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Succession of ruminal bacterial species and fermentation characteristics in preweaned Brangus calves

Succession of ruminal bacterial species and fermentation characteristics in preweaned Brangus calves Downloaded from https://academic.oup.com/tas/article-abstract/2/suppl_1/S48/5108313 by Ed 'DeepDyve' Gillespie user on 16 October 2018 Succession of ruminal bacterial species and fermentation characteristics in preweaned Brangus calves ,2 † Kathryn E. Smith,* Anna L. Garza,* Kylie M. Butterfield, * Aaron M. Dickey, † † † Amanda K. Lindholm-Perry, James E. Wells, Harvey C. Freetly, and Shanna L. Lodge-Ivey* *Department of Animal and Range Sciences, New Mexico State University, Las Cruces, NM and USDA, ARS, U.S. Meat Animal Research Center, Clay Center, NE © The Author(s) 2018. Published by Oxford University Press on behalf of the American Society of Animal Science. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com. Transl. Anim. Sci. 2018.2:S48–S52 doi: 10.1093/tas/txy043 2004). Butyrate is especially important in rumen INTRODUCTION development and has been linked to papillae Historically, rumen development has been development in the rumen (Coverdale et  al., defined by anatomical change, fermentation end 2004). Ruminal ammonia is vital for adequate products, and cultured media for identification microbial growth, therefore important for of bacterial composition (Tamate et  al., 1962; microbial and fermentation development Fonty et al., 1987). The rumen ecosystem from (Satter and Slyter, 1974). Thus, the objectives 1 d to 2 yr of age declines in aerobic and faculta- of this study are to assess the composition of tive anaerobic taxa, while increasing anaerobic the ruminal bacteria and the production of fer- taxa (Jami et al., 2013). Li et al (2012) reported mentation end products in preweaned Brangus Bacteroidetes as the prominent phylum in 42-d- calves as they age. We hypothesize that ruminal old calves, different from 14-d-old calves. This VFA, ammonia, and bacterial populations will research has focused on dairy calves, which wean increase as calves age. earlier, live in different environments, and likely has a different rumen developmental timeline in MATERIALS AND METHODS comparison to beef calves. All experimental procedures were approved The energetic efficiency of a cow convert- by New Mexico State University Institutional ing grass to milk and the calf converting milk Animal Care and Use Committee (Protocol 2017- to retained energy is less efficient than directly 001). Eleven Brangus calves born in February and converting forage to retained energy (Freetly March of 2015 were sampled at the Chihuahuan et  al., 2006). Intake of solid food increases Desert Rangeland Research Center (4.1% CP, ruminal microbial activity and higher concen- 72.5% NDF) in Las Cruces, NM. Cow–calf pairs tration of total VFAs produced that can be were housed on native rangeland with grasses utilized for retained energy (Coverdale et  al., including Black grama (Bouteloua eriopoda), three awns (Aristida), dropseeds (Sporobolus), burrograss (Scleropogon brevifolius), and tobosa USDA is an equal opportunity provider and employer. 2 (Pleuraphis mutica). Ruminal samples were col- Corresponding author: ksmith92@nmsu.edu lected via oral lavage (Lodge-Ivey et al., 2009) for Received March 16, 2018. microbiota, VFA, and ammonia analysis on 7, 35, Accepted April 14, 2018. S48 Downloaded from https://academic.oup.com/tas/article-abstract/2/suppl_1/S48/5108313 by Ed 'DeepDyve' Gillespie user on 16 October 2018 Ruminal bacterial species and fermentation characteristics in calves S49 63, 91, 119, 147, 175  ±  5 d of age. Samples were 9.4; SAS Inst. Inc., Cary, NC) with repeated meas- immediately flash frozen in liquid Nitrogen and ures for all data. Animal was the experimental unit, stored at −80 °C. and the treatment was day of age. Using Akaike’s Ruminal ammonia was analyzed using the phe- information criterion, compound symmetry was nol-hypochlorite procedure adapted to a microti- the covariance structure. Means were calculated ter plate (Broderick and Kang-Meznarich, 1980). using LSMEANS. Day effects were considered sig- Volatile fatty acid concentration was determined by nificant at a P ≤ 0.05, and mean separations were gas chromatography utilizing methods of May and performed using PDIFF. Galyean (1996). The DNA was extracted from sam- ples using a repeated beating plus column (RBB RESULTS + C) method (Yu and Morrison, 2004) followed by 1  mL of lysis buffer using the QIAmp DNA The effect of calf age at the phylum level is Stool Mini Kit (Qiagen, Valencia, CA), and qual- summarized in Table  1. Bacteroidetes, Firmicutes, ity and quantity of DNA were determined using a Proteobacteria, SRI, TM7, Tenericutes, Nanodrop (Thermo Scientific, Marietta, OH). Verrucomicrobia, and Unclassified phyla differed The V1–V3 region of 16S rRNA gene was based on age of calf (P < 0.02). Bacteroidetes was amplified, using modified universal primers most abundant at 7 (40.3%), 147 (38.2%), and 175 27F (5′ Adapter/Index/AGAGTTTGAGCCG (52.8%) d of age (P  <  0.01). The lowest Shannon GGCGCAG) and 519R (5′ Adapter/Index/ Wiener Index (SWI) and richness occurred at 7 d GTATTACCGCGGCTGCGA) including TruSeq of age and increased as the calves aged (P < 0.05). adapter sequences and indices. The PCR amplifi - Table  2 summarizes the effect of calf age at cation was performed on Bio-Rad Dyad Thermal the genera level. Richness data identified 214 gen - Cycler (Bio-Rad). Following purification, PCR era however; not all OTUs are represented in the products were diluted to approximately 4 nM, and database and therefore were not able to be classi- PCR amplicons libraries were paired-end sequenced fied at the genera level. Unidentified genera are using the Illumina MiSeq. Sequence data were pro- presented as the lowest taxonomic unit identified. cessed using MICCA pipeline for metagenomic Prevotella increased throughout the study and was analysis. Operational taxonomic units (OTUs) were most abundant at 175 d of age (P < 0.01). Family generated after removing <150 bp sequences. Final BS11, BF311, Prevotella, RF16, Fibrobacter, family OTUs were clustered by 97% similarity and clas- Lachnospiraceae, family Veillonellaceae, unclas- sified using BLASTn against a curated database sified RS1, order LD1-PB3, and unclassified gen - derived from GreenGenes. era differed based on age of calf (P < 0.02). Order All data were analyzed as a completely random Bacteroidales was not affected by age of calf design using the MIXED procedure of SAS (version (P  =  0.16). Genera richness increased (P  <  0.01) Table 1. Effect of age of Brangus calf on phyla composition, Shannon Weiner Index, and richness Days of age † ‡ Phylum , % 7 35 63 91 119 147 175 SEM P value a a b b b b a Bacteroidetes 40.3 48.3 37.6 40.0 37.8 38.2 52.8 3.2 <0.01 a,c a,c a,b b a,b a,b c Firmicutes 26.5 27.4 31.6 35.2 33.9 32.3 23.5 2.95 <0.01 a b b b b b b Proteobacteria 3.9 2.2 2.3 2.8 2.8 2.9 2.5 0.42 <0.01 a,c b b,d c c b,d d SR1 1.2 8.3 7.4 2.1 2.5 6.1 5.1 1.28 <0.01 a b a b b b a TM7 0.9 1.9 1.3 2.3 2.7 2.4 1.0 0.40 <0.01 a a a a a a a Tenericutes 4.3 3.9 4.9 4.2 4.9 5.3 2.3 0.83 0.01 a b a a a a a,b Verrucomicrobia 4.9 1.0 4.7 5.8 6.6 5.7 3.8 1.26 0.02 a b,c b b,c b,c c b,c Unclassified 10.0 4.8 6.9 5.4 6.0 4.2 4.1 1.12 <0.01 a b b,c b c,d d d Shannon Weiner Index 1.9 2.5 2.6 2.5 2.6 2.8 3.0 0.08 <0.01 a b c b,c,d d,e d,e e Richness 10.6 13.5 14.9 13.7 15.5 15.5 16.5 0.49 <0.01 Twenty-three phyla were identified. The most abundant phyla are listed in the above table. n = 11. a–e Values within rows with differing superscripts differ, P ≤ 0.05, between days of age. Translate basic science to industry innovation Downloaded from https://academic.oup.com/tas/article-abstract/2/suppl_1/S48/5108313 by Ed 'DeepDyve' Gillespie user on 16 October 2018 S50 Smith et al. Table 2. Effect of age of Brangus calf on genera composition, Shannon Weiner Index, and richness Days of age † ‡ Genera , % 7 35 63 91 119 147 175 SEM P value a,b a,b a,b a,b a a b Family BS11 1.0 1.7 2.7 1.9 3.6 3.1 8.5 1.51 <0.01 BF311 1.5 0.9 0.8 0.5 0.5 0.7 0.9 0.28 0.16 a a a a a a b Prevotella 13.4 14.1 14.9 13.9 14.8 13.9 27.1 2.6 <0.01 a,b a a,b a,b a,b a,b b Family RF16 0.9 0.8 1.4 2.3 2.2 1.4 2.6 0.72 <0.01 Family S24-7 0.8 1.8 2.6 1.1 0.9 2.0 2.3 0.69 0.35 Order Bacteroidales 12.9 16.1 9.1 10.2 10.2 14.5 8.7 2.34 0.16 CF231 6.7 5.3 3.1 4.6 4.0 3.0 1.5 1.06 0.47 YRC22 0.1 0.2 0.3 0.4 03 0.4 0.8 0.35 0.08 Fibrobacter 0.5 0.2 0.9 0.5 0.4 0.6 0.8 0.28 0.10 a a a b b a,b a Family Lachnospiracea 3.3 3.7 3.2 5.2 5.0 4.2 3.0 0.56 <0.01 Family Veillonellaceae 0.0 0.2 1.3 1.0 1.0 0.8 1.2 0.59 0.04 a a,b a,b a,b a,b a,b b RFN20 1.5 0.7 1.0 0.6 1.0 1.2 0.6 0.36 <0.01 Order RF32 0.1 0.3 0.5 0.3 0.6 0.3 0.4 0.15 0.43 a b b a a a a Unclassified RS1 1.2 8.3 7.4 1.9 2.5 5.8 3.9 1.32 <0.02 Order RF39 3.5 3.6 4.2 3.6 4.1 3.9 1.9 0.84 0.24 a a,c b,c b b b b Order LD1-PB3 0.6 0.1 3.8 4.4 5.8 4.8 1.8 1.03 <0.01 Family RFP12 2.2 0.9 0.7 0.8 0.6 0.7 1.5 0.54 0.38 a b,c b b,c b,c c c Unclassified 10.0 4.8 6.8 5.5 6.0 3.9 4.1 1.10 <0.01 a b b,d c b,c c,d c,d Shannon Weiner Index 4.0 4.6 4.7 5.3 4.9 5.0 5.1 0.14 <0.01 a a,b b,c c c c b,c Richness 40.6 44.1 48.5 50.7 51.7 52.4 48.4 1.79 <0.01 Two hundred and fourteen genera were identified. The most abundant genera are listed in the above table. n = 11. a–d Values within rows with differing superscripts differ, P ≤ 0.05, between days of age. Table 3. Age of Brangus calf effects on ruminal volatile fatty acids and ammonia Days of age Item 7 35 63 91 119 147 175 SEM P value a a,b b,c b,c c c a Total VFA, mM 45.1 61.9 76.6 79.0 82.8 85.5 49.3 6.69 <0.01 VFA, mol/100 mol a b b b b,c,d c,d d Acetate 79.3 75.7 75.9 76.4 75.0 73.4 71.9 0.83 <0.01 a,b a b b a,b a b Propionate 14.3 15.3 13.9 14.7 14.7 15.2 13.5 0.41 <0.01 a a a a a b c Isobutyrate 1.2 1.1 1.2 1.1 1.2 1.7 2.7 0.14 <0.01 a b b,c b,c b,c b,c c Buytrate 4.2 7.0 7.6 7.8 8.1 8.0 8.6 0.53 <0.01 a b a,c a,c b,c b a,b,c Acetate:Propionate 5.7 5.0 5.5 5.5 5.1 4.9 5.4 0.20 <0.01 a b b,d c c c d Ammonia, mM 10.2 6.6 5.4 2.9 2.7 3.0 4.8 0.68 <0.01 n = 11. a–d Values within rows with differing superscripts differ, P ≤ 0.05, between days of age. from 7 to 147 d of age and with a slight decrease increased as calves aged with the greatest butyr- ate concentration at day 175 (P  <  0.01). Ruminal denoted at 175 d of age. The lowest SWI occurred ammonia was greatest on day 7 and remained below at 7 d of age (P < 0.01) and the 91, 119, 147, and 6.6 mM for the remainder of the study (P < 0.01). 175 d of age did not differ (P ≥ 0.05). Table  3 summarizes the effect of calf age on DISCUSSION ruminal VFA and ammonia production. Total VFA production was similar for days 7, 35, and 175 and Limited data exist on beef calf ruminal develop- was lower than days 63, 91, 119, and 147 (P < 0.01). ment in extensive landscapes. In this study, ruminal Acetate was greatest and butyrate was lowest at day fluid was examined for bacterial composition and 7 (P < 0.01). Propionate was greater on days 7, 35, fermentation end products and generated conflict - 119, and 147 and was least on days 63, 91, and 175 ing results compared with dairy calf data. One area (P < 0.01). Acetate:Propionate was greatest on days of agreement was bacterial composition at the phy- 7, 63, 91, and 175 (P < 0.01). Butyrate production lum level. In this study, Bacteroidetes and Firmicutes Translate basic science to industry innovation Downloaded from https://academic.oup.com/tas/article-abstract/2/suppl_1/S48/5108313 by Ed 'DeepDyve' Gillespie user on 16 October 2018 Ruminal bacterial species and fermentation characteristics in calves S51 comprised approximately 72.2% of the rumen bac- raised in conventional systems have an opportu- terial population, agreeing with previously reported nity to capitalize on body weight gain and feed effi - data from Jami et  al. (2013). Approximately 70% ciency due to consumption of both available forage of the rumen bacterial population comprised and dams’ milk. Bacteroidetes and Firmicutes in 2-mo-old dairy Day 7 showed the lowest total VFA levels, and calves (Jami et  al., 2013). Rumen liquid fractions these are indicative of a low structural carbohy- commonly show increased Bacteroidetes popula- drate diet (Asai et al., 1973). However, by day 35, tions compared with Firmicutes (Pitta et al., 2010). total VFA production increases to 61.9  mM only In the current study, ruminal contents were col- to decrease to 49.3 mM on day 175. Forage quality lected via a suction strainer, and samples should be of New Mexico rangeland is greatest during July similar to the liquid fractions commonly reported and August due to monsoon-type rainfall events. in studies that strain the ruminal contents through During 2015, less than average rainfall occurred cheesecloth (Pitta et  al., 2010). Pitta et  al. (2010) during July and August; thus by September and reported increased Bacteroidetes populations com- October (day 175), forage quality declined, result- pared with Firmicutes, which is in agreement in ing in low total VFA production. with our study where Bacteroidetes population was The greatest ruminal ammonia occurred on day 3.9% to 29.3% greater than Firmicutes. 7 when calves were primarily consuming milk. Milk In 42-d-old calves, Firmicutes comprised may enter the rumen causing increased ruminal 12% of the population (Li et  al., 2012), differing ammonia (Jami et al., 2013). Ruminal ammonia con- from the present study where Firmicutes ranged centration of 2 mM is needed for maximum micro- from 27.4% to 31.6% from day 35 to 63 of age. bial growth rate (Satter and Slyter, 1974). Despite Calves fed milk replacer have decreased levels low protein and high structural carbohydrates that of Firmicutes compared with calves grazing on are typical of rangelands, calf rumen function was native rangeland (Li et al., 2012). Beef calves that not impaired for the duration of this experiment. are raised on rangelands may consume forage at Ammonia production is vital for developing calves’ earlier ages, leading to increased Firmicutes pop- nutrient synchrony. Microbial, VFA, and ammonia ulation. Bacterial genera associated with fiber data support the theory that calves are consuming degradation in the phylum Firmicutes include available forage, and microbial population makes Butyrivibrio, Ruminococcus, and Succiniclasticum use of these feedstuffs. Hollingsworth-Jenkins et al. (Latham et al., 1978). In dairy calves ranging from (1994) support this conclusion, where calves raised 14 d old to 12 mo, Ruminococcus maintained a in Sandhills region of Nebraska consumed a diet low abundance ranging from 0.3% to 3.6% of the primarily of forage prior to weaning. Calves con- population (Li et al., 2012). The low abundance of suming forage are potentially more efficient at feed Ruminococcus, in the present study, is unexpected conversion than milk (Freetly et al., 2006). By 35 d for calves on forage diet. It can be speculated that of age, calves in the present study had bacteria com- calves were selecting a higher quality diet that may position, VFA, and ammonia concentrations com- have been low in cellulose and hemicellulose. Many parable to mature ruminants on native rangeland unknown genera made up a substantial portion of (Smith et  al., 2017). According to dairy calf data, the ruminal bacteria in the present study, warrant- a functional rumen occurred at 8 wk of age. Based ing further research on what role they may play in on present data, beef calves that consume native a ruminal environment. rangeland forages develop a functional rumen at 35 At 8  wk, calves on concentrate diets have d of age. demonstrated similar VFA concentrations of adult cattle (Suárez et  al., 2006). Beef calves primarily IMPLICATIONS receive nourishment from dams’ milk and availa- ble forages. Forage inclusion in dairy calf rations We hypothesized that calf age would relate showed increased rumen weight, body weight, and to rumen maturity when considering fermenta- feed efficiency compared with nonforage fed calves tion and feed efficiency. Our data indicate that (Tamate et al., 1962), while whole milk has shown the rumen may become mature and functional at improved small intestine development (Górka an earlier age than reported in previous literature. et al., 2011). Bartle et al. (1984) reported at 9 wk of These data could lead to new management strate- lactation, milk production alone did not adequately gies in extensive landscapes such as genetic selec- support calf growth, and creep feeding was needed tion of cows for lower milk production and early to meet nutrient requirements. Thus, beef calves weaning methodologies. Translate basic science to industry innovation Downloaded from https://academic.oup.com/tas/article-abstract/2/suppl_1/S48/5108313 by Ed 'DeepDyve' Gillespie user on 16 October 2018 S52 Smith et al. Li, R.W., E.E.  Connor, C.  Li, R.L.  Baldwin Vi, and LITERATURE CITED M.E. Sparks. 2012. Characterization of the rumen micro- biota of pre-ruminant calves using metagenomic tools. Asai, T. 1973. Developmental processes of reticulorumen Environ. Microbiol. 14:129–139. doi:10.1111/j.1462- motility in calves. Nihon Juigaku zasshi. 35:239–252. 2920.2011.02543.x Bartle, S.J., J.R. Males, and R.L. Preston. 1984. Effect of energy Lodge-Ivey, S.L., J.  Browne-Silva, and M.B.  Horvath. 2009. intake on the postpartum interval in beef cows and the Technical note: bacterial diversity and fermentation adequacy of the cow’s milk production for calf growth. end products in rumen fluid samples collected via oral J. Anim. Sci. 58:1068–1074. doi:10.2527/jas1984.5851068x lavage or rumen cannula. J. Anim. Sci. 87:2333–2337. Broderick, G.A., and J.H.  Kang-Meznarich. 1980. Effect of doi:10.2527/jas.2008-1472 incremental urea supplementation on ruminal ammonia May, T., and M.  Galyean. 1996. Laboratory procedure in concentration and bacterial protein formation. J. Dairy animal nutrition research. Las Cruces, New Mexico: Sci. 63:64–75. doi:10.2527/jas1980.512422x Department of Animal and Range Sciences, New Mexico Coverdale, J.A., H.D. Tyler, J.D. Quigley, III, and J.A. Brumm. State University; p. 187. 2004. Effect of various levels of forage and form of diet Pitta, D.W., E.  Pinchak, S.E.  Dowd, J.  Osterstock, on rumen development and growth in calves. J. Dairy Sci. V. Gontcharova, E. Youn, K. Dorton, I. Yoon, B.R. Min, 87:2554–2562. doi:10.3168/jds.S0022-0302(04)73380-9 J.D. Fulford, et al. 2010. Rumen bacterial diversity dynam- Fonty, G., P.  Gouet, J.P.  Jouany, and J.  Senaud. 1987. ics associated with changing from bermudagrass hay to Establishment of the microflora and anaerobic fungi grazed winter wheat diets. Microb. Ecol. 59:511–522. in the rumen of lambs. Microbiology 133:1835–1843. doi:10.1007/s00248-009-9609-6 doi:10.1099/00221287-133-7-1835 Satter, L.D., and L.L. Slyter. 1974. Effect of ammonia concen- Freetly, H.C., J.A.  Nienaber, and T.  Brown-Brandl. 2006. tration of rumen microbial protein production in vitro. Br. Partitioning of energy during lactation of primiparous beef J. Nutr. 32:199–208. doi:10.1079/BJN19740073 cows. J. Anim. Sci. 84:2157–2162. doi:10.2527/jas.2005-534 Smith, K.E., S.L.  Rosasco, J.K.  Beard, E.R.  Oosthuysen, Górka, P., Z.M. Kowalski, P. Pietrzak, A. Kotunia, W . Jagusiak, B.  Meyerhoff, E.J.  Scholljegerdes, A.F.  Summers, and J.J. Holst, P. Guilloteau, and R. Zabielski. 2011. Effect of S.L.  Ivey. 2017. Change of season impacts ruminal fer- method of delivery of sodium butyrate on rumen devel- mentation and microbiome in heifers grazing native opment in newborn calves. J. Dairy Sci. 94:5578–5588. range. Proc. West. Sec. Amer. Soc. Anim. Sci. 63:153–156. doi:10.3168/jds.2011-4166 doi:10.2527/asasws.2017.0060 Hollingsworth-Jenkins, K.J. 1994. Escape protein, rumen Suárez, B.J., C.G.  Van Reenen, W.J.  Gerrits, N.  Stockhofe, degradable protein, or energy as the first limiting A.M. van Vuuren, and J. Dijkstra. 2006. Effects of supple- nutrient of nursing calves grazing native sandhills menting concentrates differing in carbohydrate compos- range. Lincoln, NE: ETD collection for University of ition in veal calf diets: II. Rumen development. J. Dairy Sci. Nebraska. AAI9516582. https://digitalcommons.unl.edu/ 89:4376–4386. doi:10.3168/jds.S0022-0302(06)72484-5 dissertations/AAI9516582. Tamate, H., A. McGilliard, N. Jacobson, and R. Getty. 1962. Jami, E., A. Israel, A. Kotser, and I. Mizrahi. 2013. Exploring Effect of various dietaries on the anatomical develop- the bovine rumen bacterial community from birth to ment of the stomach in the calf1. J. Dairy Sci. 45:408–420. adulthood. Isme J. 7:1069–1079. doi:10.1038/ismej.2013.2 doi:10.3168/jds.S0022-0302(62)89406-5 Latham, M.J., B.E. Brooker, G.L. Pettipher, and P.J. Harris. 1978. Yu, Z., and M. Morrison. 2004. Improved extraction of PCR- Ruminococcus flavefaciens cell coat and adhesion to cotton quality community DNA from digesta and fecal samples. cellulose and to cell walls in leaves of perennial ryegrass Biotechniques 36:808–812. (Lolium perenne). Appl. Environ. Microbiol. 35:156–165. Translate basic science to industry innovation http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Translational Animal Science Oxford University Press

Succession of ruminal bacterial species and fermentation characteristics in preweaned Brangus calves

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Downloaded from https://academic.oup.com/tas/article-abstract/2/suppl_1/S48/5108313 by Ed 'DeepDyve' Gillespie user on 16 October 2018 Succession of ruminal bacterial species and fermentation characteristics in preweaned Brangus calves ,2 † Kathryn E. Smith,* Anna L. Garza,* Kylie M. Butterfield, * Aaron M. Dickey, † † † Amanda K. Lindholm-Perry, James E. Wells, Harvey C. Freetly, and Shanna L. Lodge-Ivey* *Department of Animal and Range Sciences, New Mexico State University, Las Cruces, NM and USDA, ARS, U.S. Meat Animal Research Center, Clay Center, NE © The Author(s) 2018. Published by Oxford University Press on behalf of the American Society of Animal Science. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com. Transl. Anim. Sci. 2018.2:S48–S52 doi: 10.1093/tas/txy043 2004). Butyrate is especially important in rumen INTRODUCTION development and has been linked to papillae Historically, rumen development has been development in the rumen (Coverdale et  al., defined by anatomical change, fermentation end 2004). Ruminal ammonia is vital for adequate products, and cultured media for identification microbial growth, therefore important for of bacterial composition (Tamate et  al., 1962; microbial and fermentation development Fonty et al., 1987). The rumen ecosystem from (Satter and Slyter, 1974). Thus, the objectives 1 d to 2 yr of age declines in aerobic and faculta- of this study are to assess the composition of tive anaerobic taxa, while increasing anaerobic the ruminal bacteria and the production of fer- taxa (Jami et al., 2013). Li et al (2012) reported mentation end products in preweaned Brangus Bacteroidetes as the prominent phylum in 42-d- calves as they age. We hypothesize that ruminal old calves, different from 14-d-old calves. This VFA, ammonia, and bacterial populations will research has focused on dairy calves, which wean increase as calves age. earlier, live in different environments, and likely has a different rumen developmental timeline in MATERIALS AND METHODS comparison to beef calves. All experimental procedures were approved The energetic efficiency of a cow convert- by New Mexico State University Institutional ing grass to milk and the calf converting milk Animal Care and Use Committee (Protocol 2017- to retained energy is less efficient than directly 001). Eleven Brangus calves born in February and converting forage to retained energy (Freetly March of 2015 were sampled at the Chihuahuan et  al., 2006). Intake of solid food increases Desert Rangeland Research Center (4.1% CP, ruminal microbial activity and higher concen- 72.5% NDF) in Las Cruces, NM. Cow–calf pairs tration of total VFAs produced that can be were housed on native rangeland with grasses utilized for retained energy (Coverdale et  al., including Black grama (Bouteloua eriopoda), three awns (Aristida), dropseeds (Sporobolus), burrograss (Scleropogon brevifolius), and tobosa USDA is an equal opportunity provider and employer. 2 (Pleuraphis mutica). Ruminal samples were col- Corresponding author: ksmith92@nmsu.edu lected via oral lavage (Lodge-Ivey et al., 2009) for Received March 16, 2018. microbiota, VFA, and ammonia analysis on 7, 35, Accepted April 14, 2018. S48 Downloaded from https://academic.oup.com/tas/article-abstract/2/suppl_1/S48/5108313 by Ed 'DeepDyve' Gillespie user on 16 October 2018 Ruminal bacterial species and fermentation characteristics in calves S49 63, 91, 119, 147, 175  ±  5 d of age. Samples were 9.4; SAS Inst. Inc., Cary, NC) with repeated meas- immediately flash frozen in liquid Nitrogen and ures for all data. Animal was the experimental unit, stored at −80 °C. and the treatment was day of age. Using Akaike’s Ruminal ammonia was analyzed using the phe- information criterion, compound symmetry was nol-hypochlorite procedure adapted to a microti- the covariance structure. Means were calculated ter plate (Broderick and Kang-Meznarich, 1980). using LSMEANS. Day effects were considered sig- Volatile fatty acid concentration was determined by nificant at a P ≤ 0.05, and mean separations were gas chromatography utilizing methods of May and performed using PDIFF. Galyean (1996). The DNA was extracted from sam- ples using a repeated beating plus column (RBB RESULTS + C) method (Yu and Morrison, 2004) followed by 1  mL of lysis buffer using the QIAmp DNA The effect of calf age at the phylum level is Stool Mini Kit (Qiagen, Valencia, CA), and qual- summarized in Table  1. Bacteroidetes, Firmicutes, ity and quantity of DNA were determined using a Proteobacteria, SRI, TM7, Tenericutes, Nanodrop (Thermo Scientific, Marietta, OH). Verrucomicrobia, and Unclassified phyla differed The V1–V3 region of 16S rRNA gene was based on age of calf (P < 0.02). Bacteroidetes was amplified, using modified universal primers most abundant at 7 (40.3%), 147 (38.2%), and 175 27F (5′ Adapter/Index/AGAGTTTGAGCCG (52.8%) d of age (P  <  0.01). The lowest Shannon GGCGCAG) and 519R (5′ Adapter/Index/ Wiener Index (SWI) and richness occurred at 7 d GTATTACCGCGGCTGCGA) including TruSeq of age and increased as the calves aged (P < 0.05). adapter sequences and indices. The PCR amplifi - Table  2 summarizes the effect of calf age at cation was performed on Bio-Rad Dyad Thermal the genera level. Richness data identified 214 gen - Cycler (Bio-Rad). Following purification, PCR era however; not all OTUs are represented in the products were diluted to approximately 4 nM, and database and therefore were not able to be classi- PCR amplicons libraries were paired-end sequenced fied at the genera level. Unidentified genera are using the Illumina MiSeq. Sequence data were pro- presented as the lowest taxonomic unit identified. cessed using MICCA pipeline for metagenomic Prevotella increased throughout the study and was analysis. Operational taxonomic units (OTUs) were most abundant at 175 d of age (P < 0.01). Family generated after removing <150 bp sequences. Final BS11, BF311, Prevotella, RF16, Fibrobacter, family OTUs were clustered by 97% similarity and clas- Lachnospiraceae, family Veillonellaceae, unclas- sified using BLASTn against a curated database sified RS1, order LD1-PB3, and unclassified gen - derived from GreenGenes. era differed based on age of calf (P < 0.02). Order All data were analyzed as a completely random Bacteroidales was not affected by age of calf design using the MIXED procedure of SAS (version (P  =  0.16). Genera richness increased (P  <  0.01) Table 1. Effect of age of Brangus calf on phyla composition, Shannon Weiner Index, and richness Days of age † ‡ Phylum , % 7 35 63 91 119 147 175 SEM P value a a b b b b a Bacteroidetes 40.3 48.3 37.6 40.0 37.8 38.2 52.8 3.2 <0.01 a,c a,c a,b b a,b a,b c Firmicutes 26.5 27.4 31.6 35.2 33.9 32.3 23.5 2.95 <0.01 a b b b b b b Proteobacteria 3.9 2.2 2.3 2.8 2.8 2.9 2.5 0.42 <0.01 a,c b b,d c c b,d d SR1 1.2 8.3 7.4 2.1 2.5 6.1 5.1 1.28 <0.01 a b a b b b a TM7 0.9 1.9 1.3 2.3 2.7 2.4 1.0 0.40 <0.01 a a a a a a a Tenericutes 4.3 3.9 4.9 4.2 4.9 5.3 2.3 0.83 0.01 a b a a a a a,b Verrucomicrobia 4.9 1.0 4.7 5.8 6.6 5.7 3.8 1.26 0.02 a b,c b b,c b,c c b,c Unclassified 10.0 4.8 6.9 5.4 6.0 4.2 4.1 1.12 <0.01 a b b,c b c,d d d Shannon Weiner Index 1.9 2.5 2.6 2.5 2.6 2.8 3.0 0.08 <0.01 a b c b,c,d d,e d,e e Richness 10.6 13.5 14.9 13.7 15.5 15.5 16.5 0.49 <0.01 Twenty-three phyla were identified. The most abundant phyla are listed in the above table. n = 11. a–e Values within rows with differing superscripts differ, P ≤ 0.05, between days of age. Translate basic science to industry innovation Downloaded from https://academic.oup.com/tas/article-abstract/2/suppl_1/S48/5108313 by Ed 'DeepDyve' Gillespie user on 16 October 2018 S50 Smith et al. Table 2. Effect of age of Brangus calf on genera composition, Shannon Weiner Index, and richness Days of age † ‡ Genera , % 7 35 63 91 119 147 175 SEM P value a,b a,b a,b a,b a a b Family BS11 1.0 1.7 2.7 1.9 3.6 3.1 8.5 1.51 <0.01 BF311 1.5 0.9 0.8 0.5 0.5 0.7 0.9 0.28 0.16 a a a a a a b Prevotella 13.4 14.1 14.9 13.9 14.8 13.9 27.1 2.6 <0.01 a,b a a,b a,b a,b a,b b Family RF16 0.9 0.8 1.4 2.3 2.2 1.4 2.6 0.72 <0.01 Family S24-7 0.8 1.8 2.6 1.1 0.9 2.0 2.3 0.69 0.35 Order Bacteroidales 12.9 16.1 9.1 10.2 10.2 14.5 8.7 2.34 0.16 CF231 6.7 5.3 3.1 4.6 4.0 3.0 1.5 1.06 0.47 YRC22 0.1 0.2 0.3 0.4 03 0.4 0.8 0.35 0.08 Fibrobacter 0.5 0.2 0.9 0.5 0.4 0.6 0.8 0.28 0.10 a a a b b a,b a Family Lachnospiracea 3.3 3.7 3.2 5.2 5.0 4.2 3.0 0.56 <0.01 Family Veillonellaceae 0.0 0.2 1.3 1.0 1.0 0.8 1.2 0.59 0.04 a a,b a,b a,b a,b a,b b RFN20 1.5 0.7 1.0 0.6 1.0 1.2 0.6 0.36 <0.01 Order RF32 0.1 0.3 0.5 0.3 0.6 0.3 0.4 0.15 0.43 a b b a a a a Unclassified RS1 1.2 8.3 7.4 1.9 2.5 5.8 3.9 1.32 <0.02 Order RF39 3.5 3.6 4.2 3.6 4.1 3.9 1.9 0.84 0.24 a a,c b,c b b b b Order LD1-PB3 0.6 0.1 3.8 4.4 5.8 4.8 1.8 1.03 <0.01 Family RFP12 2.2 0.9 0.7 0.8 0.6 0.7 1.5 0.54 0.38 a b,c b b,c b,c c c Unclassified 10.0 4.8 6.8 5.5 6.0 3.9 4.1 1.10 <0.01 a b b,d c b,c c,d c,d Shannon Weiner Index 4.0 4.6 4.7 5.3 4.9 5.0 5.1 0.14 <0.01 a a,b b,c c c c b,c Richness 40.6 44.1 48.5 50.7 51.7 52.4 48.4 1.79 <0.01 Two hundred and fourteen genera were identified. The most abundant genera are listed in the above table. n = 11. a–d Values within rows with differing superscripts differ, P ≤ 0.05, between days of age. Table 3. Age of Brangus calf effects on ruminal volatile fatty acids and ammonia Days of age Item 7 35 63 91 119 147 175 SEM P value a a,b b,c b,c c c a Total VFA, mM 45.1 61.9 76.6 79.0 82.8 85.5 49.3 6.69 <0.01 VFA, mol/100 mol a b b b b,c,d c,d d Acetate 79.3 75.7 75.9 76.4 75.0 73.4 71.9 0.83 <0.01 a,b a b b a,b a b Propionate 14.3 15.3 13.9 14.7 14.7 15.2 13.5 0.41 <0.01 a a a a a b c Isobutyrate 1.2 1.1 1.2 1.1 1.2 1.7 2.7 0.14 <0.01 a b b,c b,c b,c b,c c Buytrate 4.2 7.0 7.6 7.8 8.1 8.0 8.6 0.53 <0.01 a b a,c a,c b,c b a,b,c Acetate:Propionate 5.7 5.0 5.5 5.5 5.1 4.9 5.4 0.20 <0.01 a b b,d c c c d Ammonia, mM 10.2 6.6 5.4 2.9 2.7 3.0 4.8 0.68 <0.01 n = 11. a–d Values within rows with differing superscripts differ, P ≤ 0.05, between days of age. from 7 to 147 d of age and with a slight decrease increased as calves aged with the greatest butyr- ate concentration at day 175 (P  <  0.01). Ruminal denoted at 175 d of age. The lowest SWI occurred ammonia was greatest on day 7 and remained below at 7 d of age (P < 0.01) and the 91, 119, 147, and 6.6 mM for the remainder of the study (P < 0.01). 175 d of age did not differ (P ≥ 0.05). Table  3 summarizes the effect of calf age on DISCUSSION ruminal VFA and ammonia production. Total VFA production was similar for days 7, 35, and 175 and Limited data exist on beef calf ruminal develop- was lower than days 63, 91, 119, and 147 (P < 0.01). ment in extensive landscapes. In this study, ruminal Acetate was greatest and butyrate was lowest at day fluid was examined for bacterial composition and 7 (P < 0.01). Propionate was greater on days 7, 35, fermentation end products and generated conflict - 119, and 147 and was least on days 63, 91, and 175 ing results compared with dairy calf data. One area (P < 0.01). Acetate:Propionate was greatest on days of agreement was bacterial composition at the phy- 7, 63, 91, and 175 (P < 0.01). Butyrate production lum level. In this study, Bacteroidetes and Firmicutes Translate basic science to industry innovation Downloaded from https://academic.oup.com/tas/article-abstract/2/suppl_1/S48/5108313 by Ed 'DeepDyve' Gillespie user on 16 October 2018 Ruminal bacterial species and fermentation characteristics in calves S51 comprised approximately 72.2% of the rumen bac- raised in conventional systems have an opportu- terial population, agreeing with previously reported nity to capitalize on body weight gain and feed effi - data from Jami et  al. (2013). Approximately 70% ciency due to consumption of both available forage of the rumen bacterial population comprised and dams’ milk. Bacteroidetes and Firmicutes in 2-mo-old dairy Day 7 showed the lowest total VFA levels, and calves (Jami et  al., 2013). Rumen liquid fractions these are indicative of a low structural carbohy- commonly show increased Bacteroidetes popula- drate diet (Asai et al., 1973). However, by day 35, tions compared with Firmicutes (Pitta et al., 2010). total VFA production increases to 61.9  mM only In the current study, ruminal contents were col- to decrease to 49.3 mM on day 175. Forage quality lected via a suction strainer, and samples should be of New Mexico rangeland is greatest during July similar to the liquid fractions commonly reported and August due to monsoon-type rainfall events. in studies that strain the ruminal contents through During 2015, less than average rainfall occurred cheesecloth (Pitta et  al., 2010). Pitta et  al. (2010) during July and August; thus by September and reported increased Bacteroidetes populations com- October (day 175), forage quality declined, result- pared with Firmicutes, which is in agreement in ing in low total VFA production. with our study where Bacteroidetes population was The greatest ruminal ammonia occurred on day 3.9% to 29.3% greater than Firmicutes. 7 when calves were primarily consuming milk. Milk In 42-d-old calves, Firmicutes comprised may enter the rumen causing increased ruminal 12% of the population (Li et  al., 2012), differing ammonia (Jami et al., 2013). Ruminal ammonia con- from the present study where Firmicutes ranged centration of 2 mM is needed for maximum micro- from 27.4% to 31.6% from day 35 to 63 of age. bial growth rate (Satter and Slyter, 1974). Despite Calves fed milk replacer have decreased levels low protein and high structural carbohydrates that of Firmicutes compared with calves grazing on are typical of rangelands, calf rumen function was native rangeland (Li et al., 2012). Beef calves that not impaired for the duration of this experiment. are raised on rangelands may consume forage at Ammonia production is vital for developing calves’ earlier ages, leading to increased Firmicutes pop- nutrient synchrony. Microbial, VFA, and ammonia ulation. Bacterial genera associated with fiber data support the theory that calves are consuming degradation in the phylum Firmicutes include available forage, and microbial population makes Butyrivibrio, Ruminococcus, and Succiniclasticum use of these feedstuffs. Hollingsworth-Jenkins et al. (Latham et al., 1978). In dairy calves ranging from (1994) support this conclusion, where calves raised 14 d old to 12 mo, Ruminococcus maintained a in Sandhills region of Nebraska consumed a diet low abundance ranging from 0.3% to 3.6% of the primarily of forage prior to weaning. Calves con- population (Li et al., 2012). The low abundance of suming forage are potentially more efficient at feed Ruminococcus, in the present study, is unexpected conversion than milk (Freetly et al., 2006). By 35 d for calves on forage diet. It can be speculated that of age, calves in the present study had bacteria com- calves were selecting a higher quality diet that may position, VFA, and ammonia concentrations com- have been low in cellulose and hemicellulose. Many parable to mature ruminants on native rangeland unknown genera made up a substantial portion of (Smith et  al., 2017). According to dairy calf data, the ruminal bacteria in the present study, warrant- a functional rumen occurred at 8 wk of age. Based ing further research on what role they may play in on present data, beef calves that consume native a ruminal environment. rangeland forages develop a functional rumen at 35 At 8  wk, calves on concentrate diets have d of age. demonstrated similar VFA concentrations of adult cattle (Suárez et  al., 2006). Beef calves primarily IMPLICATIONS receive nourishment from dams’ milk and availa- ble forages. Forage inclusion in dairy calf rations We hypothesized that calf age would relate showed increased rumen weight, body weight, and to rumen maturity when considering fermenta- feed efficiency compared with nonforage fed calves tion and feed efficiency. Our data indicate that (Tamate et al., 1962), while whole milk has shown the rumen may become mature and functional at improved small intestine development (Górka an earlier age than reported in previous literature. et al., 2011). Bartle et al. (1984) reported at 9 wk of These data could lead to new management strate- lactation, milk production alone did not adequately gies in extensive landscapes such as genetic selec- support calf growth, and creep feeding was needed tion of cows for lower milk production and early to meet nutrient requirements. Thus, beef calves weaning methodologies. Translate basic science to industry innovation Downloaded from https://academic.oup.com/tas/article-abstract/2/suppl_1/S48/5108313 by Ed 'DeepDyve' Gillespie user on 16 October 2018 S52 Smith et al. Li, R.W., E.E.  Connor, C.  Li, R.L.  Baldwin Vi, and LITERATURE CITED M.E. Sparks. 2012. Characterization of the rumen micro- biota of pre-ruminant calves using metagenomic tools. Asai, T. 1973. Developmental processes of reticulorumen Environ. Microbiol. 14:129–139. doi:10.1111/j.1462- motility in calves. Nihon Juigaku zasshi. 35:239–252. 2920.2011.02543.x Bartle, S.J., J.R. Males, and R.L. Preston. 1984. Effect of energy Lodge-Ivey, S.L., J.  Browne-Silva, and M.B.  Horvath. 2009. intake on the postpartum interval in beef cows and the Technical note: bacterial diversity and fermentation adequacy of the cow’s milk production for calf growth. end products in rumen fluid samples collected via oral J. Anim. Sci. 58:1068–1074. doi:10.2527/jas1984.5851068x lavage or rumen cannula. J. Anim. 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Published: Sep 27, 2018

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