Effects of feeding whole milk or milk replacer on the performance, gastrointestinal tissue development, and digestive enzyme secretions of Holstein calves

Effects of feeding whole milk or milk replacer on the performance, gastrointestinal tissue development, and digestive enzyme secretions of Holstein calves | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Effects of feeding whole milk or milk replacer on the performance, gastrointestinal tissue development, and digestive enzyme secretions of Holstein calves Xu Tian, Xuefeng Zhang This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6138081/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The purpose of the research was to compared the effects of the supplementation of whole milk or milk replacer on the performance, gastrointestinal tissue development, and digestive enzyme secretions of Holstein calves. Twelve newborn Holstein male calves (1.96 ± 0.99 d of age; 40.0 ± 4.37 kg BW) were blocked by arrival date and age, divided into two groups: whole milk group(WM) and milk replacer group(MR)(n=6) in One-way experimental design. The results showed that at 30 d, the WM group had significantly higher (P<0.05) pre-slaughter body weight (P<0.05), ADG (P<0.05) and average dry matter intake (ADMI) (P<0.05) than the MR group; and at 60 d, the ileocecal pancreatic amylase level of the WM group was significantly lower (P<0.05) than that of the MR group. Rumen development was better in the MR group than in the WM group, and small intestinal villus length development was different in each intestinal segment(P<0.05). The relative abundance of Proteobacteria in the WM group was significantly higher than that in the MR group on day 30(P<0.05) and the relative abundance of succinogens was significantly higher (P<0.05) in the MR group than in the WR group (P<0.05). At 60 d, the relative abundance of Bacteroidetes and Actinobacteria in the MR group was higher (P<0.05) than in the WM group, and the relative abundances of Prevotella (P<0.05) and Bacteroides in the WM group were greater than those in the MR group (P<0.05).Calves fed MR showed poorer growth performance early in lactation, but higher starter intake helped calves plateau in growth performance at weaning. Gastrointestinal digestive enzymes showed more positive amylase activity and more mature rumen development and teat growth. In addition, rumen microbes in MR-fed calves were more efficient at degrading starter intake.Therefore, feeding MR would improve calf digestion of solid feeds by increasing rumen development and small intestinal amylase levels. However, the evolutionary processes and specific mechanisms responsible for this outcome cannot be explained because the current study was limited to epiphenomena. Biological sciences/Zoology/Animal physiology Health sciences/Gastroenterology/Gastrointestinal system/Small intestine Whole milk milk replacement rumen microbiota Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 1. Introduction In current trends in calf nutrition, the focus of dairy calf rearing is to improve calf growth performance using whole milk (WM) or milk replacer (MR) of high nutritional quality. WM has higher energy content and contains bioactive components, such as enzymes, hormones, and growth factors compared to most commercial magnetic resonance (CMR) 1 . Compositional differences may lead to increased body weight (BW) at weaning, resulting in long-term benefits for future milk production 2 . Differences in macronutrient inclusions, minerals, vitamins, and bioactive components between MR and WM may also affect liquid feed digestibility and calf physiology. The source of dietary proteins for feeding preruminant and weaned calves modifies the SI morphology. Chymosin, elastase II, and lactase are typical postnatal enzymes, whereas pepsin, ribonuclease, and amylase are important post-weaning enzymes 3 . Numerous nutritional factors have been shown to affect pancreatic α-amylase secretion, with starch producing negative effects and casein, certain amino acids, and dietary energy producing positive effects. These factors differ between WM and MR 2 , 4 . The health and performance of calves are the primary focus areas of calf-rearing programs for dairy herds to maintain future stock. Thus, applying a suitable feeding strategy can maximize the daily weight gain and improve the health of calves. Rumen development is an important indicator of calf weaning performance 5 . Moreover, changes in the nutritional structure of MR improve the development of the rumen 6 . With adequate nutrition, starter intake has a greater effect on rumen development than WM or MR 7 . Therefore, the purpose of the current research was to compare the effects of MR and WM supplementation on the performance, gastrointestinal tissue development, and digestive enzymes in Holstein male calves and provide a theoretical basis for applying the early feeding process to calves. 2. Materials and methods All experimental designs were approved by the Animal Ethics Committee of the Jilin Agricultural University (Changchun, China) (2021)and were conducted in accordance with the recommendations of Jilin Agricultural University on animal research(Jlau-acuc2022-007). In addition, all experimental protocols were reported following the relevant guidelines and regulations reported in ARRIVE guidelines 2.0. ( https://arriveguidelines.org ). 2.1 Animals and colostrum The present study was conducted between 5 and 8 2023 on the Yijiahe Dairy Farm (Wuzhong, Ningxia Province, China). Twelve newborn Holstein male calves (1.96 ± 0.99 d of age; 40.0 ± 4.37 kg BW) were blocked by arrival date and age, divided into a whole milk group (WM) and milk replacer group (MR) (n = 6) based on a one-way experimental design. All calves were given 4 L of colostrum within 2 h of birth and 2 L of colostrum at 12 h after birth. Only colostrum that satisfied the calf colostrum feeding criteria (Brix > 22% and immunoglobulin G > 50 g/L) was provided. 2.2 Housing and feeding scheme All calves were transferred to a calf island (island: 210×145×140 cm; fence: 170×150×100 cm), and individual calves were raised on a single calf island. All islands were pre-sanitized and hayed in advance, and the hutch was embedded with rice hulls and wheat straw and equipped with a fenced outside run measuring 2.25 m 2 . From days 2 to 27 after birth, all calves were fed pasteurized WM (pasteurization at 60 ℃ for 30 min), which had an average composition of 3.98 ± 0.06% fat, 3.44 ± 0.06% protein, 4.67 ± 0.11% lactose, and 12.57 ± 0.10% total solids(Table 1 ). Milk was collected from the herd using buckets without teats twice daily (08:00 and 16:00). Calves were fed 6 L/day of milk from 2 to 6 days of age and 8 L/day from 7 to 10 days of age. On day 7, all calves were randomized into two groups (CON: whole milk-fed, MR: milk replacer-fed). The CON group was fed 10 L/day from days 7 to 10, while the MR group transitioned from milk to MR starting at day 7. The ratio of WM to MR (3:1) gradually decreased to 1:3 during the transition period and then to 0:1 on day 10. After boiling and cooling to 40–50°C, 6.5 parts of warm water were mixed with each component in the MR solids. An electric mixer was used for mixing to ensure no solid residue and no sedimentation occurred. The calves received 11 L/day of liquid feed from days 11 to 50, 8 L/day from days 51 to 55, 4 L/day from days 55 to 57, 2 L/day from day 58, and 1 L in the morning at 59 days. All calves were weaned at 60 days. This feeding program was also used for the WM treatment (Fig. 1 ). Throughout the experiment, the starter diet and water were freely accessible (Table 2 ). All calves received a calf starter manufactured by Changchun Borui Science & Technology Co. starting on day 2. Table 1 The nutrient composition of the whole milk fed to the Holstein calves Items Whole milk Nutrient constituents(% as fed) A Fat 3.98 ± 0.06 Protein 3.44 ± 0.06 Lactose 4.67 ± 0.11 Ash 0.66 ± 0.01 Solid non - fat 8.76 ± 0.19 Total solids 12.57 ± 0.10 Metabolic energy(Mcal/kg of DM) B 5.54 ± 0.02 Note: A Metabolic energy was a calculated value and others were measured values. B Calculated metabolic energy for whole milk (Mcalkg) = [0.057 x protein (%) + 0.092 x fat (%) + 0.0395 x lactose (%)]x 0.93 (NRC, 2001). Table 2 The chemical composition of the starter fed to the Holstein calves (% of diet DM) A Items Calf starter Chemical composition Dry matter (DM, % as fed) 88.75 Crude protein (CP, % DM) 24.51 Acid detergent fiber (% DM) 9.01 Neutral detergent fiber (% DM) 24.43 Ash (% DM) 6.91 Ether extract (% DM) 4.98 Starch (% DM) 32.65 Calcium (% DM) 0.78 Phosphorus (% DM) 0.53 Metabolic energy B (Mcal/kg of DM) 4.21 Note: A Metabolic energy was a calculated value and others were measured values. B Calculated metabolic energy for whole milk (Mcalkg) = [0.057 x protein (%) + 0.092 x fat (%) + 0.0395 x lactose (%)]x 0.93 (NRC, 2001). 2.3 Measurements of growth performance and nutrient utilization The test period is 60 days. MR and solid feed samples were collected monthly during the feed cycle and then analysed for nitrogen (N), ash, calcium, phosphorus, neutral detergent fibre and acid detergent fibre. Each calf was weighed separately before starting the experiment, at 30 days after starting, and at the end of the experiment, and the individual average daily weight gain (ADG) was calculated. Record the daily MR feed intake of each calf based on the difference between the allocated amount and the remaining amount, and calculate the average daily solid feed intake of each enclosure group by dividing the difference between the allocated amount and the remaining amount by the number of enclosure partners. Feed efficiency is calculated based on the ratio of ADG to average daily feed intake. The calculations were as follows: ADG: (pre-slaughter weight - newborn weight)/number of days in the trial Average daily feed intake: feed intake per head/number of days during the test period Feed-to-weight ratio: average daily feed intake/ADG 2.4 Slaughter and sample collection Calves were euthanized on days 30 and 60 after arrival at 3 h after the morning meal by injecting 6 mL tetracaine (T61; MSD Animal Health Netherlands) into the jugular vein. After slaughter, immediately lay organs such as the heart, liver, spleen, lungs, kidneys, and gallbladder were spread on a plastic sheet to measure weights. The carcass was measured and defined as the weight of the slaughtered lamb’s body without the head, lower limbs, all internal organs, and fur after being left for 30 min. The forestomach compartments, including the rumen, reticulorumen, omasum, and abomasum, were removed, emptied of digesta, and weighed. The rumen digesta were emptied into a plastic measuring cylinder and manually mixed. Approximately 30 mL of the digesta was collected and stored at − 40°C until extraction of bacterial DNA and digestive enzymes. The intestinal chyme was collected with 5 mL of the digesta and stored at − 80°C until the extraction of bacterial digestive enzyme. Four pieces of whole tissue (2 cm × 2 cm) from the rumen dorsal sac, ventral sac, caudodorsal blind sac, and caudal ventral blind sac were separately collected and rinsed in 0.9% stroke-physiological saline solutions until clear and then stored in designated 10% formalin neutral fixative solutions. The small intestine, including the duodenum, jejunum, and ileum, and large intestine, including the colon, cecum, and rectum, were spread on polystyrene plates to measure their length and weight. Pieces (2 cm in length) from the duodenum, jejunum, ileum, and cecum were washed using 0.9% stroke-physiological saline solution and fixed with 10% formalin solution for later determination of intestinal development. 2.5 Chemical analysis Concentrated feed samples were collected, and the dry matter, crude protein, ether extract, ash, and iron contents were analyzed according to the procedures of the AOAC. The feed samples were also analyzed for neutral detergent fiber content, as proposed by Van Soest et al, The chemical compositions of the MRs and solid feeds provided during fattening are reported in Table 3 . Table 3 The ingredients and chemical composition of the milk replacer (MR) fed to the Holstein calves (% of diet dry matter, DM) A Ingredients,g/kg DM Milk replacer whey powder 507.7 Whey proteins 127.3 Maltodextrin 28.0 Glucose 13.7 Modidied wheat protein 90.3 Soybean protein concentrate 14.1 Coconut oil 67.4 Palm oil 82.1 NaCl 12.0 Methionine 8.5 Lysine 3.2 Tryptophan 1.9 Threonine 1.6 Premix 14.0 Chemical composition DM,g/kg DM 974.5 Crude protein(CP),g/kg DM 130.6 Lactose,g/kg DM 521.0 Glucose,g/kg DM 14.1 Starch,g/kg DM 0.0 Ether extract,g/kg DM 167.9 Acid detergent fiber(ADF),g/kg DM 2.9 Neutral detergent fiber(NDE),g/kg DM 7.0 Ash,g/kg DM 52.5 Ca,g/kg DM 13.1 P,g/kg DM 11.7 Plant proteins,g/kg DM 95.4 Whey proteins,g/kg DM10 130.6 ME,Mcal/kg of DM B 4.47 Note: A Metabolic energy was a calculated value and others were measured values. B Calculated metabolic energy for whole milk (Mcalkg) = [0.057 x protein (%) + 0.092 x fat (%) + 0.0395 x lactose (%)]x 0.93 (NRC, 2001). 2.6 Digestive enzyme analysis Gastrointestinal chyme was thawed and rapidly centrifuged at 10,000 g for 5 min at 4°C, and 10 µL of supernatant was collected. Enzyme activity was determined using an enzyme-linked immunosorbent assay (ELISA) kit (MLbio, Shanghai, China) following the manufacturer's instructions.. Digestive enzyme activities in the chyme were determined for the crumpled stomach and in each small intestinal segment (crumpled stomach: pepsin and gastric lipase; and duodenum, jejunum, and ileum: pancreatic proteases, chymotrypsin, pancreatic lipases, pancreatic amylase, maltase, isomalto-amylase, carboxypeptidase A, carboxypeptidase B, lactase, and sucrase). 2.7 Histological analysis After fixation, four pieces of calf rumen, duodenum, jejunum, and ileum tissue samples were collected and trimmed to flatness using a scalpel. The flat tissue samples were then placed in a dehydrator (Nanjing Yanzheng Drying Equipment Co. Ltd. YZ-TS-100A), dehydrated in an ethanol series, cleared with xylene, and embedded in paraffin. An embedding machine (KH-MQ2600, Xiaogan Kohai Medical Technology Co., Ltd.) was used to embed the wax-dipped tissue samples, and corresponding labels were affixed. The samples were placed on a freezing table (KH-LQ3500, Xiaogan Kohai Medical Technology Co., Ltd., Hubei, China, -20 ℃) for cooling, and after cooling was complete, the wax blocks were trimmed. After trimming, the wax blocks were sliced on a microtome and the thickness of each slice was maintained at approximately 4 µm. The slices were spread out and fished with slides at 40 ℃, and then slides with sliced tissues were put into a 60 ℃ oven (Nanjing Yanzheng Drying Equipment Co., Ltd., YZ-KX-400S). After baking, remove the glass slide and place it at room temperature for subsequent staining. The dried tissue samples were deparaffinized with xylene and rinsed with distilled water. After paraffin removal, the sections were stained with hematoxylin staining solution for 5 min, rinsed with distilled water, and differentiated with 1% hydrochloric acid alcohol for 5 s. After rinsing, the sections were turned blue with ammonia (0.6%) and rinsed again. Sections were stained with an eosin staining solution for 2 min. Sections of stained tissue samples were transparently processed using anhydrous ethanol and xylene, and when finished, they were dried and sealed with neutral gum. Four microscopic fields per sample were selected to measure the intestinal muscle thickness, and the images were acquired and analyzed using the image analysis software Image-Pro 6.0 to determine the villus length, villus width, and crypt depth. 2.8 High-throughput sequencing analysis Microbial DNA was extracted in vitro from ruminal fluid samples (n = 5 per group) using a DNA Kit (Omega Bio-Tek, Norcross, GA, USA), which was followed by PCR amplification conducted with barcoded specific bacteria primers that targeted the variable region 3–4 (V3-V4) of the 16S rRNA gene: forward and reverse primers 338F (5’-barcode + ACTCCTACGGGAGGCAGCA-3’) and 806R (5’-GGACTACHVGGGTWTC TAAT-3’). The amplicons were extracted, purified, and quantified. A NEXTFLEX Rapid DNA-Seq Kit was used to pool the DNA products. Sequencing libraries and paired-end sequencing were performed according to standard protocols using an Illumina MiSeq platform (Shanghai BIOZERON Co., Ltd(Beijing, China) according to standard protocols. The amplicon library was then paired-end sequenced (2×250 bp) using an Illumina MiSeq platform (Shanghai BIOZERON Co., Ltd.). The raw reads were uploaded to the NCBI Sequence Read Archive database. The library amount to be loaded can be controlled between 15 and 18 pM according to the actual conditions. First, a sparse curve was plotted using QIIME2 software to assess whether the sequencing results of each sample could reflect the microbial diversity in the sample. Second, to compare the diversity of different samples, the ASV abundance matrix was randomly flattened by 95% of the sequence volume of the sample based on the lowest sequence volume among all samples to correct for the differences in diversity among samples caused by the sequencing depth. Finally, the Chao1 index, Observed species index, Shannon index, Simpson index, Faith's PD index, Pielou's evenness index, and Good's coverage index were calculated using QIIME2 software. All OTUs were assigned a taxonomic classification via a QIIME search against the Silva databases (Release 128). The beta diversity of the microbiome was compared using principal component analysis. Analysis of the ruminal microbiome abundance was performed to identify significant differences in taxon abundance at the genus level between the control and treatment groups. 3 Statistical Analysis All experimental data, including performance, gastrointestinal tissue development, and digestive enzyme secretions, were analyzed usingthe SPSS software (version 26.0; SPSS Inc., Chicago, IL, USA). The WM and MR groups were analyzed using a single-factor experimental design; data are presented as mean ± SEM. The significance level was set at P < 0.05 4 Results 4.1 Intake and growth performance Intake and growth performance, which are crucial economically important factors, were compared among the groups. As shown in Table 4 , Fig. 2 , all calves had the same initial body weight (BW). The results revealed that the pre-slaughter BW (P < 0.05), average daily gain (ADG) (P < 0.05), and average dry matter intake (ADMI) (P < 0.05) were significantly higher in the WM group at 30 days compared with the MR group. The feed-to-weight ratio of the MR group was significantly higher than that of the WM group at 30 and 60 days.(P 0.05). Table 4 Effects of milk and milk replacer on growth performance and body size index of Holstein calves Item 30d SEM p-Vaule 60d SEM p-Vaule Milk MR Milk MR Body weight/kg 39.33 39.67 0.37 0.70 39.33 40.00 0.49 0.84 Weight/kg 65.33 61.50 1.00 0.03 97.00 98.67 1.17 0.54 ADG/(kg/d) 0.87 0.73 0.03 0.01 0.96 0.98 0.01 0.58 DMI/(kg/d) 1.22 1.23 0.00 0.08 1.42 1.48 0.01 0.02 F/G 1.41 1.69 0.06 0.00 1.46 1.51 0.01 0.00 4.2 Slaughter performance and organ weight Slaughter performance was analyzed as shown in Fig. 3. The heart weight was significantly higher in the WM group than in the MR group at 30 days (P 0.05). 4.3 Digestive enzyme activity The digestive enzyme activity results are shown in Table 5 ,Table 6 and Table 7 . At 30 days, the lipase and amylase levels in the stomach were significantly higher in the WM group than in the MR group (P < 0.05). The carboxypeptidase A activity in the ileum of the calves in the MR group was significantly higher than that of the calves in the WM group (P < 0.05). However, at 60 days, the crumpled stomach lipase and amylase levels and ileal pancreatic amylase levels were significantly lower in the WM group than in the MR group (P 0.05). Table 5 Effects of milk and milk replacer on protein digestive enzyme activities in Holstein calves Item Age(day) Milk MR SEM P-Value Trypsin 30 Duodenum 22.53 25.43 1.84 0.49 Jejunum 41.07 36.67 3.39 0.58 Ileum 26.27 24.63 1.34 0.60 60 Duodenum 36.27 25.87 2.83 0.05 Jejunum 30.13 27.47 2.31 0.62 Ileum 29.87 32.67 1.10 0.24 Chymotrypsin 30 Duodenum 340.23 348.37 30.43 0.91 Jejunum 341.63 238.23 32.66 0.12 Ileum 335.50 241.10 33.68 0.18 60 Duodenum 266.03 281.73 22.87 0.77 Jejunum 258.50 212.57 30.55 0.52 Ileum 316.23 389.47 29.69 0.26 Carboxypeptidases A 30 Duodenum 101.78 123.14 14.69 0.53 Jejunum 110.16 142.54 7.69 0.01 Ileum 120.81 135.29 12.16 0.61 60 Duodenum 97.19 117.52 9.32 0.33 Jejunum 100.98 143.59 16.35 0.23 Ileum 133.14 121.72 14.52 0.74 Carboxypeptidases B 30 Duodenum 157.98 173.97 22.41 0.76 Jejunum 159.79 213.45 15.23 0.06 Ileum 146.57 176.15 15.32 0.39 60 Duodenum 163.10 170.38 6.77 0.65 Jejunum 160.75 195.70 19.96 0.44 Ileum 187.28 232.87 16.53 0.19 Table 6 Effects of milk and milk replacer on starch digestive enzyme activities in Holstein calves Item Age(day) Milk MR SEM P-Value Pancreatic amylase 30 Duodenum 343.07 243.17 33.97 0.16 Jejunum 379.60 386.14 31.11 0.93 Ileum 355.37 313.07 40.85 0.66 60 Duodenum 318.77 420.98 35.75 0.17 Jejunum 418.50 404.60 37.78 0.88 Ileum 223.37 321.20 25.13 0.02 Maltase 30 Duodenum 22.47 27.93 3.49 0.50 Jejunum 27.60 27.13 2.48 0.94 Ileum 33.00 24.57 2.58 0.10 60 Duodenum 24.33 28.37 2.91 0.55 Jejunum 17.97 26.37 2.70 0.13 Ileum 23.07 26.20 2.64 0.61 Isomaltase 30 Duodenum 19.17 23.16 4.08 0.68 Jejunum 15.74 16.87 3.47 0.89 Ileum 21.57 16.17 2.21 0.26 60 Duodenum 18.69 20.53 3.05 0.80 Jejunum 22.57 15.67 2.24 0.13 Ileum 17.80 19.87 2.03 0.66 Table 7 Effects of milk and milk replacer on Non-starch energy substances digestive enzyme activities in Holstein calves Item Age(day) Milk MR SEM P-Value Lipase 30 Duodenum 209.17 204.09 12.97 0.87 Jejunum 147.58 115.48 11.55 0.19 Ileum 198.87 211.04 6.69 0.42 60 Duodenum 156.77 132.77 6.62 0.05 Jejunum 190.63 123.05 17.62 0.03 Ileum 108.11 180.43 19.06 0.03 Lactase 30 Duodenum 13.78 13.54 1.20 0.93 Jejunum 13.11 14.35 0.74 0.46 Ileum 11.80 12.53 1.10 0.78 60 Duodenum 10.50 14.59 1.66 0.26 Jejunum 15.05 12.00 1.35 0.31 Ileum 14.86 11.93 1.34 0.32 Sucrase 30 Duodenum 8.81 8.44 0.45 0.73 Jejunum 13.28 10.67 1.99 0.57 Ileum 14.31 8.31 2.25 0.21 60 Duodenum 9.06 8.34 0.35 0.35 Jejunum 12.62 10.11 1.10 0.30 Ileum 11.27 13.44 2.15 0.67 4.4 Rumen and small bowel development Figure 4 ,Fig. 5 lists the results for rumen and small bowel development. Rumen weight and rumen papilla height were significantly higher in the MR group than in the WM group at 60 days (P < 0.05). As shown in Table 8 , the villus length and crypt depth of the duodenum, depth of the jejunal crypt, width of the ileal villi, and depth of crypts in the WM group were greater than those in the MR group at 30 days (P < 0.05). The length of the jejunal villi and weight of the empty intestine were significantly higher in the MR group than in the WM group at 60 days (P < 0.05). Table 8 Effects of milk and milk replacer on development of rumen papillae and small intestine villus in Holstein calves Item 30d 60d WM MR SEM P-Value WM MR SEM P-Value Rumen Papillae length 493.2 495.69 24.41 0.924 645.02 746.34 34.47 0.001 Papillae width 254.8 232.94 31.47 0.272 253.25 283.86 24.66 0.625 Duodenum Villus length 582.2 518.65 5.26 0.037 650.28 655.05 22.92 0.653 Villus width 151.1 154.53 10.48 0.357 176.88 172.05 11.06 0.296 Crypt depth 153.6 137.57 7.19 0.027 147.31 155.17 8.33 0.053 Jejunum Villus length 525.7 499.18 18.14 0.013 594.62 593.58 5.74 0.974 Villus width 145.3 142.20. 4.85 0.524 154.63 153.29 7.38 0.669 Crypt depth 147.7 135.62 12.74 0.032 157.73 152.39 7.35 0.126 Ileum Villus length 508.2 488.33 17.65 0.070 524.76 563.92 28.39 0.006 Villus width 134.7 114.17 16.74 0.026 143.76 140.79 5.84 0.534 Crypt depth 127.1 102.8 14.35 0.014 141.5 139.92 5.64 0.665 4.5 Rumen bacteria Alpha diversity refers to the abundance, variety, and homogeneity of species in a localized area. At 30 days, the Good’s coverage index in the WM group was higher than that in the MR group (P < 0.05) (Fig. 6 ). Figure 7 lists the profile of the ruminal microbial com munity based on an unweighted Bray–Curtis dissimilarity matrix at the genus. The composition of the rumen microbiota, which is crucial for growth performance, was assessed using 16S rRNA sequencing. The relative abundances of the dominant phyla are shown in Fig. 8 . The four most dominant phyla were Bacteroidota, Firmicutes, Actinobacteria, and Proteobacteria. The relative abundance of Proteobacteria in the WM group was significantly higher than that in the MR group on day 30 (P < 0.05). At 60 days, the relative abundances of Bacteroidetes and Actinobacteria in the MR group were higher than those in the WM group (P < 0.05). The relative abundances of dominant genera are shown in Fig. 9 . The four most dominant genera were Prevotella, Succiniclasticum, Butyrivibrio, and Bacteroides. The relative abundance of Succiniclasticum (P < 0.05) was significantly higher in the MR group than in the WR group on day 30 (P < 0.05). By day 60, the relative abundances of Prevotella (P < 0.05) and Bacteroides in the WM group were greater than those in the MR group (P < 0.05). 5 Discussion 5.1 Effects of milk and MR on the growth performance and body size index of Holstein calves The primary objective of this study was to investigate the performance, gastrointestinal tissue development, and digestive enzyme secretion of Holstein calves fed WM or MR. In a previous study, the WM group showed better growth performance 8 . Another study showed that calves in the MR group were more likely to require greater dry matter intake (DMI) than those in the WM-treated group 3 . In the present study, the DMI of the starter diet differed significantly between the WM and MR calves as their age increased 9 . The amount of liquid feed supplied gradually decreased, and the amount of the starter diet consumed by the MR calves increased relative to that of the WM calves. The DMI of the starter diets of the calves in the MR group was significantly higher than that of the calves in the WM control group, and this increase in the amount of starter feed consumed by the calves could be attributed to the better rumen development of the calves 10 . The results of rumen histomorphometry in this trial also showed that the MR group had greater rumen papilla lengths than the WM group, which is consistent with a previous study 11 . In addition, due to the significant difference in nutrient composition between the MR and WM groups, the MR-fed calves showed lower growth performance during lactation than the WM-fed calves. However, the growth disadvantage of the calves before weaning could be improved by increasing the intake of starter food. This might have been due to potential improvements in the tolerance of calves to nondairy components when fed a diet of MR, which resulted in better rumen development of calves after entering the weaning transition period 7 , 12 . Moreover, calves fed MR were able to compensate for the decrease in liquid feed supply by increasing the intake of starter feed during the weaning transition period 13 . Significant differences in ADG and G/F were observed between the WM and MR groups, which may have been due to the poorer digestion of plant-based ingredients in the MR group. Previous studies have shown that although MR powder contains less plant protein, it still reduces the digestibility of nutrients in calves and inhibits calf growth and development 14 . However, studies have shown that digestion improves as the calves ages 8 . 5.2. Effects of milk and MR on digestive enzyme activities in Holstein calves During early lactation, calves have an undeveloped rumen, which acts as the main source of nutrition through enzymatic digestion. The gastrointestinal enzyme activity of calves is key for their digestion. Khorasani et al. 15 researched pancreatic protease inhibitors and found that soy protein affects trypsin activity in calves. Moreover, at 60 days, pancreatic protease activity in the duodenum of calves in the milk group was significantly higher than that in the MR group. Another study 16 found that carboxypeptidase A activity was fairly equally distributed in the five parts of the small intestine while carboxypeptidases B activity was high in both the median and distal jejunum as well as in the ileum. In the present study, carboxypeptidase A and B activities were higher in the MR group than in the WM group, which may have been caused by the rapid entry of plant proteins into the jejunum without protein agglutination. MRs containing wheat proteins increase the abomasal emptying rate 17 . All amylase activities are low in newborn calves, while lactase activity decreases and pancreatic amylase and maltase activities gradually increase as calves grow 18 . For calves, starch enters the small intestine and is hydrolyzed to glucose by pancreatic amylase and maltase, and then glucose is absorbed and utilized by the body. A previous study 19 suggested that maltase activity limits starch digestion and that fermentation may contribute largely to the loss of total starch in milk-fed calves 20 . suggested that calves with the highest starter intake had high concentrations of serum amylase. In our study, intestinal amylase activity was higher in calves in the MR group with higher pellet feed intake. At 60 days, the WM calves had significantly higher duodenal and jejunal pancreatic lipase and significantly lower ileal pancreatic lipase than the MR calves. This may be attributed to the type of fat in the diet 21 . Compared with the WM group, the MR group used more plant-based fats and a lower fat content. Fat of lactic origin is better digested and absorbed and released more rapidly in the duodenal end of the duodenal rectum and in the jejunum. However, the vegetable fats contained in the MR and the fats in the pellets were digested slowly and flowed into the ileal segment for gradual digestion 22 . 5.3. Effects of milk and MR on gastrointestinal tract development in Holstein calves Rumen development in calves is an important indicator of weaning performance 11 . The entry of grains into the rumen promotes rumen weight and muscle tissue development through physical stimulation 23 . The MR feeding strategy based on a liquid diet as used in this trial resulted in calves consuming more open feed during lactation, which produced more physical stimulation of the rumen, resulting in significant differences between the two groups. Starter intake is critical for rumen development, and the end products of rumen fermentation, volatile fatty acids, are important stimulants of rumen papilla development 24 . In this experiment, the calves were fed liquid diets in nipple-less buckets to minimize the formation of esophageal grooves. In addition, the calves were 0–60 days old. The calves in the MR group had a larger rumen mass and longer rumen nipple lengths and exhibited certain ruminant behavior 25 . This may have been due to the stimulation of the rumen teat by the plant-based ingredients in the MR 26 . Some plant-based fats can enter the rumen and produce short-chain fatty acids through microbial fermentation, which provides more chemical stimulation to the rumen teat and promotes its rapid growth, thus providing a good foundation for the calf's growth period 27 . The small intestine is the main organ used by lactating calves to digest and absorb nutrients from their diet 28 . Previous studies have shown that increasing the proportion of plant proteins in MR powder can negatively affect calf small intestine development. In this study, the number of small intestinal villi in calves in the WM group was higher than that in the MR group at 30 days of age, which indicated that the WM group had a better absorptive capacity. This may have been one of the reasons for the higher pre-lactation growth performance of calves in the WM group 29 . A previous study 30 suggested that the type of liquid diet affects both the gut mass and epithelial microstructure differently. In this study, the ileal weight of the calves in the milk group was higher. This may be related to the greater ileocecal gastrocnemius muscle thickness in MR-fed calves 31 . We found that calves in the MR group showed significant growth of small intestinal villi compared to those in the WM group at 60 days. Compared to previous studies, MR had a negative effect on small bowel development 32 . This may be due to the influence of harmful gases produced during the microbial fermentation of granular material residues at the end of the small intestine on the small intestinal epithelium 33 . Moreover, frictional stimulation and longer intestinal retention times under conditions of increased pellet intake may be responsible for the rapid development of small intestinal villi 34 . 5.4 Effect of milk and MR on rumen flora of Holstein calves Rumen development and health status have a direct influence on calf growth. The digestive functions of the rumen are not immediately active in a newborn calf; however, after 3 days of solid feed, the rumen is stimulated to start its digestive functions, and the internal flora is established. Subsequently, the rumen of the calf starts to develop, and as the calf ages, the rumen matures 9 . Bacteria in the rumen provide the basis of its digestive function, and the composition and quantity of bacteria can directly affect the digestive ability of the rumen, which in turn directly affects the growth and development of calves 35 . For ruminants, bacterial phyla have an important role in degrading fiber and cellulose and are involved in the degradation of starch, cellulose, hemicellulose, and oligosaccharides and production of acetic, propionic, and butyric acids, which can promote absorption and metabolism in ruminants. The results of this experiment showed that the addition of MR significantly increased the relative abundance of Bacteroidetes and Actinobacteria at 60 days. Bacteroidetes and Actinobacteria are major phyla of the rumen flora and are closely related to volatile fatty acid production in the rumen 36 . Prevotella is the predominant genus in the rumen, and it reduces the inflammatory response of the organism and participates in the digestion of carbohydrates, polysaccharides, and proteins in the WM group (60 days). The genus Aspergillus includes several pathogenic bacteria that are mostly gram negative and pathogenic to the organism, and they produce LPS and irritating flagellin to promote inflammatory responses. Vibrio spp. ferment carbohydrates into succinic acid and acetic acid to increase energy utilization and meet the body's energy requirements 37 . In summary, calves in the MR group had more effective rumen flora because of their increased pellet feed intake. 6 Conclusion Calves fed MR showed poorer growth performance early in lactation than the calves fed WM; however, the higher starter intake helped the MR calves plateau in growth performance at weaning. Gastrointestinal digestive enzymes showed more positive amylase activity and more mature rumen development and teat growth. In addition, rumen microbes in MR-fed calves were more efficient at degrading starter intake. Therefore, feeding MR would improve calf digestion of solid feed by increasing rumen development and small intestinal amylase levels. However, the evolutionary processes and specific mechanisms responsible for this outcome cannot be explained by the study results because the current study was limited to epiphenomena. Declarations Competing interests The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Funding The experiment was subsidized by Changchun Borui Science & Technology Co., Ltd (Item Numberand: 20220202049NC)and Ningxia Yijiahe Animal Husbandry Development Co., Ltd are thanked for providing venue support. Acknowledgements The authors thank Jilin Agricultural University (Changchun, China) Ruminant Center members for providing help during the experiment. Author Contribution Xu Tian and Xuefeng Zhang wrote the main manuscript text .All authors reviewed the manuscript. Data Availability The flora data of this study was uploaded to the NCBI database with the primary accession code PRJNA1101260 (V3-V4) . References Lee, H. J. et al. Influence of equalizing the gross composition of milk replacer to that of whole milk on the performance of Holstein calves. J. Anim. Sci. 87 , 1129–1137 (2009). Brscic, M., Prevedello, P., Stefani, A. L., Cozzi, G. & Gottardo, F. Effects of the provision of solid feeds enriched with protein or nonprotein nitrogen on veal calf growth, welfare, and slaughter performance. J. Dairy. Sci. 97 , 4649–4657 (2014). Guilloteau, P., Zabielski, R. & Blum, J. W. Gastrointestinal tract and digestion in the young ruminant: ontogenesis, adaptations, consequences and manipulations. J. Physiol. Pharmacol. 60 (Suppl 3), 37–46 (2009). Wilms, J. N. et al. Macronutrient profile in milk replacer or a whole milk powder modulates growth performance, feeding behavior, and blood metabolites in ad libitum-fed calves. J. Dairy Sci. 105 , 6670–6692 (2022). Austin, K. J., Cunningham, H. C., Powell, S. R., Carpenter, K. T. & Cammack, K. M. Investigation of maternal breed and rearing type on the calf rumen microbiome from day 28 through weaning. Transl Anim. Sci. 2 , S125–S129 (2018). Liu, W. et al. Supplementation with sodium butyrate improves growth and antioxidant function in dairy calves before weaning. J. Anim. Sci. Biotechnol. 12 , 2 (2021). Parsons, S. D. et al. Effect of a milk byproduct-based calf starter feed on dairy calf nutrient consumption, rumen development, and performance when fed different milk levels. J. Dairy. Sci. 105 , 281–300 (2022). Huang, K. et al. Effects of protein sources for milk replacers on growth performance and serum biochemical indexes of suckling calves. Anim. Nutr. 1 , 349–355 (2015). Eckert, E., Brown, H. E., Leslie, K. E., DeVries, T. J. & Steele, M. A. Weaning age affects growth, feed intake, gastrointestinal development, and behavior in Holstein calves fed an elevated plane of nutrition during the preweaning stage. J. Dairy. Sci. 98 , 6315–6326 (2015). Yohe, T. T. et al. Effects of milk replacer allowances and levels of starch in pelleted starter on nutrient digestibility, whole gastrointestinal tract fermentation, and pH around weaning. J. Dairy. Sci. 105 , 6710–6723 (2022). Liu, X. Z. et al. Effects of the carbohydrate and protein compositions in the milk replacer on the growth performance and gastrointestinal development of the Holstein calves from 35–70 days of age. Anim. Feed Sci. Technol. 284 , 115191 (2022). Dennis, T. S. et al. Effect of milk replacer feeding rate, age at weaning, and method of reducing milk replacer to weaning on digestion, performance, rumination, and activity in dairy calves to 4 months of age. J. Dairy. Sci. 101 , 268–278 (2018). Ghorbani, G. R., Kowsar, R., Alikhani, M. & Nikkhah, A. Soymilk as a novel milk replacer to stimulate early calf starter intake and reduce weaning age and costs. J. Dairy. Sci. 90 , 5692–5697 (2007). Inabu, Y. et al. Effect of kraft pulp inclusion in calf starter on performance, health, and plasma concentration of glucagon-like peptide 2 in calves. J. Dairy. Sci. 106 , 4443–4453 (2023). Khorasani, G. R., Ozimek, L., Sauer, W. C. & Kennelly, J. J. Substitution of milk protein with isolated soy protein in calf milk replacers. J. Anim. Sci. 67 , 1634–1641 (1989). Le Huerou, I. et al. Activity distribution of seven digestive enzymes along small intestine in calves during development and weaning. Dig. Dis. Sci. 37 , 40–46 (1992). Wittek, T., Ernstberger, M., Muckenhuber, M. & Flöck, M. Effects of wheat protein in milk replacers on abomasal emptying rate in calves. J. Anim. Physiol. Anim. Nutr. (Berl) . 100 , 264–270 (2016). Harfoot, C. G. Anatomy, physiology and microbiology of the ruminant digestive tract. Prog Lipid Res. 17 , 1–19 (1978). Gilbert, M. S. et al. A titration approach to identify the capacity for starch digestion in milk-fed calves. Animal 9 , 249–257 (2015). Hill, T. M., Bateman, H. G., Aldrich, J. M. & Schlotterbeck, R. L. Effect of milk replacer program on digestion of nutrients in dairy calves. J. Dairy. Sci. 93 , 1105–1115 (2010). Castro, J. J., Hwang, G. H., Saito, A., Vermeire, D. A. & Drackley, J. K. Assessment of the effect of methionine supplementation and inclusion of hydrolyzed wheat protein in milk protein-based milk replacers on the performance of intensively fed Holstein calves. J. Dairy Sci. 99 , 6324–6333 (2016). Guo, W. et al. Survey of rumen microbiota of domestic grazing yak during different growth stages revealed novel maturation patterns of four key microbial groups and their dynamic interactions. Anim. Microbiome . 2 , 23 (2020). Raeth, M. et al. Pre- and postweaning performance and health of dairy calves fed milk replacers with differing protein sources. Prof. Anim. Sci. 32 , 833–841 (2016). Suárez, B. J., Van Reenen, C. G., Stockhofe, N., Dijkstra, J. & Gerrits, W. J. J. Effect of roughage source and roughage to concentrate ratio on animal performance and rumen development in veal calves. J. Dairy. Sci. 90 , 2390–2403 (2007). Kertz, A. F. et al. A 100-Year Review: Calf nutrition and management. J. Dairy. Sci. 100 , 10151–10172 (2017). Cannon, S. J. et al. Inclusion of psyllium in milk replacer for neonatal calves. 2. Effects on volatile fatty acid concentrations, microbial populations, and gastrointestinal tract size. J. Dairy. Sci. 93 , 4744–4758 (2010). Berends, H. et al. Effects of solid feed level and roughage-to-concentrate ratio on ruminal drinking and passage kinetics of milk replacer, concentrates, and roughage in veal calves. J. Dairy. Sci. 98 , 5621–5629 (2015). Zhang, Q. et al. Relationship between dietary amino acid release kinetics and nitrogen deposition in growing pigs. Anim. Nutr. 9 , 233–239 (2022). Eckert, E., Brown, H. E., Leslie, K. E., DeVries, T. J. & Steele, M. A. Weaning age affects growth, feed intake, gastrointestinal development, and behavior in Holstein calves fed an elevated plane of nutrition during the preweaning stage. J. Dairy. Sci. 98 , 6315–6326 (2015). Mellors, S. C. et al. Gastrointestinal structure and function of preweaning dairy calves fed a whole milk powder or a milk replacer high in fat. J. Dairy Sci. 106 , 2408–2427 (2023). Hillman, M. et al. Skim milk powder with high content of Maillard reaction products affect weight gain, organ development and intestinal inflammation in early life in rats. Food Chem. Toxicol. 125 , 78–84 (2019). Zhang, R. et al. Decreasing the pH of milk replacer containing soy flour affects nutrient digestibility, digesta pH, and gastrointestinal development of preweaned calves. J. Dairy. Sci. 100 , 236–243 (2017). Burakowska, K. et al. Canola meal or soybean meal as protein source and the effect of microencapsulated sodium butyrate supplementation in calf starter mixture. II. Development of the gastrointestinal tract. J. Dairy. Sci. 104 , 6663–6676 (2021). Oladele, P., Ngo, J., Chang, T. & Johnson, T. A. Temporal dynamics of fecal microbiota community succession in broiler chickens, calves, and piglets under aerobic exposure. Microbiol. Spectr. 12 , e04084–e04023 . Diao, Q., Zhang, R. & Fu, T. Review of Strategies to Promote Rumen Development in Calves. Anim. (Basel) . 9 , 490 (2019). Ma, L., Yang, Y., Liu, W. & Bu, D. Sodium butyrate supplementation impacts the gastrointestinal bacteria of dairy calves before weaning. Appl. Microbiol. Biotechnol. 107 , 3291–3304 (2023). Mahayri, T. M. et al. Host Species Affects Bacterial Evenness, but Not Diversity: Comparison of Fecal Bacteria of Cows and Goats Offered the Same Diet. Animals (Basel) 12, (2022). (2011). Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6138081","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":437021533,"identity":"7456303a-bf75-4ff8-b606-001f9e2930d8","order_by":0,"name":"Xu Tian","email":"","orcid":"","institution":"Jilin Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Xu","middleName":"","lastName":"Tian","suffix":""},{"id":437021534,"identity":"2298f4ba-4087-4651-ae72-21ab1fe74a0d","order_by":1,"name":"Xuefeng 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08:08:12","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6138081/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6138081/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":79765570,"identity":"6812d30b-1667-4d39-8ef5-5220fb339db8","added_by":"auto","created_at":"2025-04-02 12:09:58","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":22345,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Amounts of milk replacers and whole milk offered (L/d) by day for the Holstein calves in the MR group;(B) Amounts of WM offered (L/d) by day for the Holstein calves in the WM group.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-6138081/v1/e126cc79e1f65e18e4771e0d.png"},{"id":79765569,"identity":"c3453baf-8f76-4dd7-863d-67562d00cc5a","added_by":"auto","created_at":"2025-04-02 12:09:58","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":51344,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of whole milk and milk replacer feeding on starter intake in the Holstein calves\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-6138081/v1/68ab65b1cbc6b341151b49d3.png"},{"id":79767028,"identity":"704b6c2c-eb42-4076-a3aa-a314d26bdba6","added_by":"auto","created_at":"2025-04-02 12:33:58","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":40409,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of milk and milk replacer on organ weigh in Holstein calves\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-6138081/v1/f839e39edc1a0ab07168241b.png"},{"id":79765578,"identity":"c6c419b0-7ebe-400b-a681-51fe18827b6f","added_by":"auto","created_at":"2025-04-02 12:09:58","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":53277,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of milk and milk replacer on gastrointestinal weight in Holstein calves\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-6138081/v1/120101fd63bd81613beedf3f.png"},{"id":79766212,"identity":"191a48b2-646b-48eb-8dd7-bd804833836a","added_by":"auto","created_at":"2025-04-02 12:17:58","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":529568,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of milk and milk replacer on development of rumen papillae and small intestine villus in Holstein calves(A:WM group;B:MR group；1：rumen；2：duodenum；3：jejunm：4：ileum)\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-6138081/v1/fdfda6c8d0d24caace0ab72d.png"},{"id":79765577,"identity":"c0739206-f07d-4523-b165-6bf2bd102d73","added_by":"auto","created_at":"2025-04-02 12:09:58","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":82882,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of milk and milk replacer on rumen Alpha diversity in Holstein calves(A:30dWM;B:30dMR;C:60dWM;D:60dMR)\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-6138081/v1/52146a48f39375f69e09c5c5.png"},{"id":79766844,"identity":"e0aa666b-e7fe-4ed2-ba4f-38efb98b9b83","added_by":"auto","created_at":"2025-04-02 12:25:59","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":36250,"visible":true,"origin":"","legend":"\u003cp\u003ePrincipal coordinate analysis (PCoA) profile of the ruminal microbial com­ munity based on an unweighted Bray–Curtis dissimilarity matrix at the genus level(A:30dWM;B:30dMR;C:60dWM;D:60dMR)\u003c/p\u003e","description":"","filename":"image7.png","url":"https://assets-eu.researchsquare.com/files/rs-6138081/v1/eb1ea98125a49c4dae5f8fb2.png"},{"id":79766841,"identity":"e4a23be5-ee1a-43fc-896e-e780a28b2f1f","added_by":"auto","created_at":"2025-04-02 12:25:58","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":26191,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of feeding whole milk and milk replacer on ruminal flora phylum levels n Holstein calves\u003c/p\u003e","description":"","filename":"image8.png","url":"https://assets-eu.researchsquare.com/files/rs-6138081/v1/995b642a464eeb804a20f52e.png"},{"id":79766211,"identity":"7b9f429d-58ea-4193-b540-ad80aef2e539","added_by":"auto","created_at":"2025-04-02 12:17:58","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":30135,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of feeding whole milk and milk replacer on ruminal flora genus levels n Holstein calves\u003c/p\u003e","description":"","filename":"image9.png","url":"https://assets-eu.researchsquare.com/files/rs-6138081/v1/0a40e966469b26e5e1599e72.png"},{"id":92435803,"identity":"23750644-d29b-4516-b796-0f31e1ae6428","added_by":"auto","created_at":"2025-09-29 17:01:53","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2239388,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6138081/v1/ea953e5a-3c8d-40f0-96c2-c59cfa00c3a4.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Effects of feeding whole milk or milk replacer on the performance, gastrointestinal tissue development, and digestive enzyme secretions of Holstein calves","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eIn current trends in calf nutrition, the focus of dairy calf rearing is to improve calf growth performance using whole milk (WM) or milk replacer (MR) of high nutritional quality. WM has higher energy content and contains bioactive components, such as enzymes, hormones, and growth factors compared to most commercial magnetic resonance (CMR)\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Compositional differences may lead to increased body weight (BW) at weaning, resulting in long-term benefits for future milk production \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Differences in macronutrient inclusions, minerals, vitamins, and bioactive components between MR and WM may also affect liquid feed digestibility and calf physiology.\u003c/p\u003e \u003cp\u003eThe source of dietary proteins for feeding preruminant and weaned calves modifies the SI morphology. Chymosin, elastase II, and lactase are typical postnatal enzymes, whereas pepsin, ribonuclease, and amylase are important post-weaning enzymes \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Numerous nutritional factors have been shown to affect pancreatic α-amylase secretion, with starch producing negative effects and casein, certain amino acids, and dietary energy producing positive effects. These factors differ between WM and MR \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. The health and performance of calves are the primary focus areas of calf-rearing programs for dairy herds to maintain future stock. Thus, applying a suitable feeding strategy can maximize the daily weight gain and improve the health of calves.\u003c/p\u003e \u003cp\u003eRumen development is an important indicator of calf weaning performance \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Moreover, changes in the nutritional structure of MR improve the development of the rumen\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. With adequate nutrition, starter intake has a greater effect on rumen development than WM or MR \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eTherefore, the purpose of the current research was to compare the effects of MR and WM supplementation on the performance, gastrointestinal tissue development, and digestive enzymes in Holstein male calves and provide a theoretical basis for applying the early feeding process to calves.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cp\u003e All experimental designs were approved by the Animal Ethics Committee of the Jilin Agricultural University (Changchun, China) (2021)and were conducted in accordance with the recommendations of Jilin Agricultural University on animal research(Jlau-acuc2022-007). In addition, all experimental protocols were reported following the relevant guidelines and regulations reported in ARRIVE guidelines 2.0. (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://arriveguidelines.org\u003c/span\u003e\u003cspan address=\"https://arriveguidelines.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Animals and colostrum\u003c/h2\u003e \u003cp\u003eThe present study was conducted between 5 and 8 2023 on the Yijiahe Dairy Farm (Wuzhong, Ningxia Province, China). Twelve newborn Holstein male calves (1.96\u0026thinsp;\u0026plusmn;\u0026thinsp;0.99 d of age; 40.0\u0026thinsp;\u0026plusmn;\u0026thinsp;4.37 kg BW) were blocked by arrival date and age, divided into a whole milk group (WM) and milk replacer group (MR) (n\u0026thinsp;=\u0026thinsp;6) based on a one-way experimental design. All calves were given 4 L of colostrum within 2 h of birth and 2 L of colostrum at 12 h after birth. Only colostrum that satisfied the calf colostrum feeding criteria (Brix\u0026thinsp;\u0026gt;\u0026thinsp;22% and immunoglobulin G\u0026thinsp;\u0026gt;\u0026thinsp;50 g/L) was provided.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Housing and feeding scheme\u003c/h2\u003e \u003cp\u003eAll calves were transferred to a calf island (island: 210\u0026times;145\u0026times;140 cm; fence: 170\u0026times;150\u0026times;100 cm), and individual calves were raised on a single calf island. All islands were pre-sanitized and hayed in advance, and the hutch was embedded with rice hulls and wheat straw and equipped with a fenced outside run measuring 2.25 m\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eFrom days 2 to 27 after birth, all calves were fed pasteurized WM (pasteurization at 60 ℃ for 30 min), which had an average composition of 3.98\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06% fat, 3.44\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06% protein, 4.67\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11% lactose, and 12.57\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10% total solids(Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Milk was collected from the herd using buckets without teats twice daily (08:00 and 16:00). Calves were fed 6 L/day of milk from 2 to 6 days of age and 8 L/day from 7 to 10 days of age. On day 7, all calves were randomized into two groups (CON: whole milk-fed, MR: milk replacer-fed). The CON group was fed 10 L/day from days 7 to 10, while the MR group transitioned from milk to MR starting at day 7. The ratio of WM to MR (3:1) gradually decreased to 1:3 during the transition period and then to 0:1 on day 10. After boiling and cooling to 40\u0026ndash;50\u0026deg;C, 6.5 parts of warm water were mixed with each component in the MR solids. An electric mixer was used for mixing to ensure no solid residue and no sedimentation occurred. The calves received 11 L/day of liquid feed from days 11 to 50, 8 L/day from days 51 to 55, 4 L/day from days 55 to 57, 2 L/day from day 58, and 1 L in the morning at 59 days. All calves were weaned at 60 days. This feeding program was also used for the WM treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Throughout the experiment, the starter diet and water were freely accessible (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). All calves received a calf starter manufactured by Changchun Borui Science \u0026amp; Technology Co. starting on day 2.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe nutrient composition of the whole milk fed to the Holstein calves\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eItems\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWhole milk\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNutrient constituents(% as fed)\u003csup\u003eA\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFat\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e3.98\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eProtein\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e3.44\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLactose\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e4.67\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAsh\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e0.66\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSolid non - fat\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e8.76\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal solids\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e12.57\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMetabolic energy(Mcal/kg of DM)\u003csup\u003eB\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e5.54\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"2\"\u003eNote:\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"2\"\u003eA Metabolic energy was a calculated value and others were measured values. B Calculated metabolic energy for whole milk (Mcalkg) = [0.057 x protein (%)\u0026thinsp;+\u0026thinsp;0.092 x fat (%)\u0026thinsp;+\u0026thinsp;0.0395 x lactose\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"2\"\u003e(%)]x 0.93 (NRC, 2001).\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe chemical composition of the starter fed to the Holstein calves (% of diet DM)\u003csup\u003eA\u003c/sup\u003e\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eItems\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCalf starter\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eChemical composition\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDry matter (DM, % as fed)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e88.75\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCrude protein (CP, % DM)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e24.51\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAcid detergent fiber (% DM)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e9.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNeutral detergent fiber (% DM)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e24.43\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAsh (% DM)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e6.91\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEther extract (% DM)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4.98\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStarch (% DM)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e32.65\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCalcium (% DM)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.78\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePhosphorus (% DM)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.53\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMetabolic energy\u003csup\u003eB\u003c/sup\u003e (Mcal/kg of DM)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4.21\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"2\"\u003eNote:\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"2\"\u003eA Metabolic energy was a calculated value and others were measured values. B Calculated metabolic energy for whole milk (Mcalkg) = [0.057 x protein (%)\u0026thinsp;+\u0026thinsp;0.092 x fat (%)\u0026thinsp;+\u0026thinsp;0.0395 x lactose\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"2\"\u003e(%)]x 0.93 (NRC, 2001).\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Measurements of growth performance and nutrient utilization\u003c/h2\u003e \u003cp\u003eThe test period is 60 days. MR and solid feed samples were collected monthly during the feed cycle and then analysed for nitrogen (N), ash, calcium, phosphorus, neutral detergent fibre and acid detergent fibre.\u003c/p\u003e \u003cp\u003eEach calf was weighed separately before starting the experiment, at 30 days after starting, and at the end of the experiment, and the individual average daily weight gain (ADG) was calculated. Record the daily MR feed intake of each calf based on the difference between the allocated amount and the remaining amount, and calculate the average daily solid feed intake of each enclosure group by dividing the difference between the allocated amount and the remaining amount by the number of enclosure partners. Feed efficiency is calculated based on the ratio of ADG to average daily feed intake. The calculations were as follows:\u003c/p\u003e \u003cp\u003eADG: (pre-slaughter weight - newborn weight)/number of days in the trial\u003c/p\u003e \u003cp\u003eAverage daily feed intake: feed intake per head/number of days during the test period\u003c/p\u003e \u003cp\u003eFeed-to-weight ratio: average daily feed intake/ADG\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Slaughter and sample collection\u003c/h2\u003e \u003cp\u003eCalves were euthanized on days 30 and 60 after arrival at 3 h after the morning meal by injecting 6 mL tetracaine (T61; MSD Animal Health Netherlands) into the jugular vein.\u003c/p\u003e \u003cp\u003eAfter slaughter, immediately lay organs such as the heart, liver, spleen, lungs, kidneys, and gallbladder were spread on a plastic sheet to measure weights. The carcass was measured and defined as the weight of the slaughtered lamb\u0026rsquo;s body without the head, lower limbs, all internal organs, and fur after being left for 30 min. The forestomach compartments, including the rumen, reticulorumen, omasum, and abomasum, were removed, emptied of digesta, and weighed.\u003c/p\u003e \u003cp\u003eThe rumen digesta were emptied into a plastic measuring cylinder and manually mixed. Approximately 30 mL of the digesta was collected and stored at \u0026minus;\u0026thinsp;40\u0026deg;C until extraction of bacterial DNA and digestive enzymes. The intestinal chyme was collected with 5 mL of the digesta and stored at \u0026minus;\u0026thinsp;80\u0026deg;C until the extraction of bacterial digestive enzyme.\u003c/p\u003e \u003cp\u003eFour pieces of whole tissue (2 cm \u0026times; 2 cm) from the rumen dorsal sac, ventral sac, caudodorsal blind sac, and caudal ventral blind sac were separately collected and rinsed in 0.9% stroke-physiological saline solutions until clear and then stored in designated 10% formalin neutral fixative solutions. The small intestine, including the duodenum, jejunum, and ileum, and large intestine, including the colon, cecum, and rectum, were spread on polystyrene plates to measure their length and weight. Pieces (2 cm in length) from the duodenum, jejunum, ileum, and cecum were washed using 0.9% stroke-physiological saline solution and fixed with 10% formalin solution for later determination of intestinal development.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Chemical analysis\u003c/h2\u003e \u003cp\u003e Concentrated feed samples were collected, and the dry matter, crude protein, ether extract, ash, and iron contents were analyzed according to the procedures of the AOAC. The feed samples were also analyzed for neutral detergent fiber content, as proposed by Van Soest et al, The chemical compositions of the MRs and solid feeds provided during fattening are reported in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe ingredients and chemical composition of the milk replacer (MR) fed to the Holstein calves (% of diet dry matter, DM)\u003csup\u003eA\u003c/sup\u003e\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIngredients,g/kg DM\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMilk replacer\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ewhey powder\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e507.7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWhey proteins\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e127.3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMaltodextrin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e28.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGlucose\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e13.7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eModidied wheat protein\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e90.3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSoybean protein concentrate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e14.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCoconut oil\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e67.4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePalm oil\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e82.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNaCl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e12.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMethionine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e8.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLysine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e3.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTryptophan\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eThreonine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePremix\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e14.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eChemical composition\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDM,g/kg DM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e974.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCrude protein(CP),g/kg DM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e130.6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLactose,g/kg DM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e521.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGlucose,g/kg DM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e14.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStarch,g/kg DM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEther extract,g/kg DM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e167.9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAcid detergent fiber(ADF),g/kg DM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2.9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNeutral detergent fiber(NDE),g/kg DM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e7.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAsh,g/kg DM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e52.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCa,g/kg DM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e13.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP,g/kg DM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e11.7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePlant proteins,g/kg DM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e95.4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWhey proteins,g/kg DM10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e130.6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eME,Mcal/kg of DM\u003csup\u003eB\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4.47\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"2\"\u003eNote:\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"2\"\u003eA Metabolic energy was a calculated value and others were measured values. B Calculated metabolic energy for whole milk (Mcalkg) = [0.057 x protein (%)\u0026thinsp;+\u0026thinsp;0.092 x fat (%)\u0026thinsp;+\u0026thinsp;0.0395 x lactose\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"2\"\u003e(%)]x 0.93 (NRC, 2001).\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Digestive enzyme analysis\u003c/h2\u003e \u003cp\u003eGastrointestinal chyme was thawed and rapidly centrifuged at 10,000 \u003cem\u003eg\u003c/em\u003e for 5 min at 4\u0026deg;C, and 10 \u0026micro;L of supernatant was collected. Enzyme activity was determined using an enzyme-linked immunosorbent assay (ELISA) kit (MLbio, Shanghai, China) following the manufacturer's instructions.. Digestive enzyme activities in the chyme were determined for the crumpled stomach and in each small intestinal segment (crumpled stomach: pepsin and gastric lipase; and duodenum, jejunum, and ileum: pancreatic proteases, chymotrypsin, pancreatic lipases, pancreatic amylase, maltase, isomalto-amylase, carboxypeptidase A, carboxypeptidase B, lactase, and sucrase).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Histological analysis\u003c/h2\u003e \u003cp\u003eAfter fixation, four pieces of calf rumen, duodenum, jejunum, and ileum tissue samples were collected and trimmed to flatness using a scalpel. The flat tissue samples were then placed in a dehydrator (Nanjing Yanzheng Drying Equipment Co. Ltd. YZ-TS-100A), dehydrated in an ethanol series, cleared with xylene, and embedded in paraffin. An embedding machine (KH-MQ2600, Xiaogan Kohai Medical Technology Co., Ltd.) was used to embed the wax-dipped tissue samples, and corresponding labels were affixed. The samples were placed on a freezing table (KH-LQ3500, Xiaogan Kohai Medical Technology Co., Ltd., Hubei, China, -20 ℃) for cooling, and after cooling was complete, the wax blocks were trimmed. After trimming, the wax blocks were sliced on a microtome and the thickness of each slice was maintained at approximately 4 \u0026micro;m.\u003c/p\u003e \u003cp\u003eThe slices were spread out and fished with slides at 40 ℃, and then slides with sliced tissues were put into a 60 ℃ oven (Nanjing Yanzheng Drying Equipment Co., Ltd., YZ-KX-400S). After baking, remove the glass slide and place it at room temperature for subsequent staining. The dried tissue samples were deparaffinized with xylene and rinsed with distilled water. After paraffin removal, the sections were stained with hematoxylin staining solution for 5 min, rinsed with distilled water, and differentiated with 1% hydrochloric acid alcohol for 5 s. After rinsing, the sections were turned blue with ammonia (0.6%) and rinsed again. Sections were stained with an eosin staining solution for 2 min.\u003c/p\u003e \u003cp\u003eSections of stained tissue samples were transparently processed using anhydrous ethanol and xylene, and when finished, they were dried and sealed with neutral gum. Four microscopic fields per sample were selected to measure the intestinal muscle thickness, and the images were acquired and analyzed using the image analysis software Image-Pro 6.0 to determine the villus length, villus width, and crypt depth.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 High-throughput sequencing analysis\u003c/h2\u003e \u003cp\u003eMicrobial DNA was extracted in vitro from ruminal fluid samples (n\u0026thinsp;=\u0026thinsp;5 per group) using a DNA Kit (Omega Bio-Tek, Norcross, GA, USA), which was followed by PCR amplification conducted with barcoded specific bacteria primers that targeted the variable region 3\u0026ndash;4 (V3-V4) of the 16S rRNA gene: forward and reverse primers 338F (5\u0026rsquo;-barcode\u0026thinsp;+\u0026thinsp;ACTCCTACGGGAGGCAGCA-3\u0026rsquo;) and 806R (5\u0026rsquo;-GGACTACHVGGGTWTC TAAT-3\u0026rsquo;).\u003c/p\u003e \u003cp\u003eThe amplicons were extracted, purified, and quantified. A NEXTFLEX Rapid DNA-Seq Kit was used to pool the DNA products. Sequencing libraries and paired-end sequencing were performed according to standard protocols using an Illumina MiSeq platform (Shanghai BIOZERON Co., Ltd(Beijing, China) according to standard protocols. The amplicon library was then paired-end sequenced (2\u0026times;250 bp) using an Illumina MiSeq platform (Shanghai BIOZERON Co., Ltd.). The raw reads were uploaded to the NCBI Sequence Read Archive database.\u003c/p\u003e \u003cp\u003eThe library amount to be loaded can be controlled between 15 and 18 pM according to the actual conditions. First, a sparse curve was plotted using QIIME2 software to assess whether the sequencing results of each sample could reflect the microbial diversity in the sample. Second, to compare the diversity of different samples, the ASV abundance matrix was randomly flattened by 95% of the sequence volume of the sample based on the lowest sequence volume among all samples to correct for the differences in diversity among samples caused by the sequencing depth. Finally, the Chao1 index, Observed species index, Shannon index, Simpson index, Faith's PD index, Pielou's evenness index, and Good's coverage index were calculated using QIIME2 software. All OTUs were assigned a taxonomic classification via a QIIME search against the Silva databases (Release 128). The beta diversity of the microbiome was compared using principal component analysis. Analysis of the ruminal microbiome abundance was performed to identify significant differences in taxon abundance at the genus level between the control and treatment groups.\u003c/p\u003e \u003c/div\u003e"},{"header":"3 Statistical Analysis","content":"\u003cp\u003eAll experimental data, including performance, gastrointestinal tissue development, and digestive enzyme secretions, were analyzed usingthe SPSS software (version 26.0; SPSS Inc., Chicago, IL, USA). The WM and MR groups were analyzed using a single-factor experimental design; data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM. The significance level was set at \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/p\u003e"},{"header":"4 Results","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e4.1 Intake and growth performance\u003c/h2\u003e \u003cp\u003eIntake and growth performance, which are crucial economically important factors, were compared among the groups. As shown in Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, all calves had the same initial body weight (BW). The results revealed that the pre-slaughter BW (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), average daily gain (ADG) (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), and average dry matter intake (ADMI) (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) were significantly higher in the WM group at 30 days compared with the MR group. The feed-to-weight ratio of the MR group was significantly higher than that of the WM group at 30 and 60 days.(P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). In addition, the BW, ADG, and ADMI were not significantly different between the two groups at 60 days (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEffects of milk and milk replacer on growth performance and body size index of Holstein calves\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"9\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eItem\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e30d\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eSEM\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003ep-Vaule\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003e60d\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eSEM\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003ep-Vaule\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMilk\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMR\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eMilk\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eMR\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBody weight/kg\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e39.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e39.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e39.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e40.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0.84\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWeight/kg\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e65.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e61.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e97.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e98.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e1.17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0.54\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eADG/(kg/d)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.73\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.96\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.98\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0.58\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDMI/(kg/d)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1.42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e1.48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0.02\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eF/G\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.69\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1.46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e1.51\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0.00\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e4.2 Slaughter performance and organ weight\u003c/h2\u003e \u003cp\u003eSlaughter performance was analyzed as shown in Fig.\u0026nbsp;3. The heart weight was significantly higher in the WM group than in the MR group at 30 days (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). No significant differences were observed in the weights of the kidneys, spleen, or liver (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e4.3 Digestive enzyme activity\u003c/h2\u003e \u003cp\u003eThe digestive enzyme activity results are shown in Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e,Table\u0026nbsp;\u003cspan refid=\"Tab6\" class=\"InternalRef\"\u003e6\u003c/span\u003e and Table\u0026nbsp;\u003cspan refid=\"Tab7\" class=\"InternalRef\"\u003e7\u003c/span\u003e. At 30 days, the lipase and amylase levels in the stomach were significantly higher in the WM group than in the MR group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The carboxypeptidase A activity in the ileum of the calves in the MR group was significantly higher than that of the calves in the WM group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). However, at 60 days, the crumpled stomach lipase and amylase levels and ileal pancreatic amylase levels were significantly lower in the WM group than in the MR group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). No significant differences in the activities of other enzymes were observed between the two groups (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab5\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEffects of milk and milk replacer on protein digestive enzyme activities in Holstein calves\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eItem\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAge(day)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMilk\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMR\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eSEM\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eP-Value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"5\" rowspan=\"6\"\u003e \u003cp\u003eTrypsin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDuodenum\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e22.53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e25.43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1.84\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.49\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eJejunum\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e41.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e36.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e3.39\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.58\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eIleum\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e26.27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e24.63\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1.34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.60\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDuodenum\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e36.27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e25.87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e2.83\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.05\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eJejunum\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e30.13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e27.47\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e2.31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.62\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eIleum\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e29.87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e32.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1.10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.24\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"5\" rowspan=\"6\"\u003e \u003cp\u003eChymotrypsin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDuodenum\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e340.23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e348.37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e30.43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.91\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eJejunum\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e341.63\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e238.23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e32.66\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.12\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eIleum\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e335.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e241.10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e33.68\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.18\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDuodenum\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e266.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e281.73\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e22.87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.77\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eJejunum\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e258.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e212.57\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e30.55\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.52\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eIleum\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e316.23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e389.47\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e29.69\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.26\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"5\" rowspan=\"6\"\u003e \u003cp\u003eCarboxypeptidases A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDuodenum\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e101.78\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e123.14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e14.69\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.53\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eJejunum\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e110.16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e142.54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e7.69\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eIleum\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e120.81\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e135.29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e12.16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.61\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDuodenum\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e97.19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e117.52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e9.32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.33\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eJejunum\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e100.98\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e143.59\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e16.35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.23\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eIleum\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e133.14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e121.72\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e14.52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.74\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"5\" rowspan=\"6\"\u003e \u003cp\u003eCarboxypeptidases B\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDuodenum\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e157.98\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e173.97\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e22.41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.76\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eJejunum\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e159.79\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e213.45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e15.23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.06\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eIleum\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e146.57\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e176.15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e15.32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.39\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDuodenum\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e163.10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e170.38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e6.77\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.65\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eJejunum\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e160.75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e195.70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e19.96\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.44\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eIleum\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e187.28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e232.87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e16.53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.19\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab6\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 6\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEffects of milk and milk replacer on starch digestive enzyme activities in Holstein calves\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eItem\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAge(day)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMilk\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMR\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eSEM\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eP-Value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"5\" rowspan=\"6\"\u003e \u003cp\u003ePancreatic amylase\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDuodenum\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e343.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e243.17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e33.97\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.16\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eJejunum\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e379.60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e386.14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e31.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.93\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eIleum\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e355.37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e313.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e40.85\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.66\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDuodenum\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e318.77\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e420.98\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e35.75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.17\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eJejunum\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e418.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e404.60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e37.78\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.88\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eIleum\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e223.37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e321.20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e25.13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.02\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"5\" rowspan=\"6\"\u003e \u003cp\u003eMaltase\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDuodenum\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e22.47\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e27.93\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e3.49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.50\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eJejunum\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e27.60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e27.13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e2.48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.94\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eIleum\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e33.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e24.57\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e2.58\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.10\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDuodenum\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e24.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e28.37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e2.91\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.55\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eJejunum\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e17.97\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e26.37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e2.70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.13\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eIleum\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e23.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e26.20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e2.64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.61\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"5\" rowspan=\"6\"\u003e \u003cp\u003eIsomaltase\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDuodenum\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e19.17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e23.16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e4.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.68\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eJejunum\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e15.74\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e16.87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e3.47\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.89\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eIleum\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e21.57\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e16.17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e2.21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.26\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDuodenum\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e18.69\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e20.53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e3.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.80\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eJejunum\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e22.57\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e15.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e2.24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.13\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eIleum\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e17.80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e19.87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e2.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.66\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab7\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 7\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEffects of milk and milk replacer on Non-starch energy substances digestive enzyme activities in Holstein calves\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eItem\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAge(day)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMilk\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMR\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eSEM\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eP-Value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"5\" rowspan=\"6\"\u003e \u003cp\u003eLipase\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDuodenum\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e209.17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e204.09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e12.97\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.87\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eJejunum\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e147.58\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e115.48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e11.55\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.19\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eIleum\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e198.87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e211.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e6.69\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.42\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDuodenum\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e156.77\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e132.77\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e6.62\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.05\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eJejunum\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e190.63\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e123.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e17.62\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.03\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eIleum\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e108.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e180.43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e19.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.03\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"5\" rowspan=\"6\"\u003e \u003cp\u003eLactase\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDuodenum\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e13.78\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e13.54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1.20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.93\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eJejunum\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e13.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e14.35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.74\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.46\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eIleum\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e11.80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e12.53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1.10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.78\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDuodenum\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e10.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e14.59\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1.66\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.26\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eJejunum\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e15.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e12.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1.35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.31\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eIleum\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e14.86\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e11.93\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1.34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.32\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"5\" rowspan=\"6\"\u003e \u003cp\u003eSucrase\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDuodenum\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e8.81\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e8.44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.73\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eJejunum\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e13.28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e10.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1.99\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.57\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eIleum\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e14.31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e8.31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e2.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.21\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDuodenum\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e9.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e8.34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.35\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eJejunum\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e12.62\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e10.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1.10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.30\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eIleum\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e11.27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e13.44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e2.15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.67\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e4.4 Rumen and small bowel development\u003c/h2\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003e,Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003e lists the results for rumen and small bowel development. Rumen weight and rumen papilla height were significantly higher in the MR group than in the WM group at 60 days (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). As shown in Table\u0026nbsp;\u003cspan refid=\"Tab8\" class=\"InternalRef\"\u003e8\u003c/span\u003e, the villus length and crypt depth of the duodenum, depth of the jejunal crypt, width of the ileal villi, and depth of crypts in the WM group were greater than those in the MR group at 30 days (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The length of the jejunal villi and weight of the empty intestine were significantly higher in the MR group than in the WM group at 60 days (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab8\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 8\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEffects of milk and milk replacer on development of rumen papillae and small intestine villus in Holstein calves\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"10\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" morerows=\"1\" nameend=\"c2\" namest=\"c1\" rowspan=\"2\"\u003e \u003cp\u003eItem\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"4\" nameend=\"c6\" namest=\"c3\"\u003e \u003cp\u003e30d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c9\" namest=\"c7\"\u003e \u003cp\u003e60d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eWM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eSEM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eP-Value\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eWM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eMR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eSEM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eP-Value\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eRumen\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePapillae length\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e493.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e495.69\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e24.41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.924\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e645.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e746.34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e34.47\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePapillae width\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e254.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e232.94\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e31.47\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.272\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e253.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e283.86\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e24.66\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e0.625\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eDuodenum\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eVillus length\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e582.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e518.65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e5.26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.037\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e650.28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e655.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e22.92\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e0.653\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eVillus width\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e151.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e154.53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e10.48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.357\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e176.88\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e172.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e11.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e0.296\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCrypt depth\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e153.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e137.57\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e7.19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.027\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e147.31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e155.17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e8.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e0.053\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eJejunum\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eVillus length\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e525.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e499.18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e18.14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.013\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e594.62\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e593.58\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e5.74\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e0.974\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eVillus width\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e145.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e142.20.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4.85\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.524\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e154.63\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e153.29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e7.38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e0.669\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCrypt depth\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e147.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e135.62\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e12.74\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.032\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e157.73\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e152.39\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e7.35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e0.126\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eIleum\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eVillus length\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e508.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e488.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e17.65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.070\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e524.76\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e563.92\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e28.39\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e0.006\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eVillus width\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e134.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e114.17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e16.74\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.026\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e143.76\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e140.79\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e5.84\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e0.534\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCrypt depth\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e127.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e102.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e14.35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.014\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e141.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e139.92\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e5.64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e0.665\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e4.5 Rumen bacteria\u003c/h2\u003e \u003cp\u003eAlpha diversity refers to the abundance, variety, and homogeneity of species in a localized area. At 30 days, the Good\u0026rsquo;s coverage index in the WM group was higher than that in the MR group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Figure\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e7\u003c/span\u003e lists the profile of the ruminal microbial com munity based on an unweighted Bray\u0026ndash;Curtis dissimilarity matrix at the genus. The composition of the rumen microbiota, which is crucial for growth performance, was assessed using 16S rRNA sequencing. The relative abundances of the dominant phyla are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e8\u003c/span\u003e. The four most dominant phyla were Bacteroidota, Firmicutes, Actinobacteria, and Proteobacteria. The relative abundance of Proteobacteria in the WM group was significantly higher than that in the MR group on day 30 (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). At 60 days, the relative abundances of Bacteroidetes and Actinobacteria in the MR group were higher than those in the WM group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The relative abundances of dominant genera are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e9\u003c/span\u003e. The four most dominant genera were Prevotella, Succiniclasticum, Butyrivibrio, and Bacteroides. The relative abundance of Succiniclasticum (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) was significantly higher in the MR group than in the WR group on day 30 (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). By day 60, the relative abundances of Prevotella (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and Bacteroides in the WM group were greater than those in the MR group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"5 Discussion","content":"\u003cp\u003e \u003cb\u003e5.1 Effects of milk and MR on the growth performance and body size index of Holstein calves\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe primary objective of this study was to investigate the performance, gastrointestinal tissue development, and digestive enzyme secretion of Holstein calves fed WM or MR. In a previous study, the WM group showed better growth performance \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Another study showed that calves in the MR group were more likely to require greater dry matter intake (DMI) than those in the WM-treated group \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. In the present study, the DMI of the starter diet differed significantly between the WM and MR calves as their age increased\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. The amount of liquid feed supplied gradually decreased, and the amount of the starter diet consumed by the MR calves increased relative to that of the WM calves. The DMI of the starter diets of the calves in the MR group was significantly higher than that of the calves in the WM control group, and this increase in the amount of starter feed consumed by the calves could be attributed to the better rumen development of the calves \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. The results of rumen histomorphometry in this trial also showed that the MR group had greater rumen papilla lengths than the WM group, which is consistent with a previous study \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. In addition, due to the significant difference in nutrient composition between the MR and WM groups, the MR-fed calves showed lower growth performance during lactation than the WM-fed calves. However, the growth disadvantage of the calves before weaning could be improved by increasing the intake of starter food. This might have been due to potential improvements in the tolerance of calves to nondairy components when fed a diet of MR, which resulted in better rumen development of calves after entering the weaning transition period \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Moreover, calves fed MR were able to compensate for the decrease in liquid feed supply by increasing the intake of starter feed during the weaning transition period \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Significant differences in ADG and G/F were observed between the WM and MR groups, which may have been due to the poorer digestion of plant-based ingredients in the MR group. Previous studies have shown that although MR powder contains less plant protein, it still reduces the digestibility of nutrients in calves and inhibits calf growth and development \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. However, studies have shown that digestion improves as the calves ages \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e5.2. Effects of milk and MR on digestive enzyme activities in Holstein calves\u003c/h2\u003e \u003cp\u003eDuring early lactation, calves have an undeveloped rumen, which acts as the main source of nutrition through enzymatic digestion. The gastrointestinal enzyme activity of calves is key for their digestion. Khorasani et al. \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e researched pancreatic protease inhibitors and found that soy protein affects trypsin activity in calves. Moreover, at 60 days, pancreatic protease activity in the duodenum of calves in the milk group was significantly higher than that in the MR group. Another study \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e found that carboxypeptidase A activity was fairly equally distributed in the five parts of the small intestine while carboxypeptidases B activity was high in both the median and distal jejunum as well as in the ileum. In the present study, carboxypeptidase A and B activities were higher in the MR group than in the WM group, which may have been caused by the rapid entry of plant proteins into the jejunum without protein agglutination. MRs containing wheat proteins increase the abomasal emptying rate \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. All amylase activities are low in newborn calves, while lactase activity decreases and pancreatic amylase and maltase activities gradually increase as calves grow \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. For calves, starch enters the small intestine and is hydrolyzed to glucose by pancreatic amylase and maltase, and then glucose is absorbed and utilized by the body. A previous study \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e suggested that maltase activity limits starch digestion and that fermentation may contribute largely to the loss of total starch in milk-fed calves\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. suggested that calves with the highest starter intake had high concentrations of serum amylase. In our study, intestinal amylase activity was higher in calves in the MR group with higher pellet feed intake. At 60 days, the WM calves had significantly higher duodenal and jejunal pancreatic lipase and significantly lower ileal pancreatic lipase than the MR calves. This may be attributed to the type of fat in the diet\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Compared with the WM group, the MR group used more plant-based fats and a lower fat content. Fat of lactic origin is better digested and absorbed and released more rapidly in the duodenal end of the duodenal rectum and in the jejunum. However, the vegetable fats contained in the MR and the fats in the pellets were digested slowly and flowed into the ileal segment for gradual digestion \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e5.3. Effects of milk and MR on gastrointestinal tract development in Holstein calves\u003c/h2\u003e \u003cp\u003eRumen development in calves is an important indicator of weaning performance \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. The entry of grains into the rumen promotes rumen weight and muscle tissue development through physical stimulation \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. The MR feeding strategy based on a liquid diet as used in this trial resulted in calves consuming more open feed during lactation, which produced more physical stimulation of the rumen, resulting in significant differences between the two groups. Starter intake is critical for rumen development, and the end products of rumen fermentation, volatile fatty acids, are important stimulants of rumen papilla development\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. In this experiment, the calves were fed liquid diets in nipple-less buckets to minimize the formation of esophageal grooves. In addition, the calves were 0\u0026ndash;60 days old. The calves in the MR group had a larger rumen mass and longer rumen nipple lengths and exhibited certain ruminant behavior \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. This may have been due to the stimulation of the rumen teat by the plant-based ingredients in the MR \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Some plant-based fats can enter the rumen and produce short-chain fatty acids through microbial fermentation, which provides more chemical stimulation to the rumen teat and promotes its rapid growth, thus providing a good foundation for the calf's growth period \u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe small intestine is the main organ used by lactating calves to digest and absorb nutrients from their diet \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. Previous studies have shown that increasing the proportion of plant proteins in MR powder can negatively affect calf small intestine development. In this study, the number of small intestinal villi in calves in the WM group was higher than that in the MR group at 30 days of age, which indicated that the WM group had a better absorptive capacity. This may have been one of the reasons for the higher pre-lactation growth performance of calves in the WM group \u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. A previous study \u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e suggested that the type of liquid diet affects both the gut mass and epithelial microstructure differently. In this study, the ileal weight of the calves in the milk group was higher. This may be related to the greater ileocecal gastrocnemius muscle thickness in MR-fed calves \u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. We found that calves in the MR group showed significant growth of small intestinal villi compared to those in the WM group at 60 days. Compared to previous studies, MR had a negative effect on small bowel development \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. This may be due to the influence of harmful gases produced during the microbial fermentation of granular material residues at the end of the small intestine on the small intestinal epithelium \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. Moreover, frictional stimulation and longer intestinal retention times under conditions of increased pellet intake may be responsible for the rapid development of small intestinal villi \u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e5.4 Effect of milk and MR on rumen flora of Holstein calves\u003c/h2\u003e \u003cp\u003eRumen development and health status have a direct influence on calf growth. The digestive functions of the rumen are not immediately active in a newborn calf; however, after 3 days of solid feed, the rumen is stimulated to start its digestive functions, and the internal flora is established. Subsequently, the rumen of the calf starts to develop, and as the calf ages, the rumen matures \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Bacteria in the rumen provide the basis of its digestive function, and the composition and quantity of bacteria can directly affect the digestive ability of the rumen, which in turn directly affects the growth and development of calves \u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. For ruminants, bacterial phyla have an important role in degrading fiber and cellulose and are involved in the degradation of starch, cellulose, hemicellulose, and oligosaccharides and production of acetic, propionic, and butyric acids, which can promote absorption and metabolism in ruminants. The results of this experiment showed that the addition of MR significantly increased the relative abundance of Bacteroidetes and Actinobacteria at 60 days. Bacteroidetes and Actinobacteria are major phyla of the rumen flora and are closely related to volatile fatty acid production in the rumen \u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. \u003cem\u003ePrevotella\u003c/em\u003e is the predominant genus in the rumen, and it reduces the inflammatory response of the organism and participates in the digestion of carbohydrates, polysaccharides, and proteins in the WM group (60 days). The genus \u003cem\u003eAspergillus\u003c/em\u003e includes several pathogenic bacteria that are mostly gram negative and pathogenic to the organism, and they produce LPS and irritating flagellin to promote inflammatory responses. \u003cem\u003eVibrio\u003c/em\u003e spp. ferment carbohydrates into succinic acid and acetic acid to increase energy utilization and meet the body's energy requirements \u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. In summary, calves in the MR group had more effective rumen flora because of their increased pellet feed intake.\u003c/p\u003e \u003c/div\u003e"},{"header":"6 Conclusion","content":"\u003cp\u003eCalves fed MR showed poorer growth performance early in lactation than the calves fed WM; however, the higher starter intake helped the MR calves plateau in growth performance at weaning. Gastrointestinal digestive enzymes showed more positive amylase activity and more mature rumen development and teat growth. In addition, rumen microbes in MR-fed calves were more efficient at degrading starter intake. Therefore, feeding MR would improve calf digestion of solid feed by increasing rumen development and small intestinal amylase levels. However, the evolutionary processes and specific mechanisms responsible for this outcome cannot be explained by the study results because the current study was limited to epiphenomena.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eCompeting interests\u003c/h2\u003e \u003cp\u003eThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThe experiment was subsidized by Changchun Borui Science \u0026amp; Technology Co., Ltd (Item Numberand: 20220202049NC)and Ningxia Yijiahe Animal Husbandry Development Co., Ltd are thanked for providing venue support.\u003c/p\u003e \u003cp\u003eAcknowledgements\u003c/p\u003e \u003cp\u003eThe authors thank Jilin Agricultural University (Changchun, China) Ruminant Center members for providing help during the experiment.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eXu Tian and Xuefeng Zhang wrote the main manuscript text .All authors reviewed the manuscript.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe flora data of this study was uploaded to the NCBI database with the primary accession code PRJNA1101260 (V3-V4) .\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eLee, H. 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Host Species Affects Bacterial Evenness, but Not Diversity: Comparison of Fecal Bacteria of Cows and Goats Offered the Same Diet. \u003cem\u003eAnimals (Basel)\u003c/em\u003e 12, (2022). (2011).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Whole milk, milk replacement, rumen, microbiota","lastPublishedDoi":"10.21203/rs.3.rs-6138081/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6138081/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe purpose of the research was to compared the effects of the supplementation of whole milk or milk replacer on the performance, gastrointestinal tissue development, and digestive enzyme secretions of Holstein calves. Twelve newborn Holstein male calves (1.96 ± 0.99 d of age; 40.0 ± 4.37 kg BW) were blocked by arrival date and age, divided into two groups: whole milk group(WM) and milk replacer group(MR)(n=6) in One-way experimental design. The results showed that at 30 d, the WM group had significantly higher (P\u0026lt;0.05) pre-slaughter body weight (P\u0026lt;0.05), ADG (P\u0026lt;0.05) and average dry matter intake (ADMI) (P\u0026lt;0.05) than the MR group; and at 60 d, the ileocecal pancreatic amylase level of the WM group was significantly lower (P\u0026lt;0.05) than that of the MR group.\u003c/p\u003e\n\u003cp\u003eRumen development was better in the MR group than in the WM group, and small intestinal villus length development was different in each intestinal segment(P\u0026lt;0.05).\u003c/p\u003e\n\u003cp\u003eThe relative abundance of Proteobacteria in the WM group was significantly higher than that in the MR group on day 30(P\u0026lt;0.05) and the relative abundance of succinogens was significantly higher (P\u0026lt;0.05) in the MR group than in the WR group (P\u0026lt;0.05). At 60 d, the relative abundance of Bacteroidetes and Actinobacteria in the MR group was higher (P\u0026lt;0.05) than in the WM group, and the relative abundances of Prevotella (P\u0026lt;0.05) and Bacteroides in the WM group were greater than those in the MR group (P\u0026lt;0.05).Calves fed MR showed poorer growth performance early in lactation, but higher starter intake helped calves plateau in growth performance at weaning. Gastrointestinal digestive enzymes showed more positive amylase activity and more mature rumen development and teat growth. In addition, rumen microbes in MR-fed calves were more efficient at degrading starter intake.Therefore, feeding MR would improve calf digestion of solid feeds by increasing rumen development and small intestinal amylase levels. However, the evolutionary processes and specific mechanisms responsible for this outcome cannot be explained because the current study was limited to epiphenomena.\u003c/p\u003e","manuscriptTitle":"Effects of feeding whole milk or milk replacer on the performance, gastrointestinal tissue development, and digestive enzyme secretions of Holstein calves","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-02 12:09:54","doi":"10.21203/rs.3.rs-6138081/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"1944a1a6-ee49-4beb-82c9-b4b335e49894","owner":[],"postedDate":"April 2nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":46535550,"name":"Biological sciences/Zoology/Animal physiology"},{"id":46535551,"name":"Health sciences/Gastroenterology/Gastrointestinal system/Small intestine"}],"tags":[],"updatedAt":"2025-09-29T16:53:45+00:00","versionOfRecord":[],"versionCreatedAt":"2025-04-02 12:09:54","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6138081","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6138081","identity":"rs-6138081","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}