdominal cavity was opened, and the internal organs and digestive tract were removed and placed on a clean white porcelain plate. Rumen pH was measured immediately using an acidity meter (Sartorius PB-10, Sartorius Biotech Inc., Gottingen, Germany). Ruminal contents were mixed thoroughly and divided into three portions in 5-mL sterile tubes. One tube was stored at −80 °C for DNA extraction, and the other two tubes were stored at −20 °C for ruminal fermentation and enzymic activity analysis, respectively. Segments of the ruminal tissue (cranial ventral sac) were fixed in 4% paraformaldehyde for morphology measurements. 2.3. Analysis of diet and fecal samples MR, starter and feces were analyzed for DM (24 h at 103 °C), CP (Kjeldahl method 988.05; AOAC International, 2000), fat (alkaline treatment using Roese-Gottlieb method 932.06 for MR, diethyl ether extraction method 2003.05 for starter; AOAC International, 2000), NDF and ADF following the method of Van Soest et al. (1991) with heat-stable alpha-amylase and sodium sulfite used in the NDF procedure, and expressed inclusive of starch using a commercial assay kit (Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer’s instructions. 2.4. Measurement of histomorphology in the rumen Rumen specimens (sampled at 50 d) were embedded in paraffin, sectioned (5 μm) and stained with hematoxylin-eosin. In triplicate, 30 intact well-oriented papillae were selected for each ruminal cross section. Papilla height, and width, along with muscular thickness, were determined using an image analysis system (Version 1, Leica Imaging Systems Ltd., Cambridge, UK). Papilla height was measured from the apex to the base of the papilla along its axis, papilla width was measured at mid-papillae height (Dieho et al., 2016), and the rumen muscular thickness was measured from the junction between the submucosal and muscular layers to that between the muscular layer and the tunica serosa (Ding et al., 2018). 2.5. Measurement of metabolic phenotypes in the rumen Thawed rumen fluid was centrifuged at 15,000 g for 10 min at 4 °C. Volatile fatty acid (VFA) concentrations were determined by gas chromatography (TRACE 1300, Thermo Scientific, Milan, Italy) as described by Li et al. (2014) using a 30 m ×0.32 mm ×0.33 μm fused silica column (DB-FFAP, Agilent Technologies, USA). Total ruminal nitrogen, ammonia nitrogen and urea nitrogen were determined using a commercial assay kit (Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer’s instructions. 3

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2.6. Measurement of digestive enzymatic activity in the rumen An automatic microplate reader (Multiskan Mk3, Thermo Scientific, Wilmington, DE, USA) was used to measure the enzymatic activity of amylase, cellulase and protease in the ruminal contents. All samples were determined using commercial assay kits (Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer’s instructions. 2.7. DNA extraction, PCR amplification and sequencing Total genomic DNA was extracted from samples using the CTAB method, and DNA concentration and purity were assessed on 1% agarose gels. Concentrations of extracted DNA were determined using a Nano-Drop 2000 spectrophotometer (Thermo Scientific) after sterile water was used to dilute the DNA to 10 ng/μL. 16S rRNA genes of distinct regions were amplified using specific primers (341F: CCTAYGGGRBGCASCAG; 806R: GGACTACNNGGGTATCTAAT) with the barcode. All PCR reactions were carried out using Phusion® High-Fidelity PCR Master Mix (New England Biolabs, Essex, USA). The same volume of 1 × loading buffer was mixed with PCR products and used for electrophoresis on 2% agarose gel for detection. Samples with a bright main strip between 400–450 bp were chosen for further experiments. PCR products were mixed in ratios of the same density, then the mixture of PCR products was purified using a Qiagen Gel Extraction Kit (Qiagen, Duesseldorf, Germany). Sequencing libraries were generated using a TruSeq® DNA PCR-Free Sample Preparation Kit (Illumina, San Diego, USA) following the manufacturer's recommendations, and index codes were added. The quality of libraries were assessed on a Qubit 2.0 Fluorometer (Thermo Scientific, Wilmington, USA) and an Agilent Bioanalyzer 2100 system (Agilent Technologies, Palo Alto, USA). Finally, the libraries were sequenced on an Illumina HiSeq 2500 platform, generating 250 bp paired-end reads. 2.8. Analysis of 16S rDNA sequencing data Raw sequences were filtered through a quality control pipeline, and only those with scores above 30 were retained for further analyses. Quality filtering of the raw tags was performed under specific filtering conditions to obtain high-quality clean tags (Bokulich et al., 2013) according to the QIIME (V1.7.0, http://qiime.org/index.html) quality control process (Caporaso et al., 2010). The tags were compared to the reference database using the UCHIME algorithm (http://www.drive5.com/usearch/manual/ uchime_algo.html) (Edgar et al., 2011) to detect chimera sequences, which were then removed (Haas et al., 2011). This allowed the Effective Tags to be obtained. Sequences analysis was performed using Uparse software (v7.0.1001, http://drive5.com/uparse/) (Edgar, 2013) and sequences with ≥ 97% similarity were assigned to the same operational taxonomic units (OTUs). A representative sequence for each OTU was screened for further annotation, and the GreenGene Database (http://greengenes.lbl.gov/cgi-bin/nphindex.cgi) (DeSantis et al., 2006) was used to annotate taxonomic information based on the RDP classifier algorithm (Version 2.2, http://sourceforge.net/projects/rdp-classifier/) (Wang et al., 2007). OTU abundance information was normalized using a standard of sequence number corresponding to the sample with the fewest sequences. Subsequent analyses of alpha and beta diversities were performed based on these normalized output data. Alpha diversity was used to analyze complexity of species diversity via six indices, including Observed Species, Chao1, Shannon, Simpson, ACE. Beta diversity analysis, calculated using QIIME software, was used to evaluate differences in species complexity between samples. Non-metric multidimensional scaling (NMDS) analysis, with a conventional cut-off of < 0.2 for the stress value, was performed using the ‘vegan’ package in R software platform (v2.15.3). All sequencing data are available at NCBI (NCBI Bioproject Accession number: PRJNA432641). 2.9. Statistical analyses Data were analyzed using the SPSS software (version 19.0, Chicago, IL, USA). All data were evaluated using a 2 × 2 (weaning age and MR treatment) multi-factorial design. BW, ADG, starter intake, apparent digestibility of nutrients, rumen morphology, fermentation parameters and enzymatic activity were analyzed by ANOVA using the GLM procedure. The least significant difference (LSD) post hoc tests were used to analyze variations between groups. The bacterial abundance was analyzed using the Aligned Ranking Transform in R software platform (v2.15.3) and using the Kruskal-Wallis method to test the difference between groups when Table 2 The effects of weaning age and MR levels on rumen histomorphology of lambs. Item

Rumen Rumen Rumen Rumen Rumen

21d 2%MR

weight (g) relative weight (% of BW) papilla length (μm) papilla width (μm) muscular thickness (μm)

217 2.19 1821 326 857

21d 4%MR

241 2.27 1960 410 1031

35 d 2%MR

243 2.16 2191 373 1204

35 d 4%MR

237 1.90 1901 363 1168

BW: body weight; MR: milk replacer; SEM, standard errors of the mean. 1 W × T: Weaning age × MR treatment interaction. 4

SEM

9.12 0.05 90.46 15.89 41.23

Weaning Age

P value MR Treatment

W × T1

0.552 0.050 0.397 0.256 0.007

0.613 0.348 0.680 0.996 0.408

0.418 0.087 0.248 0.155 0.214

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the interaction was significant. Statistical significance was set at P < 0.05 and the tendency was set at P < 0. 10. 3. Results 3.1. Histomorphology of the rumen There was no significant difference (P > 0.05) among groups in rumen weight or relative weight and papilla length or papilla width. However, lambs with a later weaning age had a significantly thicker ruminal muscle (P = 0.007, Table 2). 3.2. Rumen fermentation parameters, metabolic phenotypes and digestive enzymatic activity High levels of MR significantly decreased rumen pH (P = 0.007) and enzyme activity of cellulase (P = 0.002, Table 3). However, neither fermentation parameters nor metabolic phenotypes were influenced significantly (P > 0.05) by weaning age, MR treatment or their interaction. 3.3. Ruminal microbiota diversity and community structure After data filtering, quality control, and removal of low-confidence singletons, an average of 72,295 V3–V4 16S rRNA gene sequence reads were obtained for each sample. The length of the sequences ranged between 414 to 423 bp. The library coverage of samples from the four groups was above 99%. Alpha diversity results showed that the Observed Species, Chao1, ACE, Simpson and Shannon indices were not significantly influenced (P > 0.05) by weaning age, MR treatment or their interaction (Table S1). An NMDS plot showed all points uniformly distributed in four quadrants (Figure S1). We identified the top 10 most common phyla and genera (Figure S2). The most common phyla were Bacteroidetes, Proteobacteria and Firmicutes, which were all > 20% of the total. The most common genera were Prevotella 1 and Succinivibrionaceae UCG 001, which were both > 15% of the total. The mean abundance of bacterial taxa present at > 0.5% of ruminal sequences were shown in Table 4. At the phylum level, weaning age and MR treatment had an interaction (P = 0.020) for Firmicutes. At the genus level, there was a weaning age × MR treatment interaction for Ruminococcaceae UCG 014 (P = 0.033) and Roseburia (P = 0.032). The 21 d weaning groups had a significantly higher (P = 0.035) relative abundance of Ruminococcus 1 than the 35 d weaning groups. 3.4. Nutrient apparent digestibility and growth performance The digestibility of DM, CP, fat and ADF declined progressively from 8 d to 49 d (Table S2). The weaning age and MR treatment had a negligible influence on apparent digestibility of nutrients (except NDF) at the end of the experiment (49 d). The initial BW measurements did not differ among treatments (Table 5). There was a significant effect of weaning age on ADG, final BW, carcass weight and total starter intake. Lambs weaned at 35 d had greater ADG (P = 0.001), final BW (P = 0.013) and carcass weight (P = 0.008) than 21 d weaned lambs. There was a significant effect of MR treatment on total ADG and carcass weight. Lambs given 4% MR had greater ADG (P = 0.013) and carcass weight (P = 0.036), moreover, had a trend for greater final BW (P = 0.080) than 2% MR groups. Lambs weaned at 21 d consumed more starter (P = 0.015) than 35 d weaned lambs. Lambs weaned at 35 d had significantly greater BW than 21 d weaned groups (Figure S3) at 28 d (P = 0.041), 35 d (P = 0.005), 42 d (P = 0.043) and 49 d (P = 0.013). Groups fed 4% MR had significantly higher BW than 2% MR groups at 14 d (P = 0.034), 21 d Table 3 The effects of weaning age and MR levels on rumen fermentation parameters and enzyme activity of lambs. Variable

pH Total VFA (mmol/L) Acetate (mmol/L) Propionate (mmol/L) Butyrate (mmol/L) Isobutyrate (mmol/L) Isovalerate (mmol/L) Valerate (mmol/L) Total nitrogen (mg/mL) Ammonia nitrogen (mg/mL) Urea nitrogen (mg/mL) Protein nitrogen (mg/mL) Amylase (U/mg protein) Cellulase (U/mg protein) Protease (U/mg protein)

21d 2%MR

6.98 52.03 27.22 8.14 4.17 6.14 2.31 52.03 556.20 87.47 15.04 453.70 10.95 60.86 0.37

21d 4%MR

6.85 52.87 29.33 7.25 3.19 4.45 2.91 52.87 616.13 93.63 15.63 506.87 10.03 31.43 0.25

35 d 2%MR

35 d 4%MR

7.09 52.62 28.33 7.48 3.70 5.57 2.30 52.62 614.49 86.32 16.91 511.26 10.59 69.23 0.39

6.79 53.99 25.64 8.06 3.63 6.29 2.39 53.99 595.93 84.99 18.39 492.55 12.56 42.66 0.27

MR: milk replacer; SEM: standard errors of the mean; VFA: volatile fatty acid. 1 W × T: Weaning age × MR treatment interaction. 5

SEM

0.04 0.88 0.75 0.57 0.24 0.44 0.18 0.88 43.66 3.36 1.23 40.27 1.28 4.14 0.08

Weaning Age

P value MR Treatment

W × T1

0.703 0.505 0.631 0.400 0.950 0.983 0.481 0.464 0.829 0.474 0.357 0.791 0.675 0.247 0.899

0.007 0.377 0.536 0.848 0.891 0.286 0.591 0.336 0.815 0.722 0.678 0.832 0.838 0.002 0.485

0.278 0.268 0.879 0.123 0.526 0.357 0.187 0.480 0.657 0.582 0.858 0.660 0.578 0.864 0.993

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Table 4 The effects of weaning age and MR levels on the relative abundances of bacterial taxa in the rumen of lambs. Taxon (%)

Phylum Bacteroidetes Proteobacteria Firmicutes Spirochaetes Fibrobacteres Cyanobacteria Actinobacteria Genus Prevotella 1 Succinivibrionaceae UCG 001 Others Succinivibrio Oribacterium Prevotella 7 Treponema 2 Fibrobacter Lachnospiraceae NK3A20 group Sphaerochaeta Ruminococcus 1 Ruminococcaceae UCG 014 Rikenellaceae RC9 gut group Succiniclasticum Ruminobacter Prevotellaceae UCG 001 Selenomonas Ruminococcus 2 Roseburia Sharpea Selenomonas 1

21d 2%MR

21d 4%MR

35 d 2%MR

35 d 4%MR

SEM Weaning Age

P value MR Treatment

W × T1

37.96 28.00 21.30 6.20 4.80 0.80 0.35

26.32 30.55 34.30 5.60 2.15 0.27 0.20

39.87 22.40 26.76 5.80 3.81 0.59 0.28

36.77 29.40 25.33 3.51 2.00 1.10 1.32

2.20 2.10 1.70 0.61 0.57 0.20 0.25

0.177 0.480 0.344 0.408 0.875 0.425 0.350

0.080 0.270 0.133 0.277 0.261 0.854 0.293

0.268 0.464 0.020 0.894 0.839 0.867 0.269

20.40 16.80 10.98 10.58 4.90 5.90 4.30 4.79 2.27 2.00 2.10 0.80 1.40 0.80 0.10 1.60 0.30 0.40 0.50 1.10 0.30

15.90 19.45 9.00 9.84 9.70 3.50 4.09 2.15 4.10 1.50 2.60 1.95 0.70 1.40 0.63 0.70 1.10 1.01 0.84 0.70 1.92

17.34 17.57 13.80 4.20 10.00 8.53 3.40 3.80 2.40 2.40 1.33 1.10 2.01 1.10 0.20 0.60 0.70 0.41 0.80 0.57 0.10

17.63 16.80 12.60 8.70 9.30 8.50 2.10 2.00 3.18 1.43 0.76 0.89 0.58 1.10 3.50 0.59 1.30 1.00 0.40 0.10 0.07

2.30 2.44 1.03 1.80 0.90 1.35 0.60 0.60 0.34 0.30 0.20 0.16 0.30 0.13 0.80 0.20 0.20 0.20 0.07 0.20 0.48

0.876 0.907 0.695 0.247 0.235 0.185 0.176 0.875 0.387 0.721 0.035 0.119 0.872 0.907 0.606 0.219 0.809 0.999 0.906 0.715 0.388

0.642 0.829 0.464 0.821 0.199 0.580 0.676 0.261 0.263 0.332 0.775 0.139 0.128 0.191 0.283 0.374 0.160 0.161 0.584 0.213 0.617

0.767 0.773 0.949 0.598 0.170 0.996 0.905 0.839 0.347 0.894 0.181 0.033 0.418 0.347 0.606 0.524 0.792 0.640 0.032 0.882 0.658

The mean abundance of bacterial taxa present at > 0.5% of ruminal sequences. MR: milk replacer; SEM: standard errors of the mean. 1 W × T: Weaning age × MR treatment interaction. Table 5 The effects of weaning age and MR levels on performance and DM, CP, fat, starch intake of lambs. Variable

Initial BW, kg Final BW, kg Carcass weight, kg Total MR intake, g/lamb Total ADG, g/d Total starter intake, DM g/d DM intake, g/d CP intake, g/d Starch intake, g/d Fat intake, g/d DM intake, g/kg of BW CP intake, g/kg of BW Starch intake, g/kg of BW Fat intake, g/kg of BW

21d 2%MR

4.56 9.66 4.26 1333 124.5 221.3 248.1 52.0 77.0 7.55d 31.8 6.77 9.04a 1.25c

21d 4%MR

4.54 10.69 4.53 2666 146.4 231.5 288.1 61.6 82.3 10.79c 34.8 7.51 9.02a 1.61b

35 d 2%MR

35 d 4%MR

4.64 11.26 4.75 3108 157.7 197.6 258.4 56.0 65.1 12.67b 31.6 6.94 6.92b 1.90b

4.61 12.66 5.90 6216 191.5 155.2 294.1 65.7 53.7 21.68a 32.2 7.30 4.74c 2.77a

SEM

0.17 0.38 0.16 – 5.23 9.65 9.59 1.93 3.21 0.21 0.57 0.12 0.21 0.05

Weaning Age

P value MR Treatment

W × T1

0.818 0.013 0.008 – 0.001 0.015 0.675 0.301 0.004 < 0.001 0.234 0.914 < 0.001 < 0.001

0.953 0.080 0.036 – 0.013 0.412 0.060 0.019 0.635 < 0.001 0.120 0.024 0.015 < 0.001

0.995 0.783 0.182 – 0.574 0.185 0.909 0.998 0.206 < 0.001 0.315 0.416 0.017 0.019

a–d: values in the same row with different superscript letters are significantly different (P < 0.05). ADG: average daily gain; BW: body weight; CP: crude protein; DM: dry matter; MR: milk replacer; SEM: standard errors of the mean. 1 W × T: Weaning age × MR treatment interaction.

(P = 0.002), 28 d (P = 0.027) and 35 d (P = 0.025). There were significant effects of weaning age on ADG at 22–28 d, 29–35 d and 43–49 d (Figure S4), and of MR treatment on ADG at 8–14 d, 15–21 d, 36–42 d and 43–49 d, and of weaning age and MR treatment interaction on ADG at 22–28 d (P = 0.034) and 36–42 d (P = 0.022). At 22–28 d, lambs weaned at 35 d had greater (P < 0.001) ADG than 21 d weaned lambs, and lambs in the 21 d, 4% MR group had the lowest (P < 0.05) ADG. We observed a significant weaning age and MR treatment interaction on starter intake at 35 d (P = 0.047) (Figure S5), and a 6

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significant effect of weaning age at 28 d and 35 d. The lambs weaned at 21 d had greater starter intake than 35 d weaned groups at 28 d (P = 0.001). At 35 d, lambs in the 21 d, 4% MR group had greater starter intake than that in the 35 d, 2% MR group (P = 0.001) and the 35 d, 4% MR group (P < 0.001). There was a significant effect of weaning age on absolute and mass specific starch and fat intake (Table 5). Lambs weaned at 21 d consumed more absolute and mass specific starch (P = 0.004, P < 0.001, respectively) than 35 d weaned lambs; whereas, lambs weaned at 35 d consumed more absolute and mass specific fat (P < 0.001, P < 0.001, respectively) than 21 d weaned lambs. There was a significant effect of MR treatment on CP and fat intake; and on mass specific CP, starch and fat. Groups fed 4% levels of MR had significantly higher CP intake (P = 0.019), fat intake (P < 0.001), and mass specific CP (P = 0.024) and fat (P < 0.001) than 2% MR groups. The 2% MR groups consumed more starch (P = 0.015) than the 4% MR groups. We observed a weaning age and MR treatment interaction on fat intake (P < 0.001) and on mass specific fat (P = 0.019) and starch (P = 0.017). Lambs in the 35 d, 4% MR group consumed the most absolute (P < 0.001) and mass specific (P < 0.001) fat, and the 21 d, 2% MR group consumed the least absolute (P < 0.001) and mass specific fat (P < 0.05). In contrast, lambs weaned at 21 d had the highest mass specific intake of starch (P < 0.001), and the 35 d, 4% MR group had the lowest (P < 0.01). 4. Discussion 4.1. Ruminal environment and microbiota An intensive milk feeding program can compromise solid feed intake and delay the rumen bacterial colonization in young ruminants (Gelsinger et al., 2016; Khan et al., 2016). We demonstrated a result consistent with previous studies (Henderson et al., 2015; Lima et al., 2015; Meale et al., 2016) which was the relative abundances of Firmicutes, Bacteroidetes and Proteobacteria were the highest phyla. The negligible difference in ruminal VFA profiles and microbiota diversity were likely because all lambs had accessed to the same solid feed prior to weaning, meaning that the fermentative role of the rumen had begun at a young age in all lambs. These results were in agreement with previous studies that lactation length or restricted milk intake by suckling lambs had limited influence on ruminal fermentation and microbiota (Alvarez-Rodriguez et al., 2012; Santos et al., 2018). The negligible difference suggests an important intestinal contribution to absorption of nutrients and growth. It was reported that the proportion of the small intestine and the length of ileal villi increased in ad libitum milk feeding lambs, and contributed to the absorption and utilization of nutrients (Santos et al., 2018). The ruminal pH at 50 days of age was lower in lambs fed 4% MR than 2% MR, but the ruminal pH was close to neutral pH in all groups, which was much higher than the ruminal pH of high ruminal acidosis risk lambs (Li et al., 2017). Moreover, the ruminal VFA profiles and microbiota diversity at 50 days of age were not influenced by weaning age or MR levels. Therefore, the risk of ruminal acidosis could be excluded in our study. 4.2. Ruminal histomorphology In pre-ruminants, liquid feed such as milk replacer bypasses the rumen to the abomasum. Nevertheless, these feeds play an important role in rumen development by providing ample nutrients for microbial growth (Li et al., 2012). In this study, there was no difference between groups in either papilla length or width, consistent with previous studies which found that the length and width of papillae in the ruminal ventral sac were not affected by milk allowance or lactation length in young ruminants (Roth et al., 2009; Alvarez-Rodriguez et al., 2012). These results could be because of the same rumen fermentation parameters between groups. The VFAs produced by ruminal microbes are among the determinants of ruminal papillary size and shape (Baldwin and Connor, 2017). However, lambs weaned at 35 d had greater ruminal muscular thickness, which was likely caused by an ontogenic response to rumen development, in addition to the diet-induced influences on the rumen. Clauss et al. (2003) reported that thicker ruminal muscle layers contributed to a greater motility of solid feed and increased the mixing of ruminal contents. We speculate that the prolonged MR feeding is beneficial to ruminal organ development and motility in lambs, but the effects of ontogenic control, nutrient or endocrine factors on regulatory mechanisms are still unclear. 4.3. Nutrient apparent digestibility and growth performance Generally, the digestibility of DM, protein and fat from milk-based ingredients in MR are higher than from solid feed. Consequently, it is likely that differences in digestibility of nutrients from 8 to 35 d reflected the different sources of nutrients ingested by lambs in the present study. Feeding 4% levels of MR, which was in excess of traditional recommendations, had higher apparent digestibility of DM, CP and fat than 2% MR groups from 8 to 21 d. These results are due to high digestibility of liquid feeds compared with starter that contained fiber ingredients and can explain the significantly higher ADG in the 4% MR groups and the steady decrease in nutrient (except starch and NDF) apparent digestibility throughout the trial period. It was reported that an intensive pre-weaning MR feeding program influenced the solid feed intake and DM digestibility of nutrients post-weaning (Sweeney et al., 2010; de Passille et al., 2011; Meale et al., 2015). Calves fed high-fiber starters with high amounts of MR led to higher BW gain but lower post-weaning nutrient digestion; the lower digestibility of starter post-weaning resulted in greater gut fill rather than muscle or bone growth (Hill et al., 2016). However, the pelleted starter in our study contained little fiber content, and there was negligible difference in the rumen weight and apparent digestibility of nutrients among groups at 50 d. These results fit in well with the report that ad libitum milk feeding with pelleted starter (22.7% NDF) did not affect the lambs’ rumen weight and nutrient digestibility (Santos et al., 2018). Therefore, the 4% MR treatment groups had a significant advantage on carcass weight and final BW 7

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suggesting a beneficial effect of high-level MR on lambs’ body weight gain, rather than accumulation of gut fill from poor digestion of starter. Total ADG was greater in the groups fed 4% levels of MR than 2% levels of MR, which could be attributed to the greater intake of CP and fat. The groups fed high levels of MR had a greater carcass weight and final BW, indicates that high levels of MR provide a large amount of easily absorbed nutrients, especially CP and fat. Furthermore, the higher growth rate was maintained throughout the trial, indicating a sustained effect of feeding high levels of MR on BW gain. Previous studies reported that intake of MR containing high fat concentration reduced starter intake and ADG both in pre- and post-weaning calves (Hill et al., 2009a; 2009b; 2010; 2012). In addition, a greater BW gain was reported in calves fed higher proportions of MR, but this did not occur during the post-weaning period, and the authors concluded that this result was related to lower starter intake (Chapman et al., 2016). However, in our study, although 4% level of MR provided more fat, it did not affect the total starter intake. The intensive MR feeding resulted in slight reductions in post-weaning ADG but the reduction in ADG recovered within seven days. This result is mainly due to the rapid and substantial increase of starter intake in the 4% MR groups after weaning. It was in line with previous studies that also found postweaning starter intakes were not affected by pre-weaning MR rates (Hill et al., 2007; Osorio et al., 2012; Hill et al., 2013). Weaning age affected total starter intake, total ADG, final BW and carcass weight. Starter intakes increased post weaning, and lambs weaned at 21d had an advantage to consume more starter, which was in agreement with previous studies (Hopkins, 1997; Abbas et al., 2017). Rashid et al. (2013) reported that buffalo calves weaned at 8 weeks had a greater starter intake than calves weaned at 12 weeks. Furthermore, the greater total ADG, final BW and carcass weight in lambs weaned at 35 d was consistent with previous reports in calves which showed that delaying weaning by two weeks could double growth rates in the week immediately post-weaning (Eckert et al., 2015; Meale et al., 2015). 5. Conclusions Weaning age and MR levels had limited influence on apparent digestibility of nutrients, ruminal microbiota and fermentation at 50 d. Feeding a high level of MR and later weaning age provided a large amount of easily absorbed nutrients which led to greater ADG and BW, and had a sustained positive effect on lambs’ body weight gain. 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