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Effects of antibiotic residues in milk on growth, ruminal fermentation, and microbial community of preweaning dairy calves

  • Author Footnotes
    * These authors contributed equally to this work.
    J.H. Li
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    * These authors contributed equally to this work.
    Affiliations
    State Key Laboratory of Animal Nutrition, College of Animal Science and Technology, China Agricultural University, Beijing, 100193, China
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  • Author Footnotes
    * These authors contributed equally to this work.
    M.H. Yousif
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    * These authors contributed equally to this work.
    Affiliations
    State Key Laboratory of Animal Nutrition, College of Animal Science and Technology, China Agricultural University, Beijing, 100193, China
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  • Z.Q. Li
    Affiliations
    College of Animal Science and Technology, Henan University of Science and Technology, Luoyang, Henan, 471003, China
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  • Z.H. Wu
    Affiliations
    State Key Laboratory of Animal Nutrition, College of Animal Science and Technology, China Agricultural University, Beijing, 100193, China
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  • S.L. Li
    Affiliations
    State Key Laboratory of Animal Nutrition, College of Animal Science and Technology, China Agricultural University, Beijing, 100193, China
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  • H.J. Yang
    Affiliations
    State Key Laboratory of Animal Nutrition, College of Animal Science and Technology, China Agricultural University, Beijing, 100193, China
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  • Y.J. Wang
    Affiliations
    State Key Laboratory of Animal Nutrition, College of Animal Science and Technology, China Agricultural University, Beijing, 100193, China
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  • Z.J. Cao
    Correspondence
    Corresponding author
    Affiliations
    State Key Laboratory of Animal Nutrition, College of Animal Science and Technology, China Agricultural University, Beijing, 100193, China
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  • Author Footnotes
    * These authors contributed equally to this work.
Open ArchivePublished:January 25, 2019DOI:https://doi.org/10.3168/jds.2018-15506

      ABSTRACT

      The aim of this study was to evaluate the effects of antibiotic residues in milk on growth, ruminal fermentation, and microbial community of dairy calves in their first 35 d of age. Twenty newborn Holstein bull calves were assigned to 1 of 2 treatments equally: milk replacer without antibiotics (control) and milk replacer plus 4 antibiotics: 0.024 mg/L of penicillin, 0.025 mg/L of streptomycin, 0.1 mg/L of tetracycline, and 0.33 mg/L of ceftiofur (ANT). Starter intake and fecal consistency scores of each calf were recorded on a daily basis. Body weight, withers height, body length, and heart girth were measured on d 1, 7, 14, 21, 28, and 35 before feeding in the morning. Rumen fluid was collected on d 15, 25, and 35 to determine ruminal pH, volatile fatty acids (VFA), and NH3-N concentrations. A total of 10 (5 per treatment) samples of rumen fluid taken on d 35 were analyzed for microbial community. Rumen tissues from the cranial ventral sac and cranial dorsal sac were collected from 8 calves of each group for morphology analysis on d 35 after being harvested. The results showed that calves in 2 treatments had similar starter intake, body weight, withers height, body length, heart girth, and average daily gain. The ANT group showed a lower diarrhea frequency in wk 4, and no differences were found for other weeks. Calves in the ANT group exhibited a greater concentration of acetic acid in the rumen and no differences for other VFA, total VFA, rumen pH, or NH3-N. As for rumen morphology, the length of papillae from cranial ventral sac of the ANT group was longer than that of the control group. The results of ruminal microbial community showed that antibiotic residues had minor effects on bacteria phyla and bacteria diversity. At the genus level, calves in the ANT group showed lower richness of Prevotella and higher richness of Acetitomaculum. In conclusion, antibiotic residues stimulated the development of ruminal papillae and increased the production of acetic acid in rumen, which might be caused by the influence of antibiotics on the ruminal microbial community.

      Key words

      INTRODUCTION

      Antibiotics have been an effective medical treatment since the 20th century (
      • Zaffiri L.
      • Gardner J.
      • Toledo-Pereyra L.H.
      History of antibiotics. From salvarsan to cephalosporins.
      ). However, the overuse of antibiotics causes many problems such as the disturbance of intestinal microbes and antibiotic resistance (
      • Phillips I.
      • Casewell M.
      • Cox T.
      • De Groot B.
      • Friis C.
      • Jones R.
      • Nightingale C.
      • Preston R.
      • Waddell J.
      Does the use of antibiotics in food animals pose a risk to human health? A critical review of published data.
      ). Studies have shown that the use of antibiotics during infancy increases the risk of developing allergies and obesity when babies grow up, which has a close relation to the disturbance of gut microbes due to antibiotics (
      • Johnson C.C.
      • Ownby D.R.
      • Alford S.H.
      • Havstad S.L.
      • Williams L.K.
      • Zoratti E.M.
      • Peterson E.L.
      • Joseph C.
      Antibiotic exposure in early infancy and risk for childhood atopy.
      ;
      • Ajslev T.A.
      • Andersen C.S.
      • Gamborg M.
      • Sorensen T.I.A.
      • Jess T.
      Childhood overweight after establishment of the gut microbiota: The role of delivery mode, pre-pregnancy weight and early administration of antibiotics.
      ). These side effects of antibiotics have raised a concern about using antibiotics on husbandry animals.
      Antibiotics are commonly used to treat mastitis, reproductive diseases, and hoof diseases in dairy farms. Antibiotics are injected into either vein or muscle, then end up in milk at very low concentrations. Penicillin G residue can be detected from the milk 9 d after treatment (
      • Seymour E.H.
      • Jones G.M.
      • Mcgilliard M.L.
      Persistence of residues in milk following antibiotic-treatment of dairy cattle.
      ) and 20% of milk samples showed positive results of gentamicin 6 d after treatment for mammary inflammation (
      • Martins T.
      • Santos A.F.S.
      • Miranda M.S.
      • Motta T.P.
      • Ambrósio L.A.
      • Pozzi C.R.
      • Arcaro J.R.
      Persistence of gentamicin residues in cow milk after intramammary treatment.
      ). The milk containing antibiotics along with transitional milk from fresh cows and milk containing high SCC from cows with mastitis make up waste milk, which cannot be sold commercially. Waste milk is usually used to feed calves for economic reasons. Milk is the major feedstuff for calves and plays an important role on health and growth. Many studies report consistent results that waste milk causes antibiotic resistance in gut bacteria (
      • Wray C.
      • Furniss S.
      • Benham C.L.
      Feeding antibiotic-contaminated waste milk to calves - Effects on physical performance and antibiotic-sensitivity of gut flora.
      ;
      • Aust V.
      • Knappstein K.
      • Kunz H.J.
      • Kaspar H.
      • Wallmann J.
      • Kaske M.
      Feeding untreated and pasteurized waste milk and bulk milk to calves: Effects on calf performance, health status and antibiotic resistance of faecal bacteria.
      ;
      • Maynou G.
      • Bach A.
      • Terré M.
      Feeding of waste milk to Holstein calves affects antimicrobial resistance of Escherichia coli and Pasteurella multocida isolated from fecal and nasal swabs.
      ). As for weight gain, some reported that calves fed with waste milk had higher weight gain than those fed with milk replacer (
      • Brunton L.A.
      • Reeves H.E.
      • Snow L.C.
      • Jones J.R.
      A longitudinal field trial assessing the impact of feeding waste milk containing antibiotic residues on the prevalence of ESBL-producing Escherichia coli in calves.
      ) or bulk milk (
      • Zou Y.
      • Wang Y.
      • Deng Y.
      • Cao Z.
      • Li S.
      • Wang J.
      Effects of feeding untreated, pasteurized and acidified waste milk and bunk tank milk on the performance, serum metabolic profiles, immunity, and intestinal development in Holstein calves.
      ), whereas others did not observe any differences on weight gain due to milk type (
      • Wray C.
      • Furniss S.
      • Benham C.L.
      Feeding antibiotic-contaminated waste milk to calves - Effects on physical performance and antibiotic-sensitivity of gut flora.
      ;
      • Aust V.
      • Knappstein K.
      • Kunz H.J.
      • Kaspar H.
      • Wallmann J.
      • Kaske M.
      Feeding untreated and pasteurized waste milk and bulk milk to calves: Effects on calf performance, health status and antibiotic resistance of faecal bacteria.
      ). The growth-promoting effect of waste milk is explained by higher nutrient density (
      • Zou Y.
      • Wang Y.
      • Deng Y.
      • Cao Z.
      • Li S.
      • Wang J.
      Effects of feeding untreated, pasteurized and acidified waste milk and bunk tank milk on the performance, serum metabolic profiles, immunity, and intestinal development in Holstein calves.
      ) or antibiotics (
      • Aust V.
      • Knappstein K.
      • Kunz H.J.
      • Kaspar H.
      • Wallmann J.
      • Kaske M.
      Feeding untreated and pasteurized waste milk and bulk milk to calves: Effects on calf performance, health status and antibiotic resistance of faecal bacteria.
      ) in waste milk. However, the nutrient density and antibiotics in waste milk vary in each study, which makes it difficult to determine which factor contributes to the differences in weight gain, therefore quantified antibiotics and milk replacer were used in our study to avoid the instability of waste milk. Although milk bypasses the rumen into the abomasum due to esophageal groove reflex, the disturbances of milk containing antibiotics on the rumen microbes of calves are still reported (
      • Li W.
      • Han Y.
      • Yuan X.
      • Wang G.
      • Wang Z.
      • Pan Q.
      • Gao Y.
      • Qu Y.
      Metagenomic analysis reveals the influences of milk containing antibiotics on the rumen microbes of calves.
      ), which is reasonable because the gastrointestinal ecosystem is open and integrated (
      • Savage D.C.
      Microbial ecology of the gastrointestinal tract.
      ). Rumen development is essential for young ruminants, so this study focused on how antibiotic residues affected growth performances, ruminal fermentation, and rumen microbial community of calves. Our hypothesis was that antibiotic residues would not significantly change the growth performances of calves, but would change the rumen microbe compositions, and thereby would affect the ruminal fermentation.

      MATERIALS AND METHODS

      Antibiotic Residues Testing

      This study was conducted in Dingzhou, Hebei Province, China. The experimental design and procedures were executed according to the protocols approved by the Ethical Committee of the College of Animal Science and Technology, China Agricultural University (no. 2016DR07). Waste milk samples were collected for 3 wk (3 times per week, 9 samples in total) from the dairy farm before the animal trial to determine the types and dosages of antibiotics used in this study. All samples were stored at −20°C and thawed at 4°C for 24 h before testing. According to the treatment protocols, penicillin, streptomycin, tetracycline, and ceftiofur were 4 mostly used antibiotics on the farm; therefore, the concentrations of these antibiotics were tested using a Penicillin ELISA kit (Wdwk Bio Co., Ltd., Beijing, China), Streptomycin ELISA kit (Wdwk Bio Co., Ltd.), Tetracyclines ELISA kit (Wdwk Bio Co., Ltd.), and Ceftiofur ELISA kit (Wdwk Bio Co., Ltd.) separately. The results were used to determine the amounts of antibiotics added in milk for the animal trial.

      Animals, Treatments, and Management

      The animal trial was conducted from October to December in 2016, using a randomized complete block design according to birth date. Twenty Holstein neonatal male calves (40.6 ± 3.3 kg of BW) were equally assigned to 1 of 2 treatments: control group (milk replacer with no antibiotics; CON) and antibiotic group (milk replacer plus 0.024 mg/L of penicillin, 0.025 mg/L of streptomycin, 0.1 mg/L of tetracycline, and 0.33 mg/L of ceftiofur; ANT). All the calves received 4 L of colostrum within 1 h after birth and were moved into individual hutches with free access to starter and water from d 2. Milk replacer was fed twice a day at 0730 and 1600 h from d 2 to 5 in the amount of 2 L/meal, from d 6 to 14 in the amount of 3 L/meal and from d 15 to 35 in the amount of 4 L/meal. The study ended when all calves reached d 35 of age, and 16 of them (8 per treatment) were slaughtered 2 h after the morning feeding. The milk replacer (FrieslandCampina Co., Ltd., Amersfoort, the Netherlands) used in this study contained no antibiotics. One kg of powder formed 8 L of liquid milk under 45 to 55°C; then the milk was cooled down to 38 to 40°C and antibiotics were added before feeding. Starter was offered once daily after milk feeding in the morning. The nutrient compositions of milk replacer and starter are shown in Table 1.
      Table 1Nutrient composition of milk replacer and starter
      Nutrient composition (%)Milk replacerStarter
      DM96.089.1
      CP, DM basis22.022.9
      Ether extract, DM basis20.03.5
      Ash, DM basis9.07.0
      Ca, DM basis0.61.7
      P, DM basis0.60.5
      Lactose, DM basis39.7
      NDF, DM basis21.8
      ADF, DM basis7.5

      Sample Collection

      Starter Intake and Growth Performance Measurements

      Newly fed and refused starter was recorded daily to calculate individual starter intake. Body weight, withers height, body length, and heart girth of each calf were measured on d 1, 7, 14, 28, and 35 before the morning milk feeding.

      Fecal Scoring

      Fecal consistency scores for all calves were determined daily based on a 1 to 4 system according to the guidelines suggested by Larson (
      • Larson L.L.
      • Owen F.G.
      • Albright J.L.
      • Appleman R.D.
      • Lamb R.C.
      • Muller L.D.
      Guidelines toward more uniformity in measuring and reporting calf experimental data.
      ). A fecal score of 3 and above was considered a diarrhea day. Diarrhea frequency was calculated with the following equation weekly:
      diarrhea frequency = [(number of diarrhea calves × days of diarrhea)/(total number of calves × days of trial)] × 100%.


      Rumen Fluid

      Rumen fluid was collected on d 15, 25, and 35 of age by a flexible esophageal tube (2 mm of wall thickness and 6 mm of internal diameter; Anscitech Co., Ltd., Wuhan, Hubei, China) from all calves 4 h after the morning milk feeding. The pH of rumen fluid was tested immediately after sample collection. Rumen liquid fraction was obtained by filtering rumen fluid through 4 layers of cheesecloth and 30 mL of the liquid was stored at −20°C for later analysis of VFA (
      • Erwin E.S.
      • Marco G.J.
      • Emery E.M.
      Volatile fatty acid analyses of blood and rumen fluid by gas chromatography.
      ) and NH3-N (
      • Broderick G.A.
      • Kang J.H.
      Automated simultaneous determination of ammonia and total amino-acids in ruminal fluid and in vitro media.
      ). Ten milliliters of the liquid collected on d 35 from 10 calves (5 per treatment) was stored at −80°C for later analysis of microbial composition and population.

      Rumen Tissues

      On d 35 of age, 16 calves (8 per treatment were randomly selected) were harvested. After removing the digesta, tissues from the rumen were rinsed in saline. Two 1-cm2 rumen tissue samples were removed from the center of each area of cranial ventral sac and cranial dorsal sac (
      • Lesmeister K.E.
      • Tozer P.R.
      • Heinrichs A.J.
      Development and analysis of a rumen tissue sampling procedure.
      ). The tissue samples were then fixed in 4% phosphate-buffered paraformaldehyde solution. After rinsing with water, the samples were dehydrated in a graded series of ethanol (50, 70, 80, 90, and 100%), cleared with xylene twice, and separately embedded in paraffin blocks in a vertical direction to keep the orientation of the rumen papillae the same as the cutting direction when isolating tissue slices. For each block, 5 cuts of 3- to 4-μm-thick sections were isolated and stained with hematoxylin/eosin, resulting in 10 regions of each rumen site for histology analysis. As shown in Figure 1, the 5 longest papillae were selected from each rumen tissue slice for papillae length (PL) and papillae width (PW) measurements through a computerized micrometer according to
      • Malhi M.
      • Gui H.
      • Yao L.
      • Aschenbach J.R.
      • Gäbel G.
      • Shen Z.
      Increased papillae growth and enhanced short-chain fatty acid absorption in the rumen of goats are associated with transient increases in cyclin D1 expression after ruminal butyrate infusion.
      under a microscope (Olympus CKX53, Tokyo, Japan) at 100× magnification.
      Figure thumbnail gr1
      Figure 1Papillae length (PL) and papillae width (PW) measured for rumen papillae through a computerized micrometer at 100× magnification.

      Bacteria Richness

      Ten rumen fluid samples (5 per treatment were randomly selected) on d 35 were collected for microbial analysis. The DNA from samples was extracted using the E.Z.N.A. Bacterial DNA Kit (Omega Bio-Tek, Norcross, GA) according to the manufacturer's instructions. Thirty-nanogram DNA samples were used for 50-µL PCR reaction mixtures. A pair of 2-µL primers with 10 µM forward primer (338F 5′-ACTCCTACGGGAGGCAGCAG-3′) and 10 µM reverse primer (806R 5′-GGACTACHVGGGTWTCTAAT-3′) was used to amplify a region covering the V3–V4 region of bacterial 16S rRNA genes. The PCR conditions were as follows: an initial predenaturation at 95°C for 5 min, denaturation by 28 cycles of 95°C for 30 s, annealing at 56°C for 30 s, elongation at 72°C for 40 s, and then a final extension at 72°C for 10 min and holding at 4°C. Sequencing was done on the Illumina MiSeq platform at Beijing Allwegene Technology Co., Ltd. (Beijing, China). After trimming the adaptor and primer sequences from Illumina reads, the raw sequences were assembled for each sample according to the unique barcode using QIIME (V1.8, http://qiime.org/). Quality filtering was performed under specific filtering conditions to obtain the high-quality clean tags according to QIIME (V1.8, http://qiime.org/). High-quality sequences were clustered into operational taxonomic units (OTU), defined as comprising sequences with less than a 3% difference using UPARSE (V7.0, http://drive5.com/uparse/). Chimeric OTU were removed before further analysis by UCHIME (
      • Edgar R.C.
      Search and clustering orders of magnitude faster than BLAST.
      ). Chao1, Shannon, and observed species were used to estimate α diversity. The OTU were denominated according to the Silva bacteria database (http://www.arb-silva.de).

      Statistical Analysis

      Continuous variables with repeated measurements including starter intake, BW, body measurements, rumen VFA, NH3-N, and pH were analyzed using a mixed effect model with treatment, time, and the interaction between them as fixed effects, and animal within treatment as a random effect. Variables of rumen morphological parameters were also analyzed using a mixed effect model, but with treatment alone as a fixed effect, and animal within treatment as a random effect. Diarrhea frequency was compared using the χ2 test. Variables of microbial parameters were analyzed with the Kruskal-Wallis test. MIXED procedure, FREQ procedure, and NPAR1WAY procedure of SAS 9.2 (SAS Institute Inc., Cary, NC) were used to analyze the data in this study. Significance was indicated at P < 0.05. Data were reported as least squares means. Not all the calves were used for rumen tissue sampling and bacteria richness analysis out of the consideration of budget. The sample sizes of histology analysis and microbial analysis were estimated to obtain a power of 0.8 under a significance level of 0.05.

      RESULTS

      Antibiotic Concentrations in Waste Milk

      The concentrations of penicillin, streptomycin, tetracycline, and ceftiofur residues in waste milk were 0.024 ± 0.034 mg/L, 0.019 ± 0.008 mg/L, 0.08 ± 0.05 mg/L, and 0.76 ± 0.43 mg/L (mean ± SD) separately. The large standard deviations indicated great variations of antibiotic residues; thus, medians (0.024 mg/L of penicillin, 0.025 mg/L of streptomycin, 0.10 mg/L of tetracycline, and 0.33 mg/L of ceftiofur) were used as the concentrations of antibiotics in the animal trial.

      Starter Intake, Body Weight, Body Measurements, and Diarrhea Frequency

      As shown in Table 2, calves fed with either treatment consumed similar amounts of starter (P = 0.58). Antibiotic residues did not affect the BW (P = 0.32), withers height (P = 0.41), body length (P = 0.30), heart girth (P = 0.64), or ADG (P = 0.71). Diarrhea frequency (Figure 2) for calves in the CON group was higher than the ANT group in wk 4 (P = 0.03), and no differences were found in other weeks (P > 0.05).
      Table 2The effect of antibiotic residues on starter intake and growth of dairy calves (n = 10)
      ItemTreatment
      CON = no antibiotics; ANT = antibiotics (0.024 mg/L of penicillin, 0.025 mg/L of streptomycin, 0.1 mg/L of tetracycline, and 0.33 mg/L of ceftiofur) in milk replacer.
      SEMP-value
      CONANTTreatmentTimeTreatment × time
      Starter intake (g/d)75.7692.7422.670.58<0.0010.84
      BW (kg)48.6647.241.020.32<0.0010.93
      Withers height (cm)79.1978.430.670.41<0.0010.02
      Body length (cm)72.6471.740.650.30<0.0010.60
      Heart girth (cm)86.4686.100.570.64<0.0010.99
      ADG (g)487.14503.5733.220.71
      1 CON = no antibiotics; ANT = antibiotics (0.024 mg/L of penicillin, 0.025 mg/L of streptomycin, 0.1 mg/L of tetracycline, and 0.33 mg/L of ceftiofur) in milk replacer.
      Figure thumbnail gr2
      Figure 2The effect of antibiotic residues on diarrhea frequency. CON = no antibiotics; ANT = antibiotics (0.024 mg/L of penicillin, 0.025 mg/L of streptomycin, 0.1 mg/L of tetracycline, and 0.33 mg/L of ceftiofur) in milk replacer (n = 10). *P < 0.05: difference between CON and ANT.

      Ruminal Fermentation and Morphology

      Rumen pH, VFA, and NH3-N were similar between 2 treatments (Table 3), except that calves in the ANT group exhibited a greater concentration of acetic acid than the CON group (P = 0.008). Rumen fermentation was affected by the age of calves (P < 0.01).
      Table 3The effect of antibiotic residues on rumen pH, VFA, and NH3-N (n = 10)
      ItemTreatment
      CON = no antibiotics; ANT = antibiotics (0.024 mg/L of penicillin, 0.025 mg/L of streptomycin, 0.1 mg/L of tetracycline, and 0.33 mg/L of ceftiofur) in milk replacer.
      SEMP-value
      CONANTTreatmentTimeTreatment × time
      pH6.296.320.150.86<0.0010.81
      Acetic acid (mmol/L)24.8633.731.820.008<0.0010.09
      Propionic acid (mmol/L)18.5915.262.350.32<0.0010.28
      Butyric acid (mmol/L)5.643.760.910.17<0.0010.03
      Isovaleric acid (mmol/L)0.820.650.150.470.0050.38
      Valeric acid (mmol/L)1.340.930.310.35<0.0010.41
      Total VFA (mmol/L)52.1753.964.220.77<0.0010.09
      NH3-N (mg/dL)5.745.280.920.74<0.0010.52
      1 CON = no antibiotics; ANT = antibiotics (0.024 mg/L of penicillin, 0.025 mg/L of streptomycin, 0.1 mg/L of tetracycline, and 0.33 mg/L of ceftiofur) in milk replacer.
      Effects of antibiotic residues on the PL and PW are presented in Table 4. Calves in the ANT group exhibited a longer PL at the cranial ventral sac than the CON group (P = 0.04). No differences were found for PL at the cranial dorsal sac or PW in both sacs (P > 0.05).
      Table 4The effect of antibiotic residues on rumen papillae length and width (n = 8)
      Item
      PL = papillae length; PW = papillae width; CVS = cranial ventral sac; CDS = cranial dorsal sac.
      Treatment
      CON = no antibiotics; ANT = antibiotics (0.024 mg/L of penicillin, 0.025 mg/L of streptomycin, 0.1 mg/L of tetracycline, and 0.33 mg/L of ceftiofur) in milk replacer.
      SEMP-value
      CONANT
      PL (μm)
       CVS346.54451.2732.770.04
       CDS375.66389.5836.990.79
      PW (μm)
       CVS112.26126.487.440.20
       CDS105.43120.797.200.15
      1 PL = papillae length; PW = papillae width; CVS = cranial ventral sac; CDS = cranial dorsal sac.
      2 CON = no antibiotics; ANT = antibiotics (0.024 mg/L of penicillin, 0.025 mg/L of streptomycin, 0.1 mg/L of tetracycline, and 0.33 mg/L of ceftiofur) in milk replacer.

      Diversity of Rumen Bacteria

      Chao1 index, Shannon index, and observed species were used to evaluate the diversity of rumen bacteria (Figure 3). No differences were found for Chao1 index, Shannon index, or observed species (P > 0.05).
      Figure thumbnail gr3
      Figure 3The effect of antibiotic residues on the α diversity of rumen bacteria on d 35. CON = no antibiotics; ANT = antibiotics (0.024 mg/L of penicillin, 0.025 mg/L of streptomycin, 0.1 mg/L of tetracycline, and 0.33 mg/L of ceftiofur) in milk replacer (n = 5). The central rectangle spans the first quartile to the third quartile. The segment inside the rectangle shows the median, and whiskers above and below the box show the locations of the minimum and maximum.

      Bacteria Richness

      Bacteroidetes, Firmicutes, Proteobacteria, Synergistetes, and Actinobacteria were 5 major bacterial phyla in rumen fluid of calves for both groups, accounting for more than 98% of the total rumen bacterial community (Figure 4). No differences were found in phyla relative abundance (P > 0.05) with addition of antibiotics.
      Figure thumbnail gr4
      Figure 4The effect of antibiotic residues on the relative abundance (%) of the most abundant rumen bacterial phyla. CON = no antibiotics; ANT = antibiotics (0.024 mg/L of penicillin, 0.025 mg/L of streptomycin, 0.1 mg/L of tetracycline, and 0.33 mg/L of ceftiofur) in milk replacer (n = 5).
      The rumen bacteria richness at genus level was shown in Figure 5. The 10 most common rumen bacteria at genus level for the CON group were Prevotella, Selenomonas, Succinivibrio, unidentified, U29-B03, Ruminobacter, Ruminococcaceae UCG-014, Prevotellaceae NK3B31 group, Lachnospiraceae NK3A20 group, and Succiniclasticum. The 10 most common rumen bacteria at genus level for the ANT group were Succinivibrio, Prevotella, Lachnospiraceae NK3A20 group, unidentified, Olsenella, Succiniclasticum, Atopobium, Megasphaera, Acetitomaculum, and Bacteroides. Antibiotic residues decreased the relative abundance of Prevotella (P = 0.036) and increased the relative abundance of Acetitomaculum (P = 0.046) in the rumen. No differences were observed for other species (P > 0.05).
      Figure thumbnail gr5
      Figure 5The effect of antibiotic residues on the relative abundance (%) of the most abundant rumen bacterial genera. CON = no antibiotics; ANT = antibiotics (0.024 mg/L of penicillin, 0.025 mg/L of streptomycin, 0.1 mg/L of tetracycline, and 0.33 mg/L of ceftiofur) in milk replacer. *P < 0.05 (n = 5). The error bars represent the SD.

      DISCUSSION

      Our study investigated the effects of antibiotic residues on the growth, rumen fermentation, rumen histology, and rumen bacteria, and tried to find the connections between each aspect.

      Growth and Health

      The growth-promoting effect of antibiotics was explained by the modification of intestinal microbiota for a healthy intestinal environment (
      • Gaskins H.R.
      • Collier C.T.
      • Anderson D.B.
      Antibiotics as growth promotants: Mode of action.
      ), or the inhibition of immune functions to save energy for growth purposes (
      • Roura E.
      • Homedes J.
      • Klasing K.C.
      Prevention of immunological stress contributes to the growth-permitting ability of dietary antibiotics in chicks.
      ;
      • Bhandari S.K.
      • Xu B.
      • Nyachoti C.M.
      • Giesting D.W.
      • Krause D.O.
      Evaluation of alternatives to antibiotics using an Escherichia coli K88(+) model of piglet diarrhea: Effects on gut microbial ecology.
      ). However, this effect is more likely to occur when animals are poorly managed and antibiotics are fed at high concentrations (
      • Langford F.M.
      • Weary D.M.
      • Fisher L.
      Antibiotic resistance in gut bacteria from dairy calves: A dose response to the level of antibiotics fed in milk.
      ). No significant differences were observed on growth or starter intake in our study, which was consistent with previous studies reporting that milk containing subtherapeutic antibiotics does not influence the growth of animals (
      • Langford F.M.
      • Weary D.M.
      • Fisher L.
      Antibiotic resistance in gut bacteria from dairy calves: A dose response to the level of antibiotics fed in milk.
      ;
      • Thames C.H.
      • Pruden A.
      • James R.E.
      • Ray P.P.
      • Knowlton K.F.
      Excretion of antibiotic resistance genes by dairy calves fed milk replacers with varying doses of antibiotics.
      ).
      The effect of antibiotic residues on diarrhea was minor according to previous studies (
      • Langford F.M.
      • Weary D.M.
      • Fisher L.
      Antibiotic resistance in gut bacteria from dairy calves: A dose response to the level of antibiotics fed in milk.
      ;
      • Thames C.H.
      • Pruden A.
      • James R.E.
      • Ray P.P.
      • Knowlton K.F.
      Excretion of antibiotic resistance genes by dairy calves fed milk replacers with varying doses of antibiotics.
      ).
      • Brunton L.A.
      • Reeves H.E.
      • Snow L.C.
      • Jones J.R.
      A longitudinal field trial assessing the impact of feeding waste milk containing antibiotic residues on the prevalence of ESBL-producing Escherichia coli in calves.
      reported a lower diarrhea incidence for calves fed with waste milk containing antibiotic residues compared with those fed with milk replacer, but they attributed this effect to individual differences and group housing. There is not enough evidence to support the diarrhea-reducing effect of antibiotic residues, thus the lower diarrhea frequency observed for the ANT group in wk 4 was probably due to individual differences as well.

      Rumen Fermentation and Rumen Histology

      Rumen pH is affected by several factors, including the concentration of VFA and NH3-N, the secretion of saliva, and outflow of rumen fluid. Lower pH can promote rumen epithelium to absorb VFA and accelerate rumen development (
      • Baldwin R.L.V.
      • McLeod K.R.
      Effects of diet forage:concentrate ratio and metabolizable energy intake on isolated rumen epithelial cell metabolism in vitro.
      ). In this study, the rumen pH of newborn calves was high because of low feed intake and inactivity of rumen, and it decreased with age, which was relative to the increase of starter intake (
      • Robinson P.H.
      • Tamminga S.
      • Van Vuuren A.M.
      Influence of declining level of feed intake and varying the proportion of starch in the concentrate on rumen fermentation in dairy cows.
      ).
      Antibiotics are believed to be able to inhibit rumen fermentation through suppressing rumen microbes (
      • Owens F.N.
      • Basalan M.
      Ruminal fermentation.
      ). However, we observed a greater concentration of acetic acid for calves fed with antibiotics.
      • Wasserman R.H.
      • Duncan C.W.
      • Churchill E.S.
      • Huffman C.F.
      The effect of antibiotics on in vitro cellulose digestion by rumen microorganisms.
      suggested that penicillin and streptomycin suppressed fiber degradation at high concentrations while promoting fiber degradation and increasing acetic acid concentration at low concentrations in vitro. Low concentrations of penicillin could also increase the proportion of acetic acid without significantly changing total VFA in vitro (
      • De Jong A.
      In vitro and in vivo alterations in ruminal volatile fatty acids by antimicrobial compounds.
      ).
      • Xiong Y.
      • Harb M.
      • Hong P.Y.
      Performance and microbial community variations of anaerobic digesters under increasing tetracycline concentrations.
      reported that tetracycline increased acetic acid production under anaerobic conditions. Our results along with previous studies (
      • Wasserman R.H.
      • Duncan C.W.
      • Churchill E.S.
      • Huffman C.F.
      The effect of antibiotics on in vitro cellulose digestion by rumen microorganisms.
      ;
      • De Jong A.
      In vitro and in vivo alterations in ruminal volatile fatty acids by antimicrobial compounds.
      ;
      • Xiong Y.
      • Harb M.
      • Hong P.Y.
      Performance and microbial community variations of anaerobic digesters under increasing tetracycline concentrations.
      ) showed that a low concentration of several antibiotics increased the production of acetic acid in the rumen.
      Ruminal environment has a great influence on the development of rumen papillae, therefore daily ration changes affect the development of the rumen significantly (
      • Lesmeister K.E.
      • Tozer P.R.
      • Heinrichs A.J.
      Development and analysis of a rumen tissue sampling procedure.
      ). Volatile fatty acids stimulate the development of rumen papillae and epithelia and thus are considered one of the most important factors affecting rumen development (
      • Tamate H.
      • McGilliard A.D.
      • Jacobson N.L.
      • Getty R.
      Effect of various dietaries on the anatomical development of the stomach in the calf.
      ). Acetate, propionate, and butyrate are all able to stimulate rumen development (
      • Sander E.G.
      • Warner R.G.
      • Harrison H.N.
      • Loosli J.K.
      The stimulatory effect of sodium butyrate and sodium propionate on the development of rumen mucosa in the young calf.
      ;
      • Sakata T.
      • Tamate H.
      Rumen epithelium cell proliferation accelerated by propionate and acetate.
      ). Although ANT group had higher acetic acid concentration, the total VFA concentration was not different from the CON group, and thus the longer PL at the cranial ventral sac of calves in ANT group might be due to other reasons.
      • Niwińska B.
      • Strzetelski J.
      Effects of type of liquid feed and feeding frequency on rumen development and rearing performance of calves.
      and
      • Górka P.
      • Kowalski Z.M.
      • Pietrzak P.
      • Kotunia A.
      • Jagusiak W.
      • Zabielski R.
      Is rumen development in newborn calves affected by different liquid feeds and small intestine development?.
      did not find direct connections between the concentration of rumen VFA and the development of rumen papillae as well. Insulin appears to affect the absorptive surface of the ruminal wall (
      • Hugi D.
      • Bruckmaier R.M.
      • Blum J.W.
      Insulin resistance, hyperglycemia, glucosuria, and galactosuria in intensively milk-fed calves: Dependency on age and effects of high lactose intake.
      ), so the analysis of blood insulin level could possibly help explain the longer PL caused by antibiotic residues in the future.

      Diversity of Rumen Bacteria

      We did not observe any differences on Chao1 index, Shannon index, or observed species between the 2 treatments. Similar results were reported by
      • Van Vleck Pereira R.V.V.
      • Lima S.
      • Siler J.D.
      • Foditsch C.
      • Warnick L.D.
      • Bicalho R.C.
      Ingestion of milk containing very low concentration of antimicrobials: Longitudinal effect on fecal microbiota composition in preweaned calves.
      , who fed calves with a different combination of antibiotics but at similar dosage to our study and found no difference in the diversity of feces microbiota. The inhibition of antibiotics on diversity of intestinal microbiota was reported in several studies (
      • Keesing F.
      • Belden L.K.
      • Daszak P.
      • Dobson A.
      • Harvell C.D.
      • Holt R.D.
      • Hudson P.
      • Jolles A.
      • Jones K.E.
      • Mitchell C.E.
      • Myers S.S.
      Impacts of biodiversity on the emergence and transmission of infectious diseases.
      ;
      • Livanos A.E.
      • Greiner T.U.
      • Vangay P.
      • Pathmasiri W.
      • Stewart D.
      • McRitchie S.
      • Li H.
      • Chung J.
      • Sohn J.
      • Kim S.
      • Gao Z.
      • Barber C.
      • Kim J.
      • Ng S.
      • Rogers A.B.
      • Sumner S.
      • Zhang X.
      • Cadwell K.
      • Knights D.
      • Alekseyenko A.
      • Backhed F.
      • Blaser M.J.
      Antibiotic-mediated gut microbiome perturbation accelerates development of type 1 diabetes in mice.
      ), but the dosages used in this study might not be enough to cause a difference on the diversity of rumen bacteria.

      Bacteria Richness

      The 3 most common bacteria phyla, Bacteroidetes, Firmicutes, and Proteobacteria, were observed for both groups in this study. The same rumen bacteria for dairy calves were reported as the dominant phyla by previous studies (
      • Li R.W.
      • Connor E.E.
      • Li C.
      • Baldwin V.I.
      • Ransom L.
      • Sparks M.E.
      Characterization of the rumen microbiota of pre-ruminant calves using metagenomic tools.
      ;
      • Rey M.
      • Enjalbert F.
      • Combes S.
      • Cauquil L.
      • Bouchez O.
      • Monteils V.
      Establishment of ruminal bacterial community in dairy calves from birth to weaning is sequential.
      ), and Bacteroidetes, Firmicutes, and Proteobacteria are also taxonomic groups represented within the cattle gastrointestinal tract (
      • Mao S.
      • Zhang M.
      • Liu J.
      • Zhu W.
      Characterising the bacterial microbiota across the gastrointestinal tracts of dairy cattle: Membership and potential function.
      ). Overall, a limited influence of antibiotic residues on rumen bacteria was observed at the phylum level in our study. Additionally,
      • Van Vleck Pereira R.V.V.
      • Lima S.
      • Siler J.D.
      • Foditsch C.
      • Warnick L.D.
      • Bicalho R.C.
      Ingestion of milk containing very low concentration of antimicrobials: Longitudinal effect on fecal microbiota composition in preweaned calves.
      reported that fecal bacteria phyla are not significantly changed by antibiotic residues.
      The compositions of rumen bacteria at the genus level were altered by antibiotic residues. As one of core bacterial microbiome at the genus level (
      • Henderson G.
      • Cox F.
      • Ganesh S.
      • Jonker A.
      • Young W.
      • Janssen P.H.
      Global Rumen Census Collaborators
      Rumen microbial community composition varies with diet and host, but a core microbiome is found across a wide geographical range.
      ), Prevotella was the only bacteria genus whose relative abundance was decreased by antibiotic residues, which might be due to the susceptibility of Prevotella to penicillin (
      • Falagas M.E.
      • Siakavellas E.
      Bacteroides, Prevotella, and Porphyromonas species: A review of antibiotic resistance and therapeutic options.
      ) and tetracycline (
      • Niederau W.
      • Höffler U.
      • Pulverer G.
      Susceptibility of Bacteroides melaninogenicus to 45 antibiotics.
      ). As the most abundant bacteria genus, the greatest exposure of Prevotella to the antibiotics might contribute to its lower abundance. The decreased relative abundance of Prevotella was also observed in fecal bacteria for elderly people after antibiotic treatment (
      • Bartosch S.
      • Fite A.
      • Macfarlane G.T.
      • McMurdo M.E.
      Characterization of bacterial communities in feces from healthy elderly volunteers and hospitalized elderly patients by using real-time PCR and effects of antibiotic treatment on the fecal microbiota.
      ).
      Limited studies have investigated the effect of antibiotics on Acetitomaculum.
      • Hill D.A.
      • Hoffmann C.
      • Abt M.C.
      • Du Y.
      • Kobuley D.
      • Kirn T.J.
      • Bushman F.D.
      • Artis D.
      Metagenomic analyses reveal antibiotic-induced temporal and spatial changes in intestinal microbiota with associated alterations in immune cell homeostasis.
      reported a decreased relative abundance of Acetitomaculum in the colon after antibiotic treatment for mice, which is contrary to our results, probably because they used a different combination of antibiotics and a higher dosage.
      • Carey D.E.
      • Zitomer D.H.
      • Kappell A.D.
      • Choi M.J.
      • Hristova K.R.
      • McNamara P.J.
      Chronic exposure to triclosan sustains microbial community shifts and alters antibiotic resistance gene levels in anaerobic digesters.
      added triclosan to anaerobic digesters and observed very close results to ours in terms of microbial community. They reported that high concentration of triclosan selected for the phyla Actinobacteria and Firmicutes, with Firmicutes as the most abundant phylum in triclosan-containing digesters. Firmicutes was also the most abundant phylum for calves treated with antibiotics in our study. In addition, they observed that Succinivibrio, Atopobium, Olsenella, and Acetitomaculum were enriched in the digesters treated with triclosan. In our study, Atopobium and Olsenella were among the 10 most abundant genera for the ANT group but not for the CON group, and Acetitomaculum was enriched for calves fed with antibiotic residues. As a synthetic antimicrobial agent, triclosan is not an antibiotic but has antibiotic properties (
      • Dhillon G.S.
      • Kaur S.
      • Pulicharla R.
      • Brar S.K.
      • Cledón M.
      • Verma M.
      • Surampalli R.Y.
      Triclosan: Current status, occurrence, environmental risks and bioaccumulation potential.
      ). The consistency between the 2 studies indicated a similar regulation of triclosan and the antibiotic residues in this study on the microbial community. The selectiveness of the antibiotic residues on rumen bacteria needs further study.
      The increase of the relative abundance of Acetitomaculum for the ANT group might thereby contribute to the higher concentration of acetic acid because Acetitomaculum is a member of acetogenic bacteria (
      • Le Van T.D.
      • Robinson J.A.
      • Ralph J.
      • Greening R.C.
      • Smolenski W.J.
      • Leedle J.A.
      • Schaefer D.M.
      Assessment of reductive acetogenesis with indigenous ruminal bacterium populations and Acetitomaculum ruminis.
      ). On the other hand, Prevotella is hydrogen-consuming bacterium and can produce propionate through the fermentation of sugars or lactate (
      • Li Z.P.
      • Liu H.L.
      • Li G.Y.
      • Bao K.
      • Wang K.Y.
      • Xu C.
      • Yang Y.F.
      • Yang F.H.
      • Wright A.D.G.
      Molecular diversity of rumen bacterial communities from tannin-rich and fiber-rich forage fed domestic Sika deer (Cervus nippon) in China.
      ). The decrease of the abundance of Prevotella did not significantly decrease the propionic acid concentration for the ANT group, but could possibly eliminate the difference on total VFA concentration, given that the ANT group had a higher acetic acid concentration but a similar total VFA concentration as the CON group.

      CONCLUSIONS

      The antibiotic residues had no significant effects on the growth of calves. This might suggest that antibiotic residue in waste milk is not a factor affecting the growth or health of calves. The antibiotic residues increased PL in the rumen cranial ventral sac, the reason for which is unclear. The antibiotic residues had limited effects on rumen bacteria, but increased the relative abundance of Acetitomaculum, which might contribute to the increased concentration of acetic acid in the rumen.

      ACKNOWLEDGMENTS

      This work was supported by Nutrifeed (Friesland Campina, the Netherlands; no. 2016DR07) and National Key Research and Development Program of China (2018YFD0501600). The first author is grateful for a scholarship from the Chinese Scholarship Council (2013GXZ707). We also thank SUNLUN Livestock, Dingzhou (Hebei, China) for allowing us to use their animals and facilities.

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