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Effects of yeast culture supplementation on lactation performance and rumen fermentation profile and microbial abundance in mid-lactation Holstein dairy cows

Open ArchivePublished:August 25, 2021DOI:https://doi.org/10.3168/jds.2020-19996

      ABSTRACT

      The continuous trend for a narrowing margin between feed cost and milk prices across dairy farms in the United States highlights the need to improve and maintain feed efficiency. Yeast culture products are alternative supplements that have been evaluated in terms of milk performance and feed efficiency; however, less is known about their potential effects on altering rumen microbial populations and consequently rumen fermentation. Therefore, the objective of this study was to evaluate the effect of yeast culture supplementation on lactation performance, rumen fermentation profile, and abundance of major species of ruminal bacteria in lactating dairy cows. Forty mid-lactation Holstein dairy cows (121 ± 43 days in milk; mean ± standard deviation; 32 multiparous and 8 primiparous) were used in a randomized complete block design with a 7-d adaptation period followed by a 60-d treatment period. Cows were blocked by parity, days in milk, and previous lactation milk yield and assigned to a basal total mixed ration (TMR; 1.6 Mcal/kg of dry matter, 14.6% crude protein, 21.5% starch, and 38.4% neutral detergent fiber) plus 114 g/d of ground corn (CON; n = 20) or basal TMR plus 100 g/d of ground corn and 14 g/d of yeast culture (YC; n = 20; Culture Classic HD, Cellerate Yeast Solutions, Phibro Animal Health Corp.). Treatments were top-dressed over the TMR once a day. Cows were individually fed 1 × /d throughout the trial. Blood and rumen fluid samples were collected in a subset of cows (n = 10/treatment) at 0, 30, and 60 d of the treatment period. Rumen fluid sampled via esophageal tubing was analyzed for ammonia-N, volatile fatty acids (VFA), and ruminal bacteria populations via quantitative PCR amplification of 16S ribosomal DNA genes. Milk yield was not affected by treatment effects. Energy balance was lower in YC cows than CON, which was partially explain by the trend for lower dry matter intake as % body weight in YC cows than CON. Cows fed YC had greater overall ruminal pH and greater total VFA (mM) at 60 d of treatment period. There was a contrasting greater molar proportion of isovalerate and lower acetate proportion in YC-fed cows compared with CON cows. Although the ruminal abundance of specific fiber-digesting bacteria, including Eubacterium ruminantium and Ruminococcus flavefaciens, was increased in YC cows, others such as Fibrobacter succinogenes were decreased. The abundance of amylolytic bacteria such as Ruminobacter amylophilus and Succinimonas amylolytica were decreased in YC cows than CON. Our results indicate that the yeast culture supplementation seems to promote some specific fiber-digesting bacteria while decreasing amylolytic bacteria, which might have partially promoted more neutral rumen pH, greater total VFA, and isovalerate.

      Key words

      INTRODUCTION

      Over the last decades, the focus of the dairy industry has been to maximize milk yield. This has been partially accomplished via intensive genetic selection and feeding strategies to improve feed efficiency and milk production. Feeding Saccharomyces cerevisiae yeast-based products has been reported to improve milk production (
      • Leicester H.C.
      • Robinson P.H.
      • Erasmus L.J.
      Effects of two yeast based direct fed microbials on performance of high producing dairy cows.
      ;
      • Acharya S.
      • Pretz J.P.
      • Yoon I.
      • Scott M.F.
      • Casper D.P.
      Effects of Saccharomyces cerevisiae fermentation products on the lactational performance of mid-lactation dairy cows.
      ;
      • Dias A.L.G.
      • Freitas J.A.
      • Micai B.
      • Azevedo R.A.
      • Greco L.F.
      • Santos J.E.P.
      Effect of supplemental yeast culture and dietary starch content on rumen fermentation and digestion in dairy cows.
      ) and feed efficiency (
      • Poppy G.D.
      • Rabiee A.R.
      • Lean I.J.
      • Sanchez W.K.
      • Dorton K.L.
      • Morley P.S.
      A meta-analysis of the effects of feeding yeast culture produced by anaerobic fermentation of Saccharomyces cerevisiae on milk production of lactating dairy cows.
      ;
      • Dias J.D.L.
      • Silva R.B.
      • Fernandes T.
      • Barbosa E.F.
      • Graças L.E.C.
      • Araujo R.C.
      • Pereira R.A.N.
      • Pereira M.N.
      Yeast culture increased plasma niacin concentration, evaporative heat loss, and feed efficiency of dairy cows in a hot environment.
      ), while stabilizing rumen pH and promote a constant environment for rumen fermentation (
      • Bach A.
      • Iglesias C.
      • Devant M.
      Daily rumen pH pattern of loose-housed dairy cattle as affected by feeding pattern and live yeast supplementation.
      ;
      • Chaucheyras-Durand F.
      • Walker N.D.
      • Bach A.
      Effects of active dry yeasts on the rumen microbial ecosystem: Past, present and future.
      ;
      • Dias A.L.G.
      • Freitas J.A.
      • Micai B.
      • Azevedo R.A.
      • Greco L.F.
      • Santos J.E.P.
      Effect of supplemental yeast culture and dietary starch content on rumen fermentation and digestion in dairy cows.
      ).
      Commercial yeast products and yeast-containing feed ingredients vary on many characteristics, including yeast strain, viability (i.e., active dry yeast or dead yeast), yeast culture and media associated with it, and postfermentation processing (e.g., fractionated yeast;
      • Shurson G.C.
      Yeast and yeast derivatives in feed additives and ingredients: Sources, characteristics, animal responses, and quantification methods.
      ). Yeast culture is unique among yeast products because it comprises yeast biomass and fermentation metabolites (
      • Shurson G.C.
      Yeast and yeast derivatives in feed additives and ingredients: Sources, characteristics, animal responses, and quantification methods.
      ). The composition and characteristics of fermentation metabolites are highly dependent on the medium used to grow the yeast. Yeast culture is dried in a manner to preserve the fermenting activity of the yeast and may contain small to negligible residual amounts of viable yeast cells (
      • Shurson G.C.
      Yeast and yeast derivatives in feed additives and ingredients: Sources, characteristics, animal responses, and quantification methods.
      ;
      • AAFCO
      Official Publication.
      ). The benefits of yeast culture have been attributed to the presence of functional metabolites (e.g., organic acids, B vitamins, AA, and enzymes) that may influence ruminal fermentation by supplying key nutrients otherwise scarce in the ruminal environment (
      • Oeztuerk H.
      Effects of live and autoclaved yeast cultures on ruminal fermentation in vitro.
      ;
      • Opsi F.
      • Fortina R.
      • Tassone S.
      • Bodas R.
      • López S.
      Effects of inactivated and live cells of Saccharomyces cerevisiae on in vitro ruminal fermentation of diets with different forage:concentrate ratio.
      ). At the rumen level, yeast culture seems to exert beneficial effects, preventing SARA and improving feed efficiency in dairy cows (
      • Dias J.D.L.
      • Silva R.B.
      • Fernandes T.
      • Barbosa E.F.
      • Graças L.E.C.
      • Araujo R.C.
      • Pereira R.A.N.
      • Pereira M.N.
      Yeast culture increased plasma niacin concentration, evaporative heat loss, and feed efficiency of dairy cows in a hot environment.
      ). These effects have been partially explained by yeast culture's ability to alter ruminal fermentation by stimulating growth and activity of fiber-digesting and lactate-utilizing bacteria (
      • Zhu W.
      • Wei Z.
      • Xu N.
      • Yang F.
      • Yoon I.
      • Chung Y.
      • Liu J.
      • Wang J.
      Effects of Saccharomyces cerevisiae fermentation products on performance and rumen fermentation and microbiota in dairy cows fed a diet containing low quality forage.
      ).
      The abundance of various rumen bacterial taxa has been correlated with feed efficiency and animal performance, indicating that the bacterial community plays an important role in regulating host physiological parameters (
      • Jami E.
      • White B.A.
      • Mizrahi I.
      Potential role of the bovine rumen microbiome in modulating milk composition and feed efficiency.
      ;
      • Shabat S.K.
      • Sasson G.
      • Doron-Faigenboim A.
      • Durman T.
      • Yaacoby S.
      • Berg Miller M.E.
      • White B.A.
      • Shterzer N.
      • Mizrahi I.
      Specific microbiome-dependent mechanisms underlie the energy harvest efficiency of ruminants.
      ). At the rumen level, yeast culture seems to exert beneficial effects, stabilizing ruminal pH and improving feed efficiency in dairy cows (
      • Dias J.D.L.
      • Silva R.B.
      • Fernandes T.
      • Barbosa E.F.
      • Graças L.E.C.
      • Araujo R.C.
      • Pereira R.A.N.
      • Pereira M.N.
      Yeast culture increased plasma niacin concentration, evaporative heat loss, and feed efficiency of dairy cows in a hot environment.
      ). These effects have been partially explained by yeast culture's ability to alter ruminal fermentation by stimulating growth and activity of cellulolytic (Fibrobacter and Ruminococcus species) and lactate-utilizing bacteria (Megasphaera and Selenomonas species;
      • Newbold C.J.
      • Wallace R.J.
      • Chen X.B.
      • McIntosh F.M.
      Different strains of Saccharomyces cerevisiae differ in their effects on ruminal bacterial numbers in vitro and in sheep.
      ;
      • Callaway E.S.
      • Martin S.A.
      Effects of a Saccharomyces cerevisiae culture on ruminal bacteria that utilize lactate and digest cellulose.
      ;
      • Zhu W.
      • Wei Z.
      • Xu N.
      • Yang F.
      • Yoon I.
      • Chung Y.
      • Liu J.
      • Wang J.
      Effects of Saccharomyces cerevisiae fermentation products on performance and rumen fermentation and microbiota in dairy cows fed a diet containing low quality forage.
      ). Recently,
      • Tun H.M.
      • Li S.
      • Yoon I.
      • Meale S.J.
      • Azevedo P.A.
      • Khafipour E.
      • Plaizier J.C.
      Saccharomyces cerevisiae fermentation products (SCFP) stabilize the ruminal microbiota of lactating dairy cows during periods of a depressed rumen pH.
      evaluated the effect of yeast culture supplementation both in vivo and in vitro, and their results suggested that yeast culture protected the rumen microbial diversity from the negative effect of a low rumen pH. These authors also reported that yeast culture promoted the growth of cellulolytic and lactate-utilizing bacteria in both trials. Lactate-utilizing bacteria have been linked with enhanced animal efficiency due to their involvement in channeling the metabolic pathway that converts lactate into propionate (
      • Shabat S.K.
      • Sasson G.
      • Doron-Faigenboim A.
      • Durman T.
      • Yaacoby S.
      • Berg Miller M.E.
      • White B.A.
      • Shterzer N.
      • Mizrahi I.
      Specific microbiome-dependent mechanisms underlie the energy harvest efficiency of ruminants.
      ).
      Although several studies on yeast culture products in lactating dairy cows have evaluated its effect on fermentation parameters (
      • Hristov A.N.
      • Varga G.
      • Cassidy T.
      • Long M.
      • Heyler K.
      • Karnati S.K.
      • Corl B.
      • Hovde C.J.
      • Yoon I.
      Effect of Saccharomyces cerevisiae fermentation product on ruminal fermentation and nutrient utilization in dairy cows.
      ;
      • Zhu W.
      • Wei Z.
      • Xu N.
      • Yang F.
      • Yoon I.
      • Chung Y.
      • Liu J.
      • Wang J.
      Effects of Saccharomyces cerevisiae fermentation products on performance and rumen fermentation and microbiota in dairy cows fed a diet containing low quality forage.
      ;
      • Dias A.L.G.
      • Freitas J.A.
      • Micai B.
      • Azevedo R.A.
      • Greco L.F.
      • Santos J.E.P.
      Effect of supplemental yeast culture and dietary starch content on rumen fermentation and digestion in dairy cows.
      ), only a few studies performed in lactating dairy cows have evaluated yeast culture effect in ruminal bacteria abundance, which to our knowledge were
      • Mullins C.R.
      • Mamedova L.K.
      • Carpenter A.J.
      • Ying Y.
      • Allen M.S.
      • Yoon I.
      • Bradford B.J.
      Analysis of rumen microbial populations in lactating dairy cattle fed diets varying in carbohydrate profiles and Saccharomyces cerevisiae fermentation product.
      ,
      • Zhu W.
      • Wei Z.
      • Xu N.
      • Yang F.
      • Yoon I.
      • Chung Y.
      • Liu J.
      • Wang J.
      Effects of Saccharomyces cerevisiae fermentation products on performance and rumen fermentation and microbiota in dairy cows fed a diet containing low quality forage.
      , and
      • Tun H.M.
      • Li S.
      • Yoon I.
      • Meale S.J.
      • Azevedo P.A.
      • Khafipour E.
      • Plaizier J.C.
      Saccharomyces cerevisiae fermentation products (SCFP) stabilize the ruminal microbiota of lactating dairy cows during periods of a depressed rumen pH.
      . The latter suggests that the responses to yeast culture supplementation in lactating dairy cows on ruminal microbiology remain to be elucidated, as do its associations with animal performance. We hypothesized that feeding a yeast culture product derived from a Saccharomyces cerevisiae containing yeast, yeast components, and fermentation metabolites will improve the ruminal environment and modify rumen fermentation through increased ruminal cellulolytic and lactate-utilizer bacteria. The objective of the study was to evaluate the effects of feeding yeast culture on mid-lactation performance, rumen fermentation characteristics, and rumen bacterial populations in lactating dairy cows.

      MATERIALS AND METHODS

      Animal Housing and Care

      All the procedures for this study were conducted in accordance with the protocol approved by the Institutional Animal Care and Use Committee at the South Dakota State University (Protocol no. 18–028A). The experiment was conducted from December 2018 to May 2019. Weather data from Mesonet at South Dakota State University (https://climate.sdstate.edu/) was used to evaluate the average daily temperature during the experimental period, which was −2.65 ± 6.06°C. Cows were housed in a ventilated enclosed barn with access to mattress freestalls and fed once daily at 0630 h using individual electronic admission gates with respective transponders (American Calan). Individual orts were collected once a day before feeding to determine feed intake. Feed offered was adjusted daily to achieve 5 to 10% refusal.

      Design and Treatment Diets

      Forty mid-lactation Holstein dairy cows (124 ± 42 DIM; mean ± SD), including 32 multiparous and 8 primiparous cows, were used in a randomized complete block design with a 7-d covariate period followed by a 60-d treatment period. Cows were blocked by lactation number, DIM, and milk production during the last 4 d of the adaptation period and randomly assigned to 1 of 2 treatments (n = 20 per treatment). Cows received the same basal diet as a TMR (Table 1) supplemented with 114 g/d of ground corn as a control group (CON) or 14 g/d of yeast culture (YC; Culture Classic HD, Cellerate Yeast Solutions, Phibro Animal Health Corp.) blended with 100 g/d of ground corn, as a treatment group. The treatment products were top-dressed individually over the TMR within 1 h after morning feeding. According to AAFCO definitions (
      • AAFCO
      Official Publication.
      ), the product used is a yeast culture, which contains yeast biomass and fermentation metabolites originating from Saccharomyces cerevisiae grown on a medium and dried on beneficial plant-based carriers. Cows in the YC group were fed the CON treatment during the covariate period. Diets were formulated using the CNCPS model (v. 6.55;
      • Van Amburgh M.E.
      • Collao-Saenz E.A.
      • Higgs R.J.
      • Ross D.A.
      • Recktenwald E.B.
      • Raffrenato E.
      • Chase L.E.
      • Overton T.R.
      • Mills J.K.
      • Foskolos A.
      The Cornell Net Carbohydrate and Protein System: Updates to the model and evaluation of version 6.5.
      ) contained in the NDS Professional ration formulation software (v. 6.55; RUM&N, NDS Professional).
      Table 1Ingredient and chemical composition of basal diet fed during the experiment period for all groups
      ItemBasal diet
      Ingredient, % of DM
      Ingredients included in the ration formulated by using NDS Professional based on CNCPS (Nutrition Dynamic System; RUM&N Sas.).
       Alfalfa hay10.1
       Alfalfa silage10.1
       Corn silage38.4
       Whole cottonseed with lint7.6
       Corn grain ground fine17.7
       Soybean meal4.0
       Wheat middling4.0
       Rumen-inert fat
      Energy Booster 100 (MSC).
      1.0
       Urea0.12
       Distillers grain dry4.0
       Calcium carbonate1.1
       Sodium bicarbonate1.0
       Salt white0.38
       Magnesium oxide0.18
       Vitamin E
      Vitamin E, 9,090 IU/kg.
      0.04
       Monensin
      Rumensin 90 (200 g of monensin in 1 kg of product).
      0.01
       Biotin 1%0.01
       Rumen-protected Met
      Mepron (Evonik nutrition and care GmbH).
      0.04
       Vitamin premix
      JPW dairy vitamin premix: 28.10% Ca (DM basis) 2,500 IU/kg of vitamin A, 834 IU/kg of vitamin D, and 8,334 IU/kg of vitamin E (JPW Nutrition).
      0.11
       Trace mineral premix
      JPW dairy TM premix: 10.51% Ca (DM basis), 3.70% S, 10,417 mg/kg of Fe, 62,500 mg/kg of Zn, 10,417 mg/kg of Cu, 52,084 mg/kg of Mn, 321 mg/kg of Se, 625 mg/kg of Co, and 1,042 mg/kg of I (JPW Nutrition).
      0.11
      Chemical analysis
       NEL, Mcal/kg of DM ± SD1.6 ± 0.03
       CP, % of DM14.6 ± 0.5
       ADF, % of DM24.3 ± 1.8
       NDF, % of DM38.4 ± 1.2
       NFC, % of DM33.0 ± 2.0
       Starch, % of DM21.5 ± 1.6
       Crude fat, % of DM5.1 ± 0.7
      1 Ingredients included in the ration formulated by using NDS Professional based on CNCPS (Nutrition Dynamic System; RUM&N Sas.).
      2 Energy Booster 100 (MSC).
      3 Vitamin E, 9,090 IU/kg.
      4 Rumensin 90 (200 g of monensin in 1 kg of product).
      5 Mepron (Evonik nutrition and care GmbH).
      6 JPW dairy vitamin premix: 28.10% Ca (DM basis) 2,500 IU/kg of vitamin A, 834 IU/kg of vitamin D, and 8,334 IU/kg of vitamin E (JPW Nutrition).
      7 JPW dairy TM premix: 10.51% Ca (DM basis), 3.70% S, 10,417 mg/kg of Fe, 62,500 mg/kg of Zn, 10,417 mg/kg of Cu, 52,084 mg/kg of Mn, 321 mg/kg of Se, 625 mg/kg of Co, and 1,042 mg/kg of I (JPW Nutrition).

      Data and Sample Collection

      Individual BW was recorded weekly during the adaptation and treatment periods. Body condition was scored weekly by 2 trained investigators on a 5-point scale (
      • Wildman E.E.
      • Jones G.M.
      • Wagner P.E.
      • Boman R.L.
      • Troutt Jr., H.F.
      • Lesch T.N.
      A dairy-cow body condition scoring system and its relationship to selected production characteristics.
      ). The DM of each diet ingredient was determined weekly, and the TMR was adjusted weekly to maintain the same diet formulation on a DM basis. Individual samples of diet ingredients and TMR were collected weekly and stored at −20°C until analyzed. Samples were analyzed for DM, CP, NDF, ADF, starch, and crude fat using wet chemistry methods, whereas NEL and NFC were estimated by a commercial laboratory (Dairy One, Ithaca, NY).
      Cows were milked twice a day, and the milk yield was recorded at each milking during the adaptation and experimental periods. Consecutive morning and evening milk samples were collected 1 d/wk during the experimental period. Composite milk samples were performed in proportion to milk yield at each milking, preserved with Bronopol and Natamycin (Broad Spectrum Microtabs II, Advanced Instruments), and analyzed for fat, protein, lactose, solids, MUN, and SCC using Fourier-transform infrared spectroscopy technology (Dairy One). The ECM was calculated based on milk yield and milk sample analysis as follows: ECM = [12.82 × fat yield (kg)] + [7.13 × protein yield (kg)] + [0.323 × milk yield (kg)] (
      • Tyrrell H.F.
      • Reid J.T.
      Prediction of the energy value of cow's milk.
      ).
      The energy balance (EB) for each cow was calculated based on equations from the (
      • NRC (National Research Council)
      Nutrient Requirements of Dairy Cattle.
      ). The net energy intake (NEI) was determined using daily DMI × NEL of the diet. The NEL was calculated from the TDN of the diet as NEL (Mcal/kg) = 0.0245 × TDN (%) − 0.12. The NEM was calculated as BW0.75 × 0.080. Requirements of NEL for milk production were calculated as NEMILK = (0.0929 × fat % + 0.0547 × protein % + 0.0395 × lactose %) × milk yield. The equation used to calculate EB (Mcal/d) was EB = NEI − (NEM + NEMILK) and EB (as % of requirements) = [NEI/(NEM + NEMILK)] × 100.
      Rumen fluid was collected from 20 cows (n = 10 per treatment; 16 multiparous and 4 primiparous cows) during the last day of adaptation (0 d) and at 30 and 60 d of supplementation. Cows were sampled in the afternoon via esophageal tube 3 h after morning feeding. The esophageal tubing apparatus was assembled by coupling the esophageal tube to a metal strainer (
      • Raun N.S.
      • Burroughs W.
      Suction strainer technique in obtaining rumen fluid samples from Intact Lambs.
      ) on one end, and the other end connected to the opposite handle side of a manual vacuum pump (Med-Eze stomach pump, MAI Animal Health). The rumen fluid sample was collected by passing the fluid through the pump's hollow shaft and into a plastic beaker. After discarding the first 200 mL of fluid to minimize saliva contamination, approximately 50 mL of rumen fluid was collected. After collection, the pH was immediately measured using a pH meter (Waterproof pH Testr 30, Oakton Instruments), and 2 aliquots (10 mL) were acidified with either 200 µL of 50% sulfuric acid or 2 mL of 25% meta-phosphoric acid and stored at −20°C until analysis of ammonia-N (NH3-N) and VFA, respectively. In addition, a 2-mL rumen fluid sample was collected and immediately frozen in liquid nitrogen and stored at −80°C until DNA isolation and subsequent relative abundance of bacteria species was performed via quantitative PCR (qPCR) method.
      Rumen fluid samples preserved with sulfuric acid and 25% meta-phosphoric acid were thawed and transferred into 2-mL microcentrifuge tubes. Then, samples were centrifuged at 30,000 × g for 20 min at 4°C (model 5403, Eppendorf), and the supernatant from samples in sulfuric acid was used to analyzed NH3-N using a colorimetric assay described by
      • Chaney A.L.
      • Marbach E.P.
      Modified reagents for determination of urea and ammonia.
      . The supernatant of rumen fluid containing 25% meta-phosphoric acid was analyzed for acetate, propionate, butyrate, isobutyrate, isovalerate, and valerate concentrations using an automated gas chromatograph (model 689, Hewlett-Packard) equipped with a 0.25 mm i.d × 15-m column (Nukol 24106-U, Supelco Inc.) and the internal standard used was 2-ethylbutyrate.

      Ruminal Bacteria DNA Isolation and qPCR Amplification of 16S rDNA Genes

      Ruminal bacteria DNA was isolated using the QIAamp Fast DNA Stool mini kit (Qiagen) with modifications to the protocol described by
      • Henderson G.
      • Cox F.
      • Kittelmann S.
      • Miri V.H.
      • Zethof M.
      • Noel S.J.
      • Waghorn G.C.
      • Janssen P.H.
      Effect of DNA extraction methods and sampling techniques on the apparent structure of cow and sheep rumen microbial communities.
      . Briefly, 1 mL of rumen fluid was centrifuged at 12,000 × g for 5 min at room temperature (20–25°C), and the supernatant was discarded. Pellet was resuspended in 1 mL of buffer EX, vortexed, incubated in a heat block at 95°C for 5 min, and centrifuged at 20,000 × g for 1 min at room temperature (20–25°C). Then, 600 μL of supernatant was transferred to a new microcentrifuge tube contained 25 μL of Qiagen proteinase K, followed by the addition of 600 μL of Buffer AL. The mixture was vortexed for 15 s and incubated at 70°C for 10 min. After incubation, 600 μL of 96% molecular ethanol was added and vortexed. The mixture was transferred into a QIAamp mini spin column, and the subsequent steps were performed according to manufacturer's procedures (Qiagen). The quantity and purity were measured using a NanoDrop spectrophotometer (ND 1000, NanoDrop Technologies Inc.). The extracted DNA was standardized to 8 ng/µL for qPCR.
      The primer sets used in this study to evaluate the relative abundance of 18 bacterial species have been previously reported and validated (Supplemental Table S1, https://doi.org/10.6084/m9.figshare.15145071.v1;
      • Halfen J.
      • Carpinelli N.A.
      • Pino F.A.B.D.
      • Chapman J.
      • Sharman E.
      • Anderson J.
      Effects of yeast culture supplementation on lactation performance and rumen fermentation profile and microbial abundance in mid-lactation Holstein dairy cows. figshare. Journal contribution.
      ). The quantitative PCR analysis was performed using 10 µL of qPCR mixture containing 4 µL of DNA sample, 5 µL of 1× SYBR Green master mix (Applied Biosystems), 0.4 µL of each forward and reverse primers, and 0.2 µL of DNase-RNase-free water in a MicroAmp Optical 384-well reaction plate (Applied Biosystems). Each sample was run in triplicate, and the relative abundance was determined based on a 6-point standard curve plus a no-template control. The 4-fold-dilution standard curve was created using standardized DNA from all samples. The qPCR reactions were performed with the QuantStudio 6 Flex Real-Time PCR System (Applied Biosystems) using the same conditions described by
      • Grazziotin R.C.B.
      • Halfen J.
      • Rosa F.
      • Schmitt E.
      • Anderson J.L.
      • Ballard V.
      • Osorio J.S.
      Altered rumen fermentation patterns in lactating dairy cows supplemented with phytochemicals improve milk production and efficiency.
      . A geometrical mean of 2 universal primers was used to calculate the relative abundance of bacterial species with the efficiency-corrected Δ-CT method (
      • Abdelmegeid M.K.
      • Elolimy A.A.
      • Zhou Z.
      • Lopreiato V.
      • McCann J.C.
      • Loor J.J.
      Rumen-protected methionine during the peripartal period in dairy cows and its effects on abundance of major species of ruminal bacteria.
      ). The relative abundance of each bacterial species is relative to the total bacteria abundance measured with these universal primers.

      Blood Biomarker Analyses

      Blood samples were collected from 20 cows (n = 10 per treatment; 16 multiparous and 4 primiparous cows) via coccygeal vein before morning feeding at 0, 30, and 60 d of supplementation. Samples were collected into evacuated serum tubes (BD Vacutainer, Becton Dickinson) containing either clot activator or lithium heparin for serum or plasma, respectively. After blood collection, tubes with lithium heparin were placed on ice, and tubes with clot activator were kept at 21°C until centrifugation (~30 min). Serum and plasma were obtained by centrifugation at 1,300 × g for 15 min at 21°C and 4°C, respectively. Aliquots of serum and plasma were frozen (−80°C) until further analysis. Samples were analyzed for nonesterified fatty acids (NEFA; catalog no. 99934691, Wako Chemicals Inc.) and BHB (catalog no. H7587–58, Pointe Scientific Inc.) using kits in an autoanalyzer (Vet AXCEL, Alfa Wassermann) at the Animal Disease Research and Diagnostic Laboratory at South Dakota State University. Glucose concentration was measured using a commercial kit (catalog no. 99703001, Wako Chemicals Inc.). The concentration of BUN was determined using diacetylmonoxime (catalog no. 0580250, Stanbio Laboratory)

      Statistical Analysis

      Data were analyzed as repeated measures with the MIXED procedure of SAS version 9.4 (SAS Institute Inc.) using treatment, time (day or week), parity, and 2- and 3-way interactions with treatment as fixed effects and the random effects of cow and block. Interactions with parity were tested and removed from the model when P > 0.20. Repeated measured data were modeled selecting the variance-covariance structures with least Bayesian information criterion value among compound symmetry, autoregressive 1 [AR(1)], or heterogeneous autoregressive 1 [ARH(1)]. Covariate data collected during the adaptation period, including BW, BCS, DMI, milk yield, rumen parameters, and ruminal microbial populations as well as daily average temperature, were tested and removed from the model when P > 0.20. The sample size for blood biomarkers and rumen fluid parameters (n = 10 per treatment) used in this study was based on prior studies on transition dairy cows evaluating ruminal bacteria population via PCR (
      • Abdelmegeid M.K.
      • Elolimy A.A.
      • Zhou Z.
      • Lopreiato V.
      • McCann J.C.
      • Loor J.J.
      Rumen-protected methionine during the peripartal period in dairy cows and its effects on abundance of major species of ruminal bacteria.
      ;
      • Elolimy A.A.
      • Arroyo J.M.
      • Batistel F.
      • Iakiviak M.A.
      • Loor J.J.
      Association of residual feed intake with abundance of ruminal bacteria and biopolymer hydrolyzing enzyme activities during the peripartal period and early lactation in Holstein dairy cows.
      ). However, the authors acknowledge that reducing the sample size from 20 to 10 animals per treatment cause a loss of statistical power. This subsampling was performed by randomly selecting half of the initial blocks of multiparous (8 blocks or 16 cows) and primiparous (2 blocks or 4 cows) cows. Statistical significance was considered at P ≤ 0.05, and trends were declared at 0.05 < P ≤ 0.10.

      RESULTS

      Feed Intake, BW, ADG, BCS, and EB

      Main effects and interactions of BW, BCS, DMI, DMI as % of BW, and EB parameters are presented in Table 2. A treatment effect was observed for lower EB as Mcal/d (P = 0.02) and EB as % (P = 0.03). A trend (P = 0.07) for lower DMI as % of BW in YC cows than CON was observed. The BW, BCS, and DMI were not affected by treatment effects.
      Table 2Effects of supplementing a yeast culture product (YC) on BW, BCS, DMI, DMI (% of BW), and energy balance parameters in mid-lactation dairy cows
      ItemTreatment
      Cows were assigned to a basal TMR (1.6 Mcal/kg of DM, 14.6% CP, 21.5% starch, and 38.4% NDF) plus 114 g/d of ground corn (CON; n = 20) or basal TMR plus 100 g/d of ground corn and 14 g/d of yeast culture (YC; n = 20; Culture Classic HD, Cellerate Yeast Solutions, Phibro Animal Health Corp.).
      SEM
      Largest SEM.
      P-value
      Parity was not significant (P > 0.19) in any of the parameters, and none had a parity × treatment (Trt) interaction (P > 0.20).
      CONYCTrtTimeTrt × T
      Interaction of treatment × time (week).
      BW, kg7187224.400.57<0.010.37
      BCS
      BCS based on a 1 to 5 scale (Wildman et al., 1982).
      2.852.880.030.580.020.48
      ADG, kg/d0.510.400.210.62<0.010.27
      DMI, kg/d26.0325.510.780.55<0.010.96
      DMI, % of BW3.683.490.110.07<0.010.87
      Energy balance, Mcal/d6.643.740.990.02<0.010.77
      Energy balance,
      Energy balance % = [(Net energy of intake/NEM + Net energy for milk production) × 100].
      %
      118.6110.03.210.03<0.010.76
      1 Cows were assigned to a basal TMR (1.6 Mcal/kg of DM, 14.6% CP, 21.5% starch, and 38.4% NDF) plus 114 g/d of ground corn (CON; n = 20) or basal TMR plus 100 g/d of ground corn and 14 g/d of yeast culture (YC; n = 20; Culture Classic HD, Cellerate Yeast Solutions, Phibro Animal Health Corp.).
      2 Largest SEM.
      3 Parity was not significant (P > 0.19) in any of the parameters, and none had a parity × treatment (Trt) interaction (P > 0.20).
      4 Interaction of treatment × time (week).
      5 BCS based on a 1 to 5 scale (
      • Wildman E.E.
      • Jones G.M.
      • Wagner P.E.
      • Boman R.L.
      • Troutt Jr., H.F.
      • Lesch T.N.
      A dairy-cow body condition scoring system and its relationship to selected production characteristics.
      ).
      6 Energy balance % = [(Net energy of intake/NEM + Net energy for milk production) × 100].

      Production Variables and Feed Efficiency

      Main effects and interactions for milk production variables and feed efficiency are presented in Table 3. There was a treatment (Trt) × time interaction (P = 0.03) in lactose %, which was mainly associated with a trend (P ≥ 0.08) for transient differences between treatments at 3 and 6 wk of the experiment. There was a trend (P = 0.09) for greater feed efficiency as ECM/DMI and milk/DMI in YC cows when compared with CON. Milk yield and components, MUN, SCC, and ECM, were not affected by treatment effects.
      Table 3Effects of supplementing a yeast culture product (YC) on lactation performance and feed efficiency parameters in mid-lactation dairy cows
      ItemTreatment
      Cows were assigned to a basal TMR (1.6 Mcal/kg of DM, 14.6% CP, 21.5% starch, and 38.4% NDF) plus 114 g/d of ground corn (CON; n = 20) or basal TMR plus 100 g/d of ground corn and 14 g/d of yeast culture (YC; n = 20; Culture Classic HD, Cellerate Yeast Solutions, Phibro Animal Health Corp.).
      SEM
      Largest SEM.
      P-value
      Trt × T = interaction of treatment × time (week). None of the parameters had a parity × treatment effect (P > 0.20), and all parameters had a time effect at P < 0.01.
      CONYCTrtTrt × TParity
      Milk yield, kg/d37.336.50.700.320.430.10
      Milk composition
       Fat, %3.883.900.110.880.860.35
       Fat yield, kg/d1.441.420.050.740.720.85
       Protein, %3.153.170.040.610.990.43
       Protein yield, kg/d1.171.160.030.770.590.22
       Lactose, %4.944.910.040.910.030.40
      MUN, mg/dL9.669.240.380.350.890.33
      Milk somatic cell linear score
      Somatic cell linear score = log2(SCC/100) + 3.
      2.322.500.240.540.340.08
      ECM, kg/d38.9138.370.930.610.600.38
      Milk/DMI1.431.480.030.090.720.01
      ECM/DMI1.481.550.040.090.510.08
      1 Cows were assigned to a basal TMR (1.6 Mcal/kg of DM, 14.6% CP, 21.5% starch, and 38.4% NDF) plus 114 g/d of ground corn (CON; n = 20) or basal TMR plus 100 g/d of ground corn and 14 g/d of yeast culture (YC; n = 20; Culture Classic HD, Cellerate Yeast Solutions, Phibro Animal Health Corp.).
      2 Largest SEM.
      3 Trt × T = interaction of treatment × time (week). None of the parameters had a parity × treatment effect (P > 0.20), and all parameters had a time effect at P < 0.01.
      4 Somatic cell linear score = log2(SCC/100) + 3.

      Rumen Fermentation Parameters

      Main effects and interactions for rumen fermentation characteristics are presented in Table 4. There was a Trt × time effect observed only for total VFA (P = 0.05), which was attributed to greater (P = 0.03) total VFA in YC cows than CON at 60 d (Figure 1A). Greater (P = 0.02) ruminal pH was observed in YC cows in comparison to CON. There was a lower (P < 0.01) acetate proportion in YC cows in comparison to CON. Similar to ruminal pH, a greater (P = 0.01) isovalerate proportion was observed in YC cows than CON.
      Table 4Effects of supplementing a yeast culture product (YC) on ruminal fermentation characteristics in mid-lactation dairy cows
      Rumen parameterTreatment
      Cows were assigned to a basal TMR (1.6 Mcal/kg of DM, 14.6% CP, 21.5% starch, and 38.4% NDF) plus 114 g/d of ground corn (CON; n = 20) or basal TMR plus 100 g/d of ground corn and 14 g/d of yeast culture (YC; n = 20; Culture Classic HD, Cellerate Yeast Solutions, Phibro Animal Health Corp.).
      SEM
      Largest SEM.
      P-value
      CONYCTrtTimeTrt × T
      Interaction of treatment × time (d 0, 30, and 60).
      Parity
      None of the parameters had a parity × treatment effect (P > 0.20).
      pH5.866.030.050.020.020.820.05
      NH3, mg/dL6.686.751.400.960.580.500.12
      Total VFA, mM88.590.63.80.650.040.05<0.01
      VFA (mol/100 mol)
       Acetate61.159.60.62<0.010.980.190.49
       Propionate26.627.50.900.390.620.590.61
       Butyrate10.59.90.460.350.880.410.36
       Isovalerate1.41.60.060.010.410.570.80
       Valerate1.11.00.070.35<0.010.410.12
      Acetate:Propionate2.212.090.150.340.550.630.15
      1 Cows were assigned to a basal TMR (1.6 Mcal/kg of DM, 14.6% CP, 21.5% starch, and 38.4% NDF) plus 114 g/d of ground corn (CON; n = 20) or basal TMR plus 100 g/d of ground corn and 14 g/d of yeast culture (YC; n = 20; Culture Classic HD, Cellerate Yeast Solutions, Phibro Animal Health Corp.).
      2 Largest SEM.
      3 Interaction of treatment × time (d 0, 30, and 60).
      4 None of the parameters had a parity × treatment effect (P > 0.20).
      Figure thumbnail gr1
      Figure 1Ruminal parameters of total VFA (A) and Megasphaera elsdenii (B) in mid-lactation dairy cows fed a basal diet (CON) or a control diet supplemented with a yeast culture product (YC) during 60 d of the experimental period. Mean separations between treatments at a given time point were evaluated at a treatment × time interaction (P ≤ 0.10) and differences (*) were declared at P ≤ 0.05. Values are means, and SE are represented by vertical bars.

      Abundance of Ruminal Bacteria

      Main effects and interactions for the relative abundance of selected bacterial species are presented in Table 5. Megasphaera elsdenii (Figure 1B) was the only bacteria with a Trt × time interaction (P = 0.05). This Trt × time was associated with a greater abundance of M. elsdenii in YC cows than CON at 30 d. Greater abundance of Eubacterium ruminantium (P = 0.05) and Ruminococcus flavefaciens (P = 0.03) was observed in YC cows in comparison to CON. In contrast to E. ruminantium and R. flavefaciens, a lower abundance of Fibrobacter succinogenes (P < 0.01), Ruminobacter amylophilus (P = 0.05), Selenomonas ruminantium (P = 0.02), and Succinimonas amylolytica (P = 0.04) was observed in YC cows in comparison to CON. A trend (P = 0.07) for a greater abundance of Prevotella bryantii was observed in YC cows than CON. Among all the bacteria species evaluated, only F. succinogenes had a parity × Trt (P = 0.03) interaction, which was mainly attributed to lower (P < 0.01) abundance of F. succinogenes in primiparous cows but not in multiparous cows (Supplemental Figure S1, https://doi.org/10.6084/m9.figshare.15145071.v1;
      • Halfen J.
      • Carpinelli N.A.
      • Pino F.A.B.D.
      • Chapman J.
      • Sharman E.
      • Anderson J.
      Effects of yeast culture supplementation on lactation performance and rumen fermentation profile and microbial abundance in mid-lactation Holstein dairy cows. figshare. Journal contribution.
      ).
      Table 5Effects of supplementing a yeast culture product (YC) on relative abundance (%) of microbial species in mixed ruminal fluid in mid-lactation dairy cows
      Species
      Data were log-transformed before statistics. The SEM associated with log-transformed data are in log scale.
      Treatment
      Cows were assigned to a basal TMR (1.6 Mcal/kg of DM, 14.6% CP, 21.5% starch, and 38.4% NDF) plus 114 g/d of ground corn (CON; n = 20) or basal TMR plus 100 g/d of ground corn and 14 g/d of yeast culture (YC; n = 20; Culture Classic HD, Cellerate Yeast Solutions, Phibro Animal Health Corp.).
      SEM
      Largest SEM is shown.
      P-value
      CONYCTrtTimeTrt × T
      Interaction of treatment × time (d 0, 30, and 60).
      Parity
      None of the parameters had a parity × treatment effect (P > 0.20). A parity × treatment (P = 0.03) was observed only in Fibrobacter succinogenes.
      Anaerovibrio lipolytica1.64 × 10−32.61 × 10−30.520.340.180.590.45
      Butyrivibrio fibrisolvens1.55 × 10−31.51 × 10−30.090.770.300.630.97
      Butyrivibrio proteoclasticus8.62 × 10−28.46 × 10−20.240.920.840.210.72
      Eubacterium ruminantium9.09 × 10−31.36 × 10−20.190.050.120.900.53
      Fibrobacter succinogenes6.60 × 10−24.18 × 10−20.13<0.010.020.200.84
      Megasphaera elsdenii1.10 × 10−31.57 × 10−30.440.380.030.050.97
      Prevotella albensis3.86 × 10−37.61 × 10−30.580.200.970.390.27
      Prevotella bryantii7.86 × 10−21.31 × 10−10.320.070.970.74<0.01
      Prevotella ruminicola1.10 × 1009.49 × 10−10.180.38<0.010.660.10
      Prevotella brevis2.63 × 10−12.19 × 10−10.210.350.440.680.04
      Ruminococcus albus1.04 × 10−21.12 × 10−20.260.740.290.980.89
      Ruminococcus flavefaciens5.28 × 10−48.79 × 10−40.240.030.720.940.31
      Ruminobacter amylophilus1.24 × 10−37.12 × 10−40.270.05<0.010.570.99
      Selenomonas ruminantium3.30 × 10−12.86 × 10−10.060.02<0.010.920.58
      Succinimonas amylolytica7.05 × 10−43.84 × 10−40.290.040.360.760.68
      Succinivibrio dextrinosolvens4.58 × 10−36.08 × 10−30.360.420.430.980.11
      Streptococcus bovis2.03 × 10−31.78 × 10−30.350.680.010.860.34
      Treponema bryantii1.37 × 10−21.39 × 10−20.200.940.640.710.92
      1 Data were log-transformed before statistics. The SEM associated with log-transformed data are in log scale.
      2 Cows were assigned to a basal TMR (1.6 Mcal/kg of DM, 14.6% CP, 21.5% starch, and 38.4% NDF) plus 114 g/d of ground corn (CON; n = 20) or basal TMR plus 100 g/d of ground corn and 14 g/d of yeast culture (YC; n = 20; Culture Classic HD, Cellerate Yeast Solutions, Phibro Animal Health Corp.).
      3 Largest SEM is shown.
      4 Interaction of treatment × time (d 0, 30, and 60).
      5 None of the parameters had a parity × treatment effect (P > 0.20). A parity × treatment (P = 0.03) was observed only in Fibrobacter succinogenes.

      Blood Biomarkers

      Main effects and interactions for blood biomarkers results are presented in Table 6. There was a Trt × time interaction in urea (P < 0.01) and NEFA (P = 0.05). The Trt × time in urea was related to a lower (P < 0.01) urea concentration in YC cows than CON at 60 d (Figure 2A). This was translated in an overall lower (P < 0.01) urea in YC cows than CON. The Trt × time in NEFA was associated with greater (P < 0.01) NEFA in YC cows than CON at 30 d (Figure 2B). There was a trend (P = 0.07) for greater BHB in YC-fed cows in comparison to CON. Blood glucose was not affected by treatment effects.
      Table 6Effects of supplementing a yeast culture product (YC) on blood metabolites in mid-lactation dairy cows
      ParameterTreatment
      Cows were assigned to a basal TMR (1.6 Mcal/kg of DM, 14.6% CP, 21.5% starch, and 38.4% NDF) plus 114 g/d of ground corn (CON; n = 20) or basal TMR plus 100 g/d of ground corn and 14 g/d of yeast culture (YC; n = 20; Culture Classic HD, Cellerate Yeast Solutions, Phibro Animal Health Corp.).
      SEM
      Largest SEM is shown.
      P-value
      Parity was not significant (P > 0.44) in any of the parameters, and none had a parity × treatment (Trt) interaction (P > 0.20).
      CONYCTrtTimeTrt × T
      Interaction of treatment × time (d 0, 30, and 60).
      Glucose, mg/dL45.845.21.500.790.430.65
      Urea, μmol/L16.414.40.770.060.880.02
      NEFA,
      NEFA = nonesterified fatty acids.
      mmol/L
      0.1670.2210.0400.270.950.05
      BHB, mmol/L0.2070.2660.0380.070.420.25
      1 Cows were assigned to a basal TMR (1.6 Mcal/kg of DM, 14.6% CP, 21.5% starch, and 38.4% NDF) plus 114 g/d of ground corn (CON; n = 20) or basal TMR plus 100 g/d of ground corn and 14 g/d of yeast culture (YC; n = 20; Culture Classic HD, Cellerate Yeast Solutions, Phibro Animal Health Corp.).
      2 Largest SEM is shown.
      3 Parity was not significant (P > 0.44) in any of the parameters, and none had a parity × treatment (Trt) interaction (P > 0.20).
      4 Interaction of treatment × time (d 0, 30, and 60).
      5 NEFA = nonesterified fatty acids.
      Figure thumbnail gr2
      Figure 2Blood urea (A) and nonesterified fatty acids (NEFA) (B) in mid-lactation dairy cows fed a basal diet (CON) or a control diet supplemented with a yeast culture product (YC) during 60 d of the experimental period. Mean separations between treatments at a given time point were evaluated at a treatment × time interaction (P ≤ 0.10) and differences (*) were declared at P ≤ 0.05 and trend at P ≤ 0.10. Values are means, and SE are represented by vertical bars.

      DISCUSSION

      Effects on DMI, BW, BCS, and EB

      A variety of performance parameters have been used to evaluate the potential benefits of supplementing yeast products in dairy cows' diets, with most studies focusing on DMI, milk yield, and the rumen environment (
      • Poppy G.D.
      • Rabiee A.R.
      • Lean I.J.
      • Sanchez W.K.
      • Dorton K.L.
      • Morley P.S.
      A meta-analysis of the effects of feeding yeast culture produced by anaerobic fermentation of Saccharomyces cerevisiae on milk production of lactating dairy cows.
      ;
      • Elghandour M.M.Y.
      • Khusro A.
      • Adegbeye M.J.
      • Tan Z.
      • Abu Hafsa S.H.
      • Greiner R.
      • Ugbogu E.A.
      • Anele U.Y.
      • Salem A.Z.M.
      Dynamic role of single-celled fungi in ruminal microbial ecology and activities.
      ). Reductions in DMI have been reported in dairy cows supplemented with S. cerevisiae fermentation products. For instance,
      • Shi W.
      • Knoblock C.E.
      • Murphy K.V.
      • Bruinjé T.C.
      • Yoon I.
      • Ambrose D.J.
      • Oba M.
      Effects of supplementing a Saccharomyces cerevisiae fermentation product during the periparturient period on performance of dairy cows fed fresh diets differing in starch content.
      and
      • Dias J.D.L.
      • Silva R.B.
      • Fernandes T.
      • Barbosa E.F.
      • Graças L.E.C.
      • Araujo R.C.
      • Pereira R.A.N.
      • Pereira M.N.
      Yeast culture increased plasma niacin concentration, evaporative heat loss, and feed efficiency of dairy cows in a hot environment.
      observed a decrease in DMI when early and late lactation dairy cows, respectively, were fed yeast culture products. Similarly,
      • Alshaikh M.A.
      • Alsiadi M.Y.
      • Zahran S.M.
      • Mogawer H.H.
      • Aalshowime T.A.
      Effect of feeding yeast culture from different sources on the performance of lactating Holstein cows in Saudi Arabia.
      observed lower DMI in mid-lactation dairy cows supplemented with 2 commercial sources of yeast culture. In contrast, others have reported a lack of response on feed intake in mid-lactation dairy cows supplemented with yeast culture (
      • Zhu W.
      • Wei Z.
      • Xu N.
      • Yang F.
      • Yoon I.
      • Chung Y.
      • Liu J.
      • Wang J.
      Effects of Saccharomyces cerevisiae fermentation products on performance and rumen fermentation and microbiota in dairy cows fed a diet containing low quality forage.
      ). Although no effects were observed in DMI as kg/d, a trend (P = 0.07) for a lower DMI as % BW in YC was observed (Table 2). This lower DMI as % BW can partially explain the lower EB observed in YC cow than CON. Similar to our results,
      • Dias A.L.G.
      • Freitas J.A.
      • Micai B.
      • Azevedo R.A.
      • Greco L.F.
      • Santos J.E.P.
      Effect of supplemental yeast culture and dietary starch content on rumen fermentation and digestion in dairy cows.
      reported that cows supplemented with 15 g/d of inactivated S. cerevisiae had lower EB than control cows, which was associated with increased energy requirements for milk production in yeast supplemented cows.
      Dietary treatments did not affect BW or BCS in the current study (Table 2), which agrees with previous studies with live yeast (
      • Ambriz-Vilchis V.
      • Jessop N.S.
      • Fawcett R.H.
      • Webster M.
      • Shaw D.J.
      • Walker N.
      • Macrae A.I.
      Effect of yeast supplementation on performance, rumination time, and rumen pH of dairy cows in commercial farm environments.
      ;
      • Ferreira G.
      • Richardson E.S.
      • Teets C.L.
      • Akay V.
      Production performance and nutrient digestibility of lactating dairy cows fed low-forage diets with and without the addition of a live-yeast supplement.
      ) and yeast culture (
      • Dias J.D.L.
      • Silva R.B.
      • Fernandes T.
      • Barbosa E.F.
      • Graças L.E.C.
      • Araujo R.C.
      • Pereira R.A.N.
      • Pereira M.N.
      Yeast culture increased plasma niacin concentration, evaporative heat loss, and feed efficiency of dairy cows in a hot environment.
      ). Yeast supplementation effects on BCS and BW are seldomly reported in adult dairy cows and seem to have a more consistent effect when supplemented to young ruminants (
      • Alugongo G.M.
      • Xiao J.
      • Wu Z.
      • Li S.
      • Wang Y.
      • Cao Z.
      Review: Utilization of yeast of Saccharomyces cerevisiae origin in artificially raised calves.
      ).

      Production Variables and Feed Efficiency

      Several studies utilizing yeast culture products have shown inconsistent results in milk performance parameters. For instance,
      • Poppy G.D.
      • Rabiee A.R.
      • Lean I.J.
      • Sanchez W.K.
      • Dorton K.L.
      • Morley P.S.
      A meta-analysis of the effects of feeding yeast culture produced by anaerobic fermentation of Saccharomyces cerevisiae on milk production of lactating dairy cows.
      performed a meta-analysis on 36 yeast culture supplementation studies and observed a positive effect on milk yield; however, yeast culture had the greatest influence on milk yield in early lactation. In a recent study in mid-lactation dairy cows, an increase of 3.3 kg/d of milk yield was observed when cows were fed an inactivated dry S. cerevisiae yeast culture (
      • Dias A.L.G.
      • Freitas J.A.
      • Micai B.
      • Azevedo R.A.
      • Greco L.F.
      • Santos J.E.P.
      Effect of supplemental yeast culture and dietary starch content on rumen fermentation and digestion in dairy cows.
      ). However, other authors did not observe effects on milk yield in mid (
      • Schingoethe D.J.
      • Linke K.N.
      • Kalscheur K.F.
      • Hippen A.R.
      • Rennich D.R.
      • Yoon I.
      Feed efficiency of mid-lactation dairy cows fed yeast culture during summer.
      ) and early lactation (
      • Shi W.
      • Knoblock C.E.
      • Murphy K.V.
      • Bruinjé T.C.
      • Yoon I.
      • Ambrose D.J.
      • Oba M.
      Effects of supplementing a Saccharomyces cerevisiae fermentation product during the periparturient period on performance of dairy cows fed fresh diets differing in starch content.
      ) dairy cows supplemented with yeast culture. Similar to those results reported in the literature, YC supplementation in the current study did not cause any effect on milk yield adding to the contrasting effects of yeast culture supplementation on lactation performance.
      Although ECM and milk yield did not differ between the treatments, YC cows tended (P = 0.09) to be more efficient in producing milk in terms of ECM/DMI and Milk/DMI ratios (Table 3). The increase in feed efficiency with no effects on milk yield has been reported previously in late (
      • Dias J.D.L.
      • Silva R.B.
      • Fernandes T.
      • Barbosa E.F.
      • Graças L.E.C.
      • Araujo R.C.
      • Pereira R.A.N.
      • Pereira M.N.
      Yeast culture increased plasma niacin concentration, evaporative heat loss, and feed efficiency of dairy cows in a hot environment.
      ) and early (
      • Shi W.
      • Knoblock C.E.
      • Murphy K.V.
      • Bruinjé T.C.
      • Yoon I.
      • Ambrose D.J.
      • Oba M.
      Effects of supplementing a Saccharomyces cerevisiae fermentation product during the periparturient period on performance of dairy cows fed fresh diets differing in starch content.
      ) lactation dairy cows, whereas others demonstrated a trend for greater milk yield without differences on ECM and ECM/DMI (
      • Zhu W.
      • Wei Z.
      • Xu N.
      • Yang F.
      • Yoon I.
      • Chung Y.
      • Liu J.
      • Wang J.
      Effects of Saccharomyces cerevisiae fermentation products on performance and rumen fermentation and microbiota in dairy cows fed a diet containing low quality forage.
      ). Similar to the current study,
      • Schingoethe D.J.
      • Linke K.N.
      • Kalscheur K.F.
      • Hippen A.R.
      • Rennich D.R.
      • Yoon I.
      Feed efficiency of mid-lactation dairy cows fed yeast culture during summer.
      evaluated the effect of 60 g/d yeast culture supplementation on mid-lactation dairy cows and observed no effect in either milk yield or ECM. However, a significant increase in feed efficiency as ECM/DMI was reported in cows fed yeast culture.
      • Dias J.D.L.
      • Silva R.B.
      • Fernandes T.
      • Barbosa E.F.
      • Graças L.E.C.
      • Araujo R.C.
      • Pereira R.A.N.
      • Pereira M.N.
      Yeast culture increased plasma niacin concentration, evaporative heat loss, and feed efficiency of dairy cows in a hot environment.
      showed an increase in feed efficiency in late lactation cows supplemented with 15 g/d of inactivated yeast culture, attributed to lower DMI in yeast culture fed cows. The trend for improved feed efficiency observed in YC cows in the present study may be from the contribution of yeast metabolites present in the YC. Although the mechanisms of action of yeast culture have not been clearly established, its effects have been attributed to functional metabolites such as growth factors, B vitamins, AA, organic acids, enzymes, and other fermentation products (
      • Ghazanfar S.
      • Khalid N.
      • Ahmed I.
      • Imran M.
      Probiotic yeast: Mode of action and its effects on ruminant nutrition.
      ;
      • Elghandour M.M.Y.
      • Khusro A.
      • Adegbeye M.J.
      • Tan Z.
      • Abu Hafsa S.H.
      • Greiner R.
      • Ugbogu E.A.
      • Anele U.Y.
      • Salem A.Z.M.
      Dynamic role of single-celled fungi in ruminal microbial ecology and activities.
      ). These metabolites are thought to influence ruminal fermentation by increasing VFA concentration and stimulating the growth of cellulolytic bacteria (
      • Opsi F.
      • Fortina R.
      • Tassone S.
      • Bodas R.
      • López S.
      Effects of inactivated and live cells of Saccharomyces cerevisiae on in vitro ruminal fermentation of diets with different forage:concentrate ratio.
      ;
      • Zhu W.
      • Wei Z.
      • Xu N.
      • Yang F.
      • Yoon I.
      • Chung Y.
      • Liu J.
      • Wang J.
      Effects of Saccharomyces cerevisiae fermentation products on performance and rumen fermentation and microbiota in dairy cows fed a diet containing low quality forage.
      ), improving DM digestibility (
      • Miller-Webster T.
      • Hoover W.H.
      • Holt M.
      • Nocek J.E.
      Influence of yeast culture on ruminal microbial metabolism in continuous culture.
      ;
      • Dias A.L.G.
      • Freitas J.A.
      • Micai B.
      • Azevedo R.A.
      • Greco L.F.
      • Santos J.E.P.
      Effect of supplemental yeast culture and dietary starch content on rumen fermentation and digestion in dairy cows.
      ) and consequently, feed efficiency.

      Blood Biomarkers

      In ruminants, the concentration of urea in blood and milk is highly correlated with N recycling and ruminal protein degradation due to the conversion of ruminal NH3 into urea in the liver (
      • Colmenero J.J.O.
      • Broderick G.A.
      Effect of dietary crude protein concentration on milk production and nitrogen utilization in lactating dairy cows.
      ). The supplementation with yeast culture has been associated with a more efficient conversion of ruminal NH3 into microbial protein and decrease NH3 concentration in the rumen while reducing the N excretion as urinary urea or MUN (
      • Hristov A.N.
      • Varga G.
      • Cassidy T.
      • Long M.
      • Heyler K.
      • Karnati S.K.
      • Corl B.
      • Hovde C.J.
      • Yoon I.
      Effect of Saccharomyces cerevisiae fermentation product on ruminal fermentation and nutrient utilization in dairy cows.
      ). In the present study, the ruminal NH3 was similar between groups (Table 4), and by the end of the experiment, YC cows had lower blood urea ~13 µmol/L, whereas CON cows had much higher levels (~18 µmol/L), indicating a higher level of ureagenesis. This increase in urea in CON cows was not matched by an increase in MUN, suggesting that blood urea was either excreted in urine or recycled back to the rumen. Perhaps a more intensive evaluation of ruminal, metabolic, and milk parameters related to N recycling will confirm these potential effects of the yeast culture used in this study.
      The greater NEFA in YC cows at 30 d (Figure 2B) coupled with the trend (P = 0.07) for greater BHB in YC cows than CON (Table 6) are in line with the lower EB in YC cows than CON (Table 2). However, although elevated NEFA and BHB are commonly associated with a lipolytic state due to low EB (
      • Ospina P.A.
      • McArt J.A.
      • Overton T.R.
      • Stokol T.
      • Nydam D.V.
      Using nonesterified fatty acids and beta-hydroxybutyrate concentrations during the transition period for herd-level monitoring of increased risk of disease and decreased reproductive and milking performance.
      ), our data does not indicate cows were in a highly active lipolytic state but suggests that YC and CON cows have different metabolic demands as a result of the lower EB in YC cows.

      Rumen Fermentation Parameters

      Lactation diets are commonly formulated to contain greater amounts of starch in comparison to late lactation or dry cow diets to increase energy intake to support high milk production; however, this dietary condition can alter the normal microbial ecology in the rumen and increase the risk to develop digestive disorders, such as ruminal acidosis (
      • Enemark J.M.
      The monitoring, prevention and treatment of sub-acute ruminal acidosis (SARA): A review.
      ;
      • Abdela N.
      Sub-acute ruminal acidosis (SARA) and its consequence in dairy cattle: A review of past and recent research at global prospective.
      ). In this context, there have been studies showing a lack of response of yeast culture supplementation on rumen pH (
      • Hristov A.N.
      • Varga G.
      • Cassidy T.
      • Long M.
      • Heyler K.
      • Karnati S.K.
      • Corl B.
      • Hovde C.J.
      • Yoon I.
      Effect of Saccharomyces cerevisiae fermentation product on ruminal fermentation and nutrient utilization in dairy cows.
      ;
      • Zhu W.
      • Wei Z.
      • Xu N.
      • Yang F.
      • Yoon I.
      • Chung Y.
      • Liu J.
      • Wang J.
      Effects of Saccharomyces cerevisiae fermentation products on performance and rumen fermentation and microbiota in dairy cows fed a diet containing low quality forage.
      ). In contrast, others have observed an effect of yeast culture on maintaining rumen pH, which has been attributed to a reduction in ruminal lactate production (
      • Dias A.L.G.
      • Freitas J.A.
      • Micai B.
      • Azevedo R.A.
      • Greco L.F.
      • Santos J.E.P.
      Effect of supplemental yeast culture and dietary starch content on rumen fermentation and digestion in dairy cows.
      ) by stimulating the growth of lactate-utilizing bacteria such as M. elsdenii (
      • Tun H.M.
      • Li S.
      • Yoon I.
      • Meale S.J.
      • Azevedo P.A.
      • Khafipour E.
      • Plaizier J.C.
      Saccharomyces cerevisiae fermentation products (SCFP) stabilize the ruminal microbiota of lactating dairy cows during periods of a depressed rumen pH.
      ). A similar effect was observed in the present study, where YC-fed cows maintained a greater ruminal pH at 3 h postfeeding than CON (Table 4). When collecting rumen fluid taken at 4 h postfeeding via esophageal tubing,
      • Plaizier J.C.
      Replacing chopped alfalfa hay with alfalfa silage in barley grain and alfalfa-based total mixed rations for lactating dairy cows.
      used a threshold pH of 6.0 for SARA condition. Although differences in ruminal pH between CON and YC cows were small (5.86 vs. 6.03), the biological relevance of these data are underscored when compared with
      • Plaizier J.C.
      Replacing chopped alfalfa hay with alfalfa silage in barley grain and alfalfa-based total mixed rations for lactating dairy cows.
      SARA threshold pH of 6.0. Suggesting that cows in CON group were at a greater risk of developing SARA than YC cows.
      It is important to note that there was a small inclusion rate of monensin, an ionophore with antimicrobial properties, in the basal diet in the present study (Table 1). This feed additive commonly leads to alterations in the ruminal parameters, including acetate and propionate (
      • Bergen W.G.
      • Bates D.B.
      Ionophores: Their effect on production efficiency and mode of action.
      ). However, according to
      • Erasmus L.J.
      • Robinson P.H.
      • Ahmadi A.
      • Hinders R.
      • Garrett J.E.
      Influence of prepartum and postpartum supplementation of a yeast culture and monensin, or both, on ruminal fermentation and performance of multiparous dairy cows.
      , no extent or complementary effects were observed between yeast culture and monensin by comparing their individual and combined effect on performance and ruminal parameters of dairy cows.
      In the current study, cows fed YC had greater total VFA than CON at 60 d (Figure 1A), despite the lack of effect on total VFA indicated by other studies in mid-lactation dairy cows supplemented with yeast culture (
      • Hristov A.N.
      • Varga G.
      • Cassidy T.
      • Long M.
      • Heyler K.
      • Karnati S.K.
      • Corl B.
      • Hovde C.J.
      • Yoon I.
      Effect of Saccharomyces cerevisiae fermentation product on ruminal fermentation and nutrient utilization in dairy cows.
      ;
      • Li S.
      • Yoon I.
      • Scott M.
      • Khafipour E.
      • Plaizier J.C.
      Impact of Saccharomyces cerevisiae fermentation product and subacute ruminal acidosis on production, inflammation, and fermentation in the rumen and hindgut of dairy cows.
      ;
      • Dias A.L.G.
      • Freitas J.A.
      • Micai B.
      • Azevedo R.A.
      • Greco L.F.
      • Santos J.E.P.
      Effect of supplemental yeast culture and dietary starch content on rumen fermentation and digestion in dairy cows.
      ). This greater total VFA has been attributed to improved stability of rumen fermentation due to yeast culture supplementation, coupled with an increase in rumen fiber-degrading bacteria (i.e., R. flavefaciens;
      • Zhu W.
      • Wei Z.
      • Xu N.
      • Yang F.
      • Yoon I.
      • Chung Y.
      • Liu J.
      • Wang J.
      Effects of Saccharomyces cerevisiae fermentation products on performance and rumen fermentation and microbiota in dairy cows fed a diet containing low quality forage.
      ). Similarly,
      • Oeztuerk H.
      Effects of live and autoclaved yeast cultures on ruminal fermentation in vitro.
      reported greater total VFA production during an in vitro trial with either live or autoclaved yeast. Therefore, the increase in total VFA in YC cows at 60 d can be partially ascribed to enhanced rumen benefits on increasing cellulolytic bacteria.
      Yeast culture supplementation has been reported to alter not only total VFA but also the profile of VFA. For instance,
      • Miller-Webster T.
      • Hoover W.H.
      • Holt M.
      • Nocek J.E.
      Influence of yeast culture on ruminal microbial metabolism in continuous culture.
      reported greater propionate and lower acetate in a continuous culture study with yeast fermentation products. Similarly, a lower molar proportion of acetate was observed in YC cows (Table 4).
      • Acharya S.
      • Pretz J.P.
      • Yoon I.
      • Scott M.F.
      • Casper D.P.
      Effects of Saccharomyces cerevisiae fermentation products on the lactational performance of mid-lactation dairy cows.
      observed that cows fed different yeast culture products had greater propionate and lower acetate than control cows, whereas others have observed lower ruminal lactate in dairy cows supplemented with yeast culture (
      • Dias A.L.G.
      • Freitas J.A.
      • Micai B.
      • Azevedo R.A.
      • Greco L.F.
      • Santos J.E.P.
      Effect of supplemental yeast culture and dietary starch content on rumen fermentation and digestion in dairy cows.
      ). The lower acetate coupled with lower lactate was partially explained by S. cerevisiae culture's stimulatory growth effect on rumen microbes that metabolize lactate into VFA, such as propionate (
      • Callaway E.S.
      • Martin S.A.
      Effects of a Saccharomyces cerevisiae culture on ruminal bacteria that utilize lactate and digest cellulose.
      ;
      • Tun H.M.
      • Li S.
      • Yoon I.
      • Meale S.J.
      • Azevedo P.A.
      • Khafipour E.
      • Plaizier J.C.
      Saccharomyces cerevisiae fermentation products (SCFP) stabilize the ruminal microbiota of lactating dairy cows during periods of a depressed rumen pH.
      ). In the current study, the increase in total VFA in YC cows could be explained by the greater R. flavefaciens in YC cows than CON, a fiber-digesting bacterium that responds to yeast culture supplementation (
      • Zhu W.
      • Wei Z.
      • Xu N.
      • Yang F.
      • Yoon I.
      • Chung Y.
      • Liu J.
      • Wang J.
      Effects of Saccharomyces cerevisiae fermentation products on performance and rumen fermentation and microbiota in dairy cows fed a diet containing low quality forage.
      ). In addition, ruminal R. flavefaciens has been reported to require isovalerate (
      • Allison M.J.
      • Bryant M.P.
      • Doetsch R.N.
      Studies on the metabolic function of branched-chain volatile fatty acids, growth factors for ruminococci. I. Incorporation of isovalerate into leucine.
      ), which might help explain the greater abundance of R. flavefaciens in YC cows because a greater isovalerate was observed in YC cows (Table 4).
      The decrease in acetate in YC cows can partially explain by the lower abundance of Fibrobacter succinogenes in YC cows, which is an important fiber-digesting bacterium. The F. succinogenes abundance was further described by a parity × Trt, where lower F. succinogenes in YC cows was only observed in primiparous but not multiparous cows (Supplemental Figure S1, https://doi.org/10.6084/m9.figshare.15145071.v1;
      • Halfen J.
      • Carpinelli N.A.
      • Pino F.A.B.D.
      • Chapman J.
      • Sharman E.
      • Anderson J.
      Effects of yeast culture supplementation on lactation performance and rumen fermentation profile and microbial abundance in mid-lactation Holstein dairy cows. figshare. Journal contribution.
      ). The lack of a similar parity × Trt in acetate renders this connection with F. succinogenes inconclusive especially when the abundance of other fiber-digesting bacteria such as E. ruminantium and R. flavefaciens was increased in YC cows.

      Abundance of Ruminal Bacteria

      The positive effects of yeast supplementation on R. flavefaciens growth have been documented before (
      • Callaway E.S.
      • Martin S.A.
      Effects of a Saccharomyces cerevisiae culture on ruminal bacteria that utilize lactate and digest cellulose.
      ;
      • Mao H.L.
      • Mao H.L.
      • Wang J.K.
      • Liu J.X.
      • Yoon I.
      Effects of Saccharomyces cerevisiae fermentation product on in vitro fermentation and microbial communities of low-quality forages and mixed diets.
      ). Such effects are related to yeast culture growth factors (i.e., organic acids, B vitamins, and AA) that stimulate fiber-digesting bacteria (
      • Callaway E.S.
      • Martin S.A.
      Effects of a Saccharomyces cerevisiae culture on ruminal bacteria that utilize lactate and digest cellulose.
      ), thus, benefiting fiber-digesting bacteria such as R. flavefaciens. This is in line with results in the current study, where a greater R. flavefaciens abundance was observed in the YC group. Additionally, an in vitro study comparing 2 levels of rumen pH (5.8 vs. 6.5) observed a lower concentration of R. flavefaciens at 5.8 pH in comparison to 6.5 (
      • Jiao P.
      • Wei C.
      • Sun Y.
      • Xie X.
      • Zhang Y.
      • Wang S.
      • Hu G.
      • AlZahal O.
      • Yang W.
      Screening of live yeast and yeast derivatives for their impact of strain and dose on in vitro ruminal fermentation and microbial profiles with varying media pH levels in high-forage beef cattle diet.
      ), confirming prior data suggesting that fiber-degrading bacteria are sensitive to low rumen pH (
      • Chaucheyras-Durand F.
      • Walker N.D.
      • Bach A.
      Effects of active dry yeasts on the rumen microbial ecosystem: Past, present and future.
      ). Then, it is likely that the greater abundance of R. flavefaciens in YC cows was partially driven by a more neutral rumen pH observed in this group.
      The E. ruminantium, a gram-positive bacterium, which also plays a cellulolytic role in the rumen, tended (P = 0.09) to be more predominant in YC-fed cows (Table 4). Similar to the present study, a greater relative abundance of E. ruminantium was reported in beef cows classified as most efficient based on low residual feed intake (RFI), and such efficiency was linked to E. ruminantium, improving fiber degradation and feed digestibility (
      • Elolimy A.A.
      • Abdelmegeid M.K.
      • McCann J.C.
      • Shike D.W.
      • Loor J.J.
      Residual feed intake in beef cattle and its association with carcass traits, ruminal solid-fraction bacteria, and epithelium gene expression.
      ).
      The Ruminobacter genus is comprised of the main amylolytic bacteria, including Ruminobacter amylophilus, a gram-negative bacterium, which primarily relies on starch, maltose, and maltodextrins as an energy source (
      • Anderson K.L.
      Biochemical analysis of starch degradation by Ruminobacter amylophilus 70.
      ). The YC supplementation promoted a lower proportion of R. amylophilus. According to
      • Jiang Y.
      • Ogunade I.M.
      • Qi S.
      • Hackmann T.J.
      • Staples C.R.
      • Adesogan A.T.
      Effects of the dose and viability of Saccharomyces cerevisiae. 1. Diversity of ruminal microbes as analyzed by Illumina MiSeq sequencing and quantitative PCR.
      , the addition of live yeast instead of inactivated yeast to the diet of late lactation dairy cows decreased the relative abundance of Ruminobacter genus, which could at least support the case for a lower abundance of R. amylophilus in YC cows in the current study. Another amylolytic bacteria which abundance was depressed by yeast culture supplementation was Succinimonas amylolytica. This bacterium has been related to SARA conditions in dairy goats, which, along with results on R. amylophilus, might help explain the overall greater ruminal pH observed in YC cows in the current study. Similar to the current study, others have correlated a lower ruminal abundance of S. amylolytica with feed efficiency in beef cattle (
      • Elolimy A.A.
      • Abdelmegeid M.K.
      • McCann J.C.
      • Shike D.W.
      • Loor J.J.
      Residual feed intake in beef cattle and its association with carcass traits, ruminal solid-fraction bacteria, and epithelium gene expression.
      ) and dairy cows (
      • Shabat S.K.
      • Sasson G.
      • Doron-Faigenboim A.
      • Durman T.
      • Yaacoby S.
      • Berg Miller M.E.
      • White B.A.
      • Shterzer N.
      • Mizrahi I.
      Specific microbiome-dependent mechanisms underlie the energy harvest efficiency of ruminants.
      ;
      • Elolimy A.A.
      • Arroyo J.M.
      • Batistel F.
      • Iakiviak M.A.
      • Loor J.J.
      Association of residual feed intake with abundance of ruminal bacteria and biopolymer hydrolyzing enzyme activities during the peripartal period and early lactation in Holstein dairy cows.
      ).
      Selenomonas ruminantium has been commonly associated with starch digestion while producing lactic, acetic, and propionic acids (
      • Zhou M.
      • Chen Y.
      • Guan L.L.
      Rumen bacteria.
      ). This microbial species is a propionate producer through decarboxylation of succinate and utilizes a wide range of substrates, including lactate (
      • Fernando S.C.
      • Purvis 2nd, H.T.
      • Najar F.Z.
      • Sukharnikov L.O.
      • Krehbiel C.R.
      • Nagaraja T.G.
      • Roe B.A.
      • Desilva U.
      Rumen microbial population dynamics during adaptation to a high-grain diet.
      ). In a study where they evaluate RFI in dairy cows, they observed that the acrylate pathway that utilizes lactate to propionate was enriched in the low RFI (most efficient) dairy cows (
      • Shabat S.K.
      • Sasson G.
      • Doron-Faigenboim A.
      • Durman T.
      • Yaacoby S.
      • Berg Miller M.E.
      • White B.A.
      • Shterzer N.
      • Mizrahi I.
      Specific microbiome-dependent mechanisms underlie the energy harvest efficiency of ruminants.
      ). Such pathway was further evaluated at the genomic level with lactate-utilizing bacteria, including A. lipolytica, S. ruminantium, and M. elsdenii. From these, only M. elsdenii presented an enrichment of the acrylate pathway. This indicates that highly efficient dairy cows will rely on M. elsdenii to channel propionate production from lactate. In the current study, the higher abundance of M. elsdenii at 30 d in YC cows might partially explain the trend for greater feed efficiency in this group in comparison to CON.
      Taken together, these data indicate that YC supplementation can positively affect key microbial species related to fiber digestion and lactate-utilizing through the supply of functional metabolites (e.g., AA, peptides, vitamins, and organic acids) and promote essential and consistent conditions such as a more neutral rumen pH.

      CONCLUSIONS

      The findings observed in this study suggested that YC supplementation improves the rumen environment by promoting a more neutral rumen pH and increasing total VFA and decreasing ruminal acetate. Additionally, our results suggest that feeding YC enhanced specific ruminal bacteria populations, including lactate-utilizers (i.e., M. elsdenii) and fiber digesters (i.e., E. ruminantium and Ruminococcus flavefaciens). The lower abundance of amylolytic bacteria in YC cows, including Ruminobacter amylophylus and Succinimonas amylolytica, could partially explain the ruminal lower pH in YC cows. In contrast to CON cows, YC cows had lower EB, which could be partly described by the trend for lower DMI as % of BW. Overall, the changes in ruminal bacteria species evaluated in this study by yeast culture supplementation seem to promote specific fiber-digesting and lactate-utilizing bacteria while decreasing amylolytic bacteria, which might have partially promoted more neutral rumen pH, greater total VFA, and isovalerate. A more powerful study with multiple diurnal rumen collections would be required to clearly demonstrate the level of improvement on these rumen parameters and ruminal bacteria populations.

      ACKNOWLEDGMENTS

      The authors gratefully acknowledge Phibro Animal Health Corporation (Teaneck, NJ) for partial financial support of this research. The authors thank Peter Linke and Danielle Tews of the Dairy Research and Training Facility (South Dakota State University, Brookings) staff for help with animal management. J. D. Chapman and E. D. Sharman are employees of Phibro Animal Health Corporation, which had a role in the study design and provided financial support to cover costs of animal use, data collection, and sample analysis. The authors have not stated any other conflicts of interest.

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