Evaluation of the protective effect of probiotics fed to dairy cows during a subacute ruminal acidosis challenge

Animal Feed Science and Technology 153 (2009) 278–291

Contents lists available at ScienceDirect

Animal Feed Science and Technology journal homepage: www.elsevier.com/locate/anifeedsci

Evaluation of the protective effect of probiotics fed to dairy cows during a subacute ruminal acidosis challenge夽 J. Chiquette ∗ Agriculture and Agri-Food Canada, Dairy and Swine Research and Development Centre, Sherbrooke, QC, Canada, J1 M 1Z3

a r t i c l e

i n f o

Article history: Received 26 September 2008 Received in revised form 7 July 2009 Accepted 9 July 2009 Keywords: Probiotic Subacute ruminal acidosis Dairy cow

a b s t r a c t Subacute ruminal acidosis (SARA) can be extremely costly when it occurs in dairy cows. The use of probiotic supplements to stabilize rumen pH during the transition period following parturition could attenuate the symptoms of this metabolic disorder. Four ruminally fistulated Holstein dairy cows in mid to late lactation were assigned to the following experimental treatments in a 4 × 4 Latin square design: (1) control (C); (2) 0.6 g/cow/d of a fermentation extract of Aspergillus oryzae (AO-0.6); (3) 3 g/cow/d of A. oryzae (AO-3.0); and (4) 2 g/cow/d of a probiotic combination consisting of Enterococcus faecium and Saccharomyces cerevisiae (ES). Each period of the Latin square consisted of three weeks of adaptation to the respective treatments followed by four days of induction of SARA and three days of rest. During the four days of induction of SARA, 0.30 of ad libitum intake of the total mixed ration (TMR) was replaced with wheat and barley pellets (WBP) containing 0.5 ground wheat and 0.5 ground barley. Ruminal pH was recorded continuously using an indwelling pH probe over a 24 h period for each week of the adaptation period and continuously over the four days of SARA induction. Average ruminal pH was lower (P=0.0001) during SARA than during the weeks of adaptation (5.6 versus 6.1). When the cows were fed ES, the minimum pH recorded was higher (P=0.05) (5.0) than when they were in the control (4.4). The proportion of time during which pH was between 5.6 and 6.0 was higher (P=0.05) with ES (0.31) than with C (0.22). Mean pH tended (P=0.06) to be higher with ES (5.84)

Abbreviations: AO, Aspergillus oryzae; C, control; DIM, days in milk; DM, dry matter; EF, Enterococcus faecium; ES, combination of Enterococcus faecium and Saccharomyces cerevisiae; SARA, subacute ruminal acidosis; SC, Saccharomyces cerevisiae; TMR, total mixed ration; VFA, volatile fatty acids; WBP, wheat and barley pellets. 夽 Contribution number 1000 from Agriculture and Agri-Food Canada. ∗ Tel.: +1 819 565 9171; fax: +1 819 564 5507. E-mail address: [email protected]. 0377-8401/$ – see front matter. Crown Copyright © 2009 Published by Elsevier B.V. All rights reserved. doi:10.1016/j.anifeedsci.2009.07.001

J. Chiquette / Animal Feed Science and Technology 153 (2009) 278–291

279

than with C (5.41), and the proportion of time during which pH was below 5.2 tended (P=0.09) to be lower with ES (0.09) than with C (0.31). AO-0.6 tended (P=0.07) to increase minimum pH (4.96) compared with C (4.43) and tended (P=0.08) to decrease the percentage of time during which pH was below 5.2 (0.08 versus 0.31 for AO-0.6 and C, respectively). The addition of probiotics during SARA had no effect on the number of cellulolytic bacteria or on the population of Megasphaera elsdenii in the rumen. Crown Copyright © 2009 Published by Elsevier B.V. All rights reserved.

1. Introduction Ruminal acidosis is a serious digestive disorder that occurs when large quantities of rapidly fermentable carbohydrates, exceeding the buffering capacity of the rumen, are fed to ruminants. In the most acute forms, pH drops below 5.2 (Mutsvangwa et al., 2002), whereas in the subacute form, pH is between 5.2 and 5.6 (Keunen et al., 2002). If pH drops below 6.0, fibre digestibility is impaired (Stewart, 1977) and, when pH drops between 5.2 and 5.6, the animal may show clinical signs of subacute ruminal acidosis (SARA), which causes physical discomfort and decreased production (Duffield et al., 2004). Clinical signs of SARA vary and may include mild transient anorexia, intermittent diarrhea, dehydration, poor body condition, depression, decreased rumen motility, laminitis, unexplained abscesses and decreased milk production (Duffield et al., 2004). The culling rate can be as high as 0.45 in a dairy herd having SARA problems, which is 0.20 higher than the ideal culling rate (Programme d’analyse des troupeaux laitiers du Québec, 1999). Costs of SARA resulting from lost production were estimated at US $1.12 per cow per day in a 500-cow herd diagnosed with SARA (Stone, 1999). Several dietary strategies proposed for use during this critical period were reported in the review by Krause and Oetzel (2006), including feeding unprocessed grains that are less fermentable, providing total mixed rations (TMR) instead of separate ingredients, feeding smaller meals more frequently and at regular intervals, and introducing grains progressively so that the rumen microbial population can adapt. It is also recommended that ration formulations provide a minimum content of total and physically effective fibre. This is not always sufficient, however, because cows are able to sort out long feed particles (Bach, 2007). In spite of these precautions, some cattle are more susceptible to a pH drop, and additional measures to prevent SARA could be beneficial. Several studies investigated the role of monensin (an antibiotic ionophore) in preventing SARA. Some have reported an elevation of ruminal pH with monensin through a reduction in concentrations of volatile fatty acids (VFA), with no effect on ruminal lactate (Burrin and Britton, 1986). Others have reported no effect on ruminal pH (Mutsvangwa et al., 2002; Osborne et al., 2004), but neither of these studies reported lactate concentrations. Mutsvangwa et al. (2002) reported no effect of monensin on VFA concentrations. However, owing to general concerns about antibiotics, recent research evaluated the use of live microorganisms to maintain higher ruminal pH during feeding of early lactation dairy cows (Nocek et al., 2002; Beauchemin et al., 2003). Supplementation with live yeast has been associated with stabilization of ruminal pH through promotion of the use of lactic acid by lactate-utilizing bacteria, fostering microbial growth and competition with rumen bacteria for rapidly fermentable carbohydrates (Bach, 2007). Saccharomyces cells are believed to eliminate traces of oxygen in the rumen, thus helping oxygen sensitive bacteria to grow, as well as resulting in an increase in viable anaerobic bacteria (Marden et al., 2008). In vitro experiments demonstrated that culture filtrates of Saccharomyces cerevisiae (SC) stimulated the rate of cellulose digestion by the predominant cellulolytics Fibrobacter succinogenes and Ruminococcus flavefaciens and the utilization of lactate by Megasphaera elsdenii and Selenomonas ruminantium (Callaway and Martin, 1997). Aspergillus oryzae (AO) fermentation extracts provide additional cellulases and hemicellulases that contribute to fibre digestion. These fungal fibrolytic enzymes may act synergistically with fibrolytic rumen bacterial and fungal glycohydrolases to improve the efficiency and/or rate of plant cell wall degradation (Varel et al., 1993). Chiquette (1995) reported increased milk efficiency when dairy cows

280

J. Chiquette / Animal Feed Science and Technology 153 (2009) 278–291

Table 1 Composition of the diets during the project (g/kg).

Total mixed ration Hay Wheat and barley pellets (0.5/0.5)

Adaptation

Challenge

Resting

950 50 –

700 – 300

950 50 –

received AO alone or in combination with SC. Williams and Newbold (1990) reported that AO reduced the lactate peak 2 h after sheep were fed hay and barley grain. They also observed an increased bacterial population, which they attributed to a better stabilization of ruminal pH. The response to AO has been highly variable, however, and other authors saw no improvements (Oellermann et al., 1990; Sievert and Shaver, 1993). Because all these studies reporting no effects used the commercially recommended dose, the present study added a treatment consisting of five times the recommended dose. When incorporated into the diet, certain probiotics that synthesize lactic acid may sustain a higher and more stable level of lactic acid in the rumen which would stimulate the growth of lactic acid utilizing bacteria. The lactic acid utilizing bacteria would then be in a greater number when a lactic acid surge occurs following ingestion of large quantities of rapidly fermentable carbohydrates (Nocek et al., 2002). In a study by, Nocek et al. (2002), a combination of SC and two lactate producers, Lactobacillus plantarum and Enterococcus faecium (EF), that was fed daily to dairy cows at 105 cfu/ml of rumen fluid resulted in slightly less acidic rumen conditions and a higher digestion of ear corn dry matter (DM) and corn silage. The present study reports effects of daily addition of AO at two dose levels, and a combination of EF and SC, on rumen fermentation characteristics and milk production during a SARA challenge. 2. Materials and methods 2.1. Animals, feeding and sampling procedure All animals in this experiment were cared for according to the standards set by the Canadian Council on Animal Care (1993). Four ruminally fistulated Holstein dairy cows averaging 741 ± 55.1 kg body weight and 212 ± 19.4 days in milk (DIM) were assigned to the following experimental treatments in a 4 × 4 Latin square design: (1) control (C); (2) 0.6 g/cow/d of AO (AO-0.6) (Probiotec Inc., St-Eustache, QC, Canada); (3) 3 g/cow/d of AO (AO-3.0); and (4) 2 g/cow/d of a probiotic combination (ES) providing 5 × 109 cells of two lactic acid producing strains of EF and 2 × 109 cells of SC (Probios TC, Chr. Hansen, Milwaukee, WI, USA). The composition of the diets during the project is in Table 1. The Latin square Table 2 Composition of the total mixed ration. g/kg DMa Grass silage Corn silage Corn grain Protein supplementb Soybean meal, solvent (450 g/kg CP) Vitamin–mineral mixturec a

251 255 302 71 101 20

Dry matter. Protein supplement contained the following ingredients (g/kg): corn distiller’s grain (250), wheat distiller’s grain (150), canola meal (150), and SoyPLUS (450). c Vitamin–mineral mixture contained the following major minerals (g/kg): Ca (95), P (55), Mg (55), Na (130), Cl (150), K (14), S (21); the following minor minerals (mg/kg): Fe (2745), Mn (2065), Zn (3000), Cu (495), I (69), Co (33), Se (20); and the following vitamins (UI/kg): Vitamin A (501,859), Vitamin D (65,000), Vitamin E (2600). b

J. Chiquette / Animal Feed Science and Technology 153 (2009) 278–291

281

Table 3 Nutrient composition (g/kg DMa ). TMRb

WBPc

Hay

Dry matter

500

892

893

105 ◦ C dry matter (g/kg) Ash Crude protein aNDFd ADFe Ether extract NSCf

61 150 324 229 44 420

29 151 165 84 19 636

57 91 650 404 17 185

a

Dry matter.

b

Total mixed ration. Wheat and barley pellets (0.5/0.5). Neutral detergent fibre assayed with a heat stable amylase. Acid detergent fibre.

c d e f

Non-structural carbohydrates = 100 − (NDF + ether extract + crude protein + ash).

consisted of three weeks of adaptation to the respective treatments during which the animals were fed ad libitum twice a day at 08:00 and 15:00 h of a conventional early lactation TMR (Table 2) plus 2 kg of dry hay; four days of lactic acidosis challenge (the feeding procedure for which is described below); and three days of rest during which they received the same TMR as that given during adaptation, except that no probiotic supplements were provided. The TMR was formulated to meet the metabolizable protein, net energy, mineral and vitamin requirements for lactating Holstein cows weighing 625 kg and producing 40 kg of 39 g/kg fat-corrected milk when consuming 24 kg/d of DM (NRC, 1989). The procedure described by Keunen et al. (2002) was followed for induction of SARA. During the four days of induction of SARA, 0.30 of ad libitum intake of the TMR was replaced with wheat and barley pellets (WBP) containing 0.50 ground wheat and 0.50 ground barley. The cows were fed 2 kg of the TMR at 07:00 h and two-thirds of the WBP at 09:00 h. Between 11:00 and 11:30 h, the cows were given access to their TMR. At 13:00 h, they received the remainder of the WBP. From 15:00 to 15:30 h, the cows again had access to their TMR. At 17:00 h, the remainder of the TMR was fed to the animals. Grain pellets that were not consumed within 1 h of feeding were placed into the rumen via the fistula. No hay was provided during the week of the challenge. Feed intake was recorded daily, and samples of TMR and WBP were collected weekly and pooled by period. On the last day of SARA induction, 1 L of rumen contents (solid + liquid) was sampled prior to feeding (07:00 h) as well as at 10:00, 14:00 and 20:00 h. Rumen content samples were squeezed through four layers of cheesecloth to remove large feed particles. For bacterial DNA quantification, a mixture of solid and liquid content was sampled at the same times except for 20:00 h. Samples were kept at −20 ◦ C until further analyses. Samples for VFA analysis were acidified prior to freezing [5 ml filtered rumen fluid and 1 ml H2 SO4 (0.5 M)]. Physical exams (i.e. temperature, general appearance, visual fecal appearance) were completed daily. One cow showing clinical signs of acidosis at the beginning of project was withdrawn and replaced. 2.2. Analyses of feed nutrients The nutrient composition of the TMR, WBP and hay is in Table 3. The DM content of the TMR was determined by oven drying at 105 ◦ C for 48 h (AOAC, 1990, I.D. No. 930.15), and the ash content was determined by incineration at 550 ◦ C overnight. Total N was measured by thermal conductivity (LECO model FP-428 Nitrogen Determinator, LECO, St. Joseph, MI, USA). The concentration of neutral detergent fibre (aNDF) was determined as described by Van Soest et al. (1991) without the use of sodium sulphite and with the inclusion of heat-stable ␣-amylase. It was expressed inclusive of residual ash. The NDF procedure was adapted for use in an ANKOM200 Fiber Analyzer (ANKOM Technology Corp., Fairport, NY, USA). The ether extract content of the TMR, silages and orts was measured using a Soxtec

282

J. Chiquette / Animal Feed Science and Technology 153 (2009) 278–291

HT 6 apparatus (Tecator, Fisher Scientific, Montreal, QC, Canada) according to AOAC, 1990, I.D. No. 920.39. 2.3. Milk production and composition Milk production was recorded daily during the adaptation weeks and the challenge period. Milk samples were collected during four consecutive milkings (i.e. Monday evening, Tuesday morning, Tuesday evening, Wednesday morning) during the last week of adaptation only. Milk samples were kept at 4 ◦ C using bronopol as a preservative and were shipped weekly to Valacta (Centre d’expertise en production laitière du Québec, Ste-Anne-de-Bellevue, QC, Canada). The milk fat, crude protein and urea N content were determined using a near-infrared analyzer (Foss Electric, Denmark) according to AOAC, 1990, I.D. No. 972.16. 2.4. Ruminal pH Ruminal pH was recorded every 10 min over a 24 h period during each of the three weeks of adaptation, and continuously during the four days of SARA induction. A submersible electrode (Cole-Parmer, Mississauga, ON, Canada) was inserted through the rumen fistula and suspended in the ventral sac of the rumen. The electrode was protected by a wire shield and attached to a 0.5 kg weight. The electrodes were connected to a pH meter with a RS-232 recorder output (Oakton 1000, Fisher Scientific, Nepean, ON, Canada), which was connected to a computer with the Balance Talk software program (Labtronics Inc., Guelph, ON, Canada). The electrodes were calibrated with pH 4 and 7 buffer solutions (Fisher Scientific, Nepean, ON, Canada) before insertion into the rumen and were not withdrawn during pH measurement unless unrealistic readings were recorded. Calibration was checked at the end of data collection, but no drifts in calibration occurred. 2.5. Ruminal VFA and lactate determination Upon thawing, rumen fluid samples were centrifuged (29,000 × g, 20 min, 4 ◦ C). Approximately 1 ml of resin (Dowex 50 WX8-100, Sigma–Aldrich, St. Louis, MO, USA) was added and incubated for 10 min, and samples were filtered through a 0.22 ␮m syringe filter. Subsamples (0.5 ␮l) were analyzed using a Hewlett Packard model 6890 gas chromatograph. A Stabilwax-DA column (30 m × 0.53 mm × 0.50 ␮m) (Chromatographic Specialties, Brockville, ON, Canada) was used. Injector and detector temperatures were 250 ◦ C and 300 ◦ C, respectively. Filtered rumen fluid (5 ml) was added to duplicate vials and analyzed for lactate using the colorimetric assay of Taylor (1996). 2.6. Quantification of bacteria 2.6.1. Bacterial strains and growth Strains were obtained from the American Type Culture Collection (ATCC). The following strains of rumen bacteria were used as reference: Ruminococcus flavefaciens (strain C52, ATCC 49949), Ruminococcus albus (strain 7, ATCC 27210), Fibrobacter succinogenes (strain GC5, ATCC 51216) and Megasphaera elsdenii (ATCC 25940). They were cultured in a rumen fluid based medium as previously reported (Klieve et al., 1989). For M. elsdenii, the medium was modified so that lactic acid replaced both glucose and cellobiose as the major substrate for growth. 2.6.2. DNA extraction and purification from pure bacterial cultures and mixed rumen contents Rumen samples were thawed in a 37 ◦ C water bath and diluted with an equal volume of mineral salts buffer prior to DNA extraction (Attwood and Reilly, 1996). DNA from rumen fluid samples and from bacterial cultures was extracted in duplicate by physical disruption using a bead beater (Mini-Beater, BioSpec Products, Bartlesville, OK, USA) following the protocol of Reilly and Attwood (1998). Extracted DNA was subsequently purified using the QIAamp DNA Mini Kit from Qiagen Inc. (Mississauga, ON, Canada) according to the manufacturer’s instructions. The concentration and purity of the DNA were determined spectrophotometrically by measuring the absorbance at 260 and 280 nm (A260/280 ) with

J. Chiquette / Animal Feed Science and Technology 153 (2009) 278–291

283

a NanoDrop ND-1000 (NanoDrop Technologies, Inc., Wilmington, DE, USA). The DNA used for these analyses had an A260/280 ratio of 1.8–2.0. 2.6.3. Real-time primers, probes and operating conditions The development and application of a real-time Taq nuclease assay for the enumeration of R. flavefaciens, R. albus, F. succinogenes and M. elsdenii were reported previously (Ouwerkerk et al., 2002; Chiquette et al., 2007). Quantification of the DNA of each bacterial species in total rumen DNA used an ABI 7500 Fast Real-Time PCR System (PE Applied Biosystems, Foster City, CA, USA). Optimal PCR reactions (10 ␮l total volume) contained: 5 ␮l of TaqMan Universal PCR Master Mix (PE Applied Biosystems, Foster City, CA, USA); 2 ␮l of diluted DNA; forward primers; reverse primers; and probes at 300 nM, 300 nM and 200 nM for R. flavefaciens, 900 nM, 900 nM and 200 nM for R. albus, 300 nM, 300 nM and 200 nM for F. succinogenes, and 300 nM, 300 nM and 200 nM for M. elsdenii. The analyses were in triplicate. The cycling parameters were 20 s at 95 ◦ C, followed by 40 cycles of 3 s at 95 ◦ C and 30 s at 60 ◦ C. Real-time TaqMan assays were calibrated by the cycle threshold method (Heid et al., 1996). Serial dilutions of purified genomic DNA from the reference strains cited previously were used to construct species-specific calibration curves. The standard curves for each primer/probe set were linear (correlation coefficients > 0.998) with target cell concentrations ranging from 7.6 × 103 cells/ml to 7.6 × 107 cells/ml for R. flavefaciens, 8.6 × 102 cells/ml to 8.6 × 106 cells/ml for R. albus, 1.3 × 102 cells/ml to 1.3 × 106 cells/ml for F. succinogenes and 1.1 × 104 cells/ml to 1.1 × 108 cells/ml for M. elsdenii. Amplification efficiencies, calculated from the slopes, ranged from 0.81 to 0.94. A standard curve was made for each analysis run. The number of cells in fresh overnight cultures was determined by microscopic enumeration using a Petroff–Hausser counting chamber at ×400 magnification and dilution in the Norris–Powell solution (Norris and Powell, 1961), as described by Koch (1994). Counts were repeated four times for each bacterial group. The quantity of bacterial groups was expressed as cells per millilitre of rumen contents. Possible PCR inhibitors such as reagents used during nucleic acid extraction or co-purified components from rumen contents were tested using serial dilution of several unknown samples and assessment of the efficiency of amplification (Nolan et al., 2006). 2.7. Statistical analysis Variables were summarized to one data point per cow per period and statistically analysed according to a 4 × 4 Latin square design balanced for residual effects, with cow, period and treatment as fixed factors. Variables were analyzed in repeated measurements. The MIXED procedure of SAS (2002) was used for all analyses. Treatments AO-0.06, AO-3.0 and ES were further compared to C using Dunnett’s correction for multiple comparisons. Tukey’s test was used to compare all combinations of sampling times. Lactate concentration and M. elsdenii population data were log-transformed to obtain a normal distribution and homogeneous residual error. Significance was declared at P≤0.05, and trends were accepted if P≤0.10. 3. Results 3.1. Ruminal pH A 24 h rumen pH profile is shown for one representative cow under all treatments during the adaptation period (Fig. 1) and during SARA (Fig. 2). Maximum pH values and proportion of time during which rumen pH was between 5.6 and 6.0 were similar during adaptation and SARA. All other pH characteristics were indicative of more (P=0.0001) acidic conditions in the rumen during SARA (Table 4). The effects of probiotics on ruminal pH during SARA are in Table 5. The average daily pH tended (P=0.06) to be higher with ES (5.84) than with C (5.41). The minimum pH attained during SARA was higher (P=0.05) with ES (5.02) than with C (4.43). The proportion of time during which pH was between 5.6 and 6.0 was greater (P=0.05) with ES (0.310) than with C (0.221). Supplementing with ES tended to decrease the proportion of time during which pH was less than 5.6 (0.280 with ES and 0.660 with C) (P=0.11) and less than 5.2 (0.09 with ES and 0.31 with C) (P=0.09). Supplementing with AO at 3 g/cow/d

284

J. Chiquette / Animal Feed Science and Technology 153 (2009) 278–291

Fig. 1. A 24 h rumen pH profile in cow 2002 under all probiotic treatments during the adaptation period. C: control; ES: mixture of Enterococcus faecium and Saccharomyces cerevisiae at 2 g/cow/day; AO-3.0: Aspergillus oryzae at 3.0 g/cow/d; AO-0.6: Aspergillus oryzae at 0.6 g/cow/d.

Fig. 2. A 24 h rumen pH profile in cow 2002 under all probiotic treatments during the challenge period. C: control; ES: mixture of Enterococcus faecium and Saccharomyces cerevisiae at 2 g/cow/day; AO-3.0: Aspergillus oryzae at 3.0 g/cow/d; AO-0.6: Aspergillus oryzae at 0.6 g/cow/d. Table 4 Ruminal pH characteristics during adaptation weeks compared to SARAa week. pH

Adaptation weeks

Mean Minb Maxc <6.0d >5.6, <6.0 <5.6 >5.2, <5.6 <5.2 a b c d

1

2

3

6.14 5.56 6.71 0.32 0.22 0.11 0.10 0.01

6.10 5.55 6.61 0.34 0.25 0.09. 0.08 0.01

6.03 5.41 6.62 0.41 0.29 0.13 0.09 0.04

SARA

SEM

Probability adaptation versus SARA (P≤)

5.64 4.82 6.65 0.75 0.27 0.48 0.32 0.17

0.07 0.08 0.07 0.007 0.005 0.006 0.004 0.003

0.0001 0.0001 0.95 0.0001 0.75 0.0001 0.0001 0.0001

Subacute ruminal acidosis. Minimum pH recorded. Maximum pH recorded. Proportion of time during which pH values were below 6.0.

J. Chiquette / Animal Feed Science and Technology 153 (2009) 278–291

285

Table 5 Effects of probiotics on ruminal pH during induction of SARAa . pH

Mean Ming Maxh <6.0i >5.6, <6.0 <5.6 >5.2, <5.6 <5.2 a b c d e f g h i

P (Trtb versus Cc )

Treatment d

e

f

C

AO-0.6

AO-3.0

ES

SEM

AO-0.6

AO-3.0

ES

5.41 4.43 6.53 0.88 0.22 0.66 0.35 0.31

5.72 4.96 6.53 0.70 0.25 0.45 0.37 0.08

5.59 4.88 6.85 0.82 0.28 0.54 0.36 0.18

5.84 5.02 6.69 0.59 0.31 0.28 0.19 0.09

0.099 0.134 0.094 0.092 0.021 0.108 0.097 0.060

0.17 0.07 1.00 0.45 0.64 0.45 0.99 0.08

0.50 0.12 0.13 0.95 0.17 0.80 0.99 0.37

0.06 0.05 0.52 0.16 0.05 0.11 0.55 0.09

Subacute ruminal acidosis. Treatment. Control. Aspergillus oryzae at 0.6 g/cow/d. Aspergillus oryzae at 3.0 g/cow/d. Mixture of Enterococcus faecium and Saccharomyces cerevisiae at 2 g/cow/day. Minimum pH recorded. Maximum pH recorded. Proportion of time during which pH values were below 6.0.

had no effect on ruminal pH, whereas supplementing with 0.6 g/cow/d tended (P=0.07) to increase minimum pH (4.96) compared with C (4.43). Proportion of time during which pH was less than 5.2 tended (P=0.08) to be less with AO-0.6 (0.08) than with C (0.31). There was no significant carryover effects of SARA, or addition of probiotics, on ruminal pH characteristics from one experimental period to the next. There was no effect of day on ruminal pH profile. 3.2. General animal health, dry matter intake, milk production and composition The addition of AO or ES had no effect on feed DM intake in either the adaptation weeks or the challenge week. Replacing 0.30 of the DM in the TMR with WBP resulted in an average daily consumption of 5.1 kg (DM basis) of WBP during SARA. Induction of SARA did not affect milk production. When considering the overall experimental period (adaptation and challenge), AO-0.6 tended (P=0.11) to increase milk production (27.3 kg/d) compared with C (25.7 kg/d). At 3 g/cow/d, AO decreased (P=0.04) milk production (23.4 kg/d) compared with C (25.7 kg/d). No change in milk composition occurred and milk fat, protein and urea N averaged 40 and 37 g/kg, and 11.3 mg/100 ml. 3.3. Volatile fatty acids and lactate concentrations Neither VFA concentrations in the rumen nor proportions of individual VFA relative to total VFA were affected by probiotics when each probiotic was compared with the control. However acetate concentrations tended (P=0.10) to be lower with ES (57.9 mM) than with C (62.7 mM). As the relative proportions of individual VFA changed with time after feeding during SARA, VFA data are presented over sampling times and averaged over probiotic treatments (Table 6). There was a shift toward higher proportions of propionate, butyrate and valerate at the expense of acetate and isoacids, which decreased with time after feeding during SARA. The effects of probiotics and time of sampling on ruminal lactate concentrations are in Table 7. Lactate concentration increased with time and, for all treatments except ES, returned to pre-feeding values by 13 h post-feeding. When cows were supplemented with ES, ruminal concentrations of lactate remained elevated and higher (P=0.01) than the concentrations in C 13 h post-feeding, indicating that lactate production was exceeding the capacity of the lactate utilizing bacteria. However lactate was eventually metabolized, as evidenced by the similar basal pre-feeding lactate concentrations observed for all treatments. Lactate concentrations were very variable with time and among cows.

286

J. Chiquette / Animal Feed Science and Technology 153 (2009) 278–291

Table 6 Effect of sampling time post-feeding on proportions (mol/mol) of ruminal volatile fatty acids (VFA) during induction of SARAa . Sampling time (h)

Acetate (A) Propionate (P) Butyrate Valerate Isobutyrate Isovalerate A/P

SEM

0

3

7

13

60.4a b 25.9a 10.2a 1.40a 0.82a 1.28a 2.45a

60.7a 25.7a 10.4a 1.31a 0.77a 1.14a 2.46a

57.6b 28.0b 11.5ab 1.47a 0.54b 0.85b 2.19b

55.9c 28.4b 12.7b 1.67b 0.52b 0.88b 2.10b

a

Subacute ruminal acidosis.

b

Within lines, values with different superscripts (a–c) differ (P < 0.05).

0.56 0.68 0.58 0.077 0.041 0.072 0.072

3.4. Quantification of bacteria Probiotics had no effect on R. flavefaciens, R. albus, F. succinogenes and M. elsdenii populations when each treatment was compared to the control during the challenge. Therefore data were averaged over probiotic treatments and presented over time after feeding during the SARA challenge (Table 8). The R. albus and F. succinogenes populations increased 3 h following feeding and remained stable 7 h post-feeding, whereas the M. elsdenii population increased more slowly. R. flavefaciens did not differ with time of sampling. The R. flavefaciens population was the largest (3.2 × 108 ), followed by R. albus (5.1 × 106 ), M. elsdenii (1.5 × 106 ) and F. succinogenes (5.5 × 105 ). 4. Discussion 4.1. Ruminal pH Although the dairy cows were in mid to late lactation, SARA was successfully induced, in accordance with the study objective, in order to compare the effectiveness of probiotics on ruminal pH regulation. There were no clinical signs of acute ruminal acidosis during or after the study, although ruminal pH depression was more severe than that reported in similar studies, where mean ruminal pH ranged from 5.85 to 6.11 during SARA (Keunen et al., 2002; Krause and Oetzel, 2005; Gozho et al., 2007). Also, the average duration of ruminal pH below 5.6 was 506 min/d and 309 min/d in Krause and Oetzel (2005) and Gozho et al. (2007), respectively, compared with 691 min/d in the present study. That difference is probably due to direct introduction of unmasticated grain into the rumen via the fistula. Feeds placed directly into the rumen do not stimulate saliva production that would otherwise occur during chewing. Keunen et al. (2002) also placed unconsumed grain in the rumen, but at the level of 0.25 of the TMR as opposed to 0.30 in the present study. This could be part of the reason for the difference in rumen acidic conditions between the studies. The effect of ES on pH stabilization was more pronounced in the present study than in the study by Nocek et al. (2002), which used a different experimental design and no SARA challenge. But the present data are similar to those of Bach et al. (2007), who supplemented loose-housed dairy cattle with SC strain CNCM I-1077 (Levucell SC2, Lallemand, Toulouse, France). In the present study, mean ruminal pH was lower (P=0.0001) during SARA (5.64) than during adaptation (6.09), even though it was reported in earlier studies that mean pH was not a good indicator of SARA (Krause and Combs, 2003). Keunen et al. (2002) also reported lower mean pH during SARA (6.11) than during C weeks (6.25). It is interesting to note that none of the treatments in the present study had an effect on the maximum daily pH recorded. This finding was also reported by Keunen et al. (2002) and is indicative of recovery to normal pH values within 24 h. The present study is the first to report on the effect of probiotic fungi such as AO on the regulation of ruminal pH in cows subjected to a SARA challenge. Such information was found to be lacking in the literature (Bach, 2007).

Time after feeding (h)

Control (C)

AO-0.6b

AO-3.0c

ESd

0 3 7 13

0.17Ae [0.11–0.25] 0.19A [0.04–0.88] 0.53B [0.10–2.68] 0.30AB [0.13–0.65]

0.26A [0.17–0.39] 1.03AB [0.23–4.66] 1.25B [0.25–6.33] 0.60AB [0.27–1.31]

0.20a 0.55a 0.63a 0.35a

0.24A [0.16–0.35] 0.83AB [0.18–3.74] 1.24B [0.24–6.27] 1.77B [0.81–3.89]

a

[0.14–0.30] [0.12–2.48] [0.12–3.19] [0.16–0.76]

Lactate concentrations were log-transformed, so confidence limits are presented instead of SEM.

b

Aspergillus oryzae at 0.6 g/head/d.

c

Aspergillus oryzae at 3.0 g/head/d.

d

Enterococcus faecium and Saccharomyces cerevisiae. Within columns, means with different subscripts differ: (A, B) (P ≤ 0.05) or (a, b) (P < 0.10).

e

P< AO-0.6 vs. C

AO-3.0 vs. C

ES vs. C

0.24 0.22 0.37 0.30

0.79 0.52 0.98 0.96

0.40 0.30 0.38 0.01

J. Chiquette / Animal Feed Science and Technology 153 (2009) 278–291

Table 7 Effect of time and probiotics on rumen lactate concentrations (mM)a .

287

288

J. Chiquette / Animal Feed Science and Technology 153 (2009) 278–291

Table 8 Effect of time on specific rumen bacterial populations during induction of SARAa . Time after feeding (h) Bacterial populations

0

3

7

SEM

R. flavefaciensb R. albusc F. succinogenese M. elsdeniif

2.9 4.6a d 4.0a 1.2a [0.4–3.6]

3.7 5.5b 6.0b 1.2a [0.4–3.6]

2.9 5.3ab 6.6b 2.0b [0.7–5.9]

0.31 0.37 0.76 –

a b c d e f

Subacute ruminal acidosis. Ruminococcus flavefaciens, ×108 cfu/ml. Ruminococcus albus, ×106 cfu/ml. Within rows, means with different subscripts (a, b) differ (P ≤ 0.05). Fibrobacter succinogenes, ×105 cfu/ml. Megasphaera elsdenii, ×106 cfu/ml (data were log-transformed, so confidence limits are presented instead of SEM values).

4.2. Dry matter intake, milk production and composition The DM intake was not affected by SARA. Different DM intake responses following SARA induction have been reported. Bradford and Allen (2007) suggested that factors such as the plasma insulin concentration of the individual cows prior to SARA would play a role on intake regulation during SARA. This could partly explain the discrepancy in the response to feed intake in cows with SARA. Similar milk production results were reported by Gozho et al. (2007) when SARA was induced in dairy cows at 121 ± 8 DIM. Those authors reported average milk yields of 27.5 kg/d and 28.6 kg/d during the C and SARA periods, respectively. In a previous study (Chiquette, 1995) in which AO was fed as a supplement to dairy cows in weeks 7–12 of lactation, a 4% increase in feed efficiency (kg milk/kg DMI) occurred. Decreased milk fat proportion has often been associated with SARA (Enemark et al., 2004) but does not always occur, especially when the duration of SARA is not long enough to allow metabolic adaptation to affect milk composition (Stone, 1999; Krause and Oetzel, 2005). In a similar study by Chiquette (2008), milk fat proportion was not affected during a four day SARA challenge. 4.3. Volatile fatty acids and lactate concentrations In the present study, rumen contents were sampled on the last day of SARA. Because no effect of days was detected on ruminal pH profile over 24 h periods, the rumen fermentation characteristics on Day 4 can be considered representative of those prevailing during the first days of SARA. To the author’s knowledge, no other studies have reported the effect of ES or AO on VFA concentrations during a SARA challenge. Beauchemin et al. (2003) reported higher propionate concentrations and reduced butyrate concentrations in feedlot cattle supplemented with ES, and several studies reported a change in the relative proportion of each VFA during SARA. That total VFA concentration was numerically less with ES versus C might corroborate the increased rumen pH when cows were fed ES. It is difficult however to establish a link between rumen pH and VFA concentrations as sampling frequency was much less for VFA than for pH. Goad et al. (1998), who induced SARA in hay adapted or grain adapted steers, reported a similar shift in VFA proportions. In that study, the magnitude of the decrease in the proportion of acetate (62.7–54.2%) and the increase in the proportion of propionate (19.1–26.9%) from 0 to 12 h following feeding was higher than the magnitude in the present study (60.4–55.9% and 25.9–28.4% for acetate and propionate, respectively). In the present study, the increase in the proportion of butyrate (10.2–12.7%) in the first 13 h following feeding was higher than the 13.8–14.7% increase reported by Goad et al. (1998). The increase in the proportion of valerate with time in Goad et al. (1.0–1.3%) was similar to that in the present study (1.4–1.7%). More acidic rumen conditions in the study by Goad et al. (1998), with steers fed an 0.80 grain diet, might explain differences observed in the magnitude of the response with particular VFA.

J. Chiquette / Animal Feed Science and Technology 153 (2009) 278–291

289

These fermentation changes during SARA have been associated with rumen microbial changes. Martin et al. (2006) reported that when the ruminal pH drop is moderate (5.5 < pH < 6.0), protozoa are still active, resulting in butyrate production but, when pH drops to between 5.0 and 5.5, protozoal activity is impaired and fermentation orients toward propionate production. Concomitantly, there is an increase in lactate utilizing bacteria, which is believed to prevent accumulation of lactate, and also contributes to butyrate production via lactate utilization (Goad et al., 1998). The lactate concentrations in the present study were within the limits of what is considered normal, which is less than 5.0 mM. For example, Keunen et al. (2002) reported lactate levels of 0.45 mM and 0.74 mM in cows with SARA and Martin et al. (2006) indicated that lactate concentrations are generally less than 10 mM during SARA, but occasionally concentrations as high as 50 mM have been observed. It has been generally reported that the ruminal pH decline during SARA is a consequence of increased VFA production, while the lactate concentration remains controlled by lactate utilizing bacteria (Jouany, 2006). With the ES treatment, however, it seems that more lactate was produced, and it took longer to re-establish the baseline concentration following feeding than with the other treatments. 4.4. Bacterial populations There is a paucity of data on the effect of SARA on rumen microbial populations. Cellulolytics are particularly sensitive to ruminal pH. Although rumen conditions were generally less acidic in the treated cows than in the control cows, the difference was not sufficient to maintain a higher cellulolytic population in the treated cows. Cellulolytic activity is decreased when rumen pH reaches the threshold value of 6.0. Although ES maintained higher pH conditions during SARA, that 0.60 of the time rumen pH was below 6.0 with ES might have been detrimental to the cellulolytic populations and could explain the absence of a probiotic effect on cellulolytic populations. Because populations of R. flavefaciens, R. albus and F. succinogenes are generally in the range of 107 to 108 in high concentrate fed cattle (Chiquette et al., 2007), it appears that the F. succinogenes population, which was as low as 105 in the present study, was particularly affected by SARA. When Holstein cows were changed from a high forage to a high concentrate diet, Tajima et al. (2001) observed a reduction in F. succinogenes DNA that was 10 times greater than the reduction in R. flavefaciens DNA. However data on ruminal pH conditions were not provided in that study. When Hereford steers were gradually introduced to a 0.75 rolled barley diet, Klieve et al. (2003) reported that the M. elsdenii population was slow to increase, and it took 12 days for M. elsdenii to reach a population of 106 cells/ml, which is similar to the population observed in the present study, and 21 days to reach 108 cells/ml. It is possible that M. elsdenii had not attained its maximum population in the present study. M. elsdenii is a lactate-utilizing bacteria and E. faecium from ES is believed to sustain higher lactate concentrations in the rumen to foster the growth of lactate-utilizing bacteria. 5. Conclusion ES used as a probiotic mitigated SARA symptoms when mid to late lactation cows were submitted to a SARA challenge. AO used at the recommended dose gave an intermediate response between ES and C in terms of pH control. At five times the recommended AO dose, there was no increase in pH. More research is needed to confirm the role of probiotics in the prevention of SARA and/or in the alleviation of its symptoms. Understanding the interaction of SARA with indigenous rumen microbial populations is of particular interest for the development of new and more efficient probiotics. Acknowledgements The author acknowledges the technical work performed by Francine Markwell and Caroline Roy and the animal care provided by Denis Thibault and Franc¸ois Dehours. They also wish to thank the barn project coordinator, Chantal Bolduc. The statistical support of Steve Methot is greatly appreciated. Lastly, the authors wish to thank Probiotec Inc. (Ivan Girard) for providing the AO and Chr. Hansen (Stephen A. Graham) for providing the ES.

290

J. Chiquette / Animal Feed Science and Technology 153 (2009) 278–291

References AOAC, 1990. Association of Official Analytical Chemists. Official Methods of Analysis, 15th ed. AOAC, Arlington, VA, USA. Attwood, G.T., Reilly, K., 1996. Characterization of proteolytic activities of rumen bacterial isolates from forage-fed cattle. J. Appl. Bacteriol. 81, 545–552. Bach, A., 2007. The importance of ruminal pH and the impact of probiotics on reducing the incidence of subacute acidosis. In: Proceedings of the 43rd Eastern Nutrition Conference, QC, Canada, pp. 7–20. Bach, A., Iglesias, C., Devant, M., 2007. Daily rumen pH pattern of loose-housed dairy cattle as affected by feeding pattern and live yeast supplementation. Anim. Feed Sci. Technol. 136, 146–153. Beauchemin, K.A., Yang, W.Z., Morgavi, D.P., Ghorbani, G.R., Kautz, W., Leedle, J.A.Z., 2003. Effects of bacterial direct-fed microbials and yeast on site and extent of digestion, blood chemistry, and subclinical ruminal acidosis in feedlot cattle. J. Anim. Sci. 81, 1628–1640. Bradford, B.J., Allen, M.S., 2007. Depression in feed intake by a highly fermentable diet is related to plasma insulin concentration and insulin response to glucose infusion. J. Dairy Sci. 90, 3838–3845. Burrin, D.G., Britton, R.A., 1986. Response to monensin in cattle during subacute acidosis. J. Anim. Sci. 63, 888–893. Callaway, E.S., Martin, S.A., 1997. Effects of a Saccharomyces cerevisiae culture on ruminal bacteria that utilize lactate and digest cellulose. J. Dairy Sci. 80, 2035–2044. Canadian Council on Animal Care, 1993. In: Olfert, E.D., Cross, B.M., McWilliams, A.A. (Eds.), Guide to the Care and Use of Experimental Animals, vol. 1, 2nd ed. CCAC, Ottawa, ON, Canada. Chiquette, J., 1995. Saccharomyces cerevisiae and Aspergillus oryzae, used alone or in combination, as a feed supplement for beef and dairy cattle. Can. J. Anim. Sci. 75, 405–415. Chiquette, J., 2008. Effect of dosing the rumen with Enterococcus faecium and Saccharomyces cerevisiae on rumen pH, milk production and composition during a subacute acidosis challenge in early lactation dairy cows. In: Proceedings of the 6th Joint Symposium INRA-RRI, Clermont-Ferrand, France, pp. 89–90. Chiquette, J., Talbot, G., Markwell, F., Nili, N., Forster, R.J., 2007. Repeated ruminal dosing of Ruminococcus flavefaciens NJ along with a probiotic mixture in forage or concentrate-fed dairy cows: effect on ruminal fermentation, cellulolytic populations and in sacco digestibility. Can. J. Anim. Sci. 87, 237–249. Duffield, T., Plaizier, J.C., Fairfield, A., Bagg, R., Vessie, G., Dick, P., Wilson, J., Aramini, P., McBride, B.W., 2004. Comparison of techniques for measurement of rumen pH in lactating dairy cows. J. Dairy Sci. 87, 59–66. Enemark, J.M.D., Jørgensen, R.J., Kristensen, N.B., 2004. An evaluation of parameters for the detection of subclinical rumen acidosis in dairy herds. Vet. Res. Commun. 28, 687–709. Goad, D.W., Goad, C.L., Nagaraja, T.G., 1998. Ruminal microbial and fermentative changes associated with experimentally induced subacute acidosis in steers. J. Anim. Sci. 76, 234–241. Gozho, G.N., Krause, D.O., Plaizier, J.C., 2007. Ruminal lipopolysaccharide concentration and inflammatory response during grain-induced subacute ruminal acidosis in dairy cows. J. Dairy Sci. 90, 856–866. Heid, C.A., Stevens, J., Livak, K.J., Williams, P.M., 1996. Real-time quantitative PCR. Genome Res. 6, 986–994. Jouany, J.P., 2006. Optimizing rumen functions in the close-up transition period and early lactation to drive dry matter intake and energy balance in cows. Anim. Prod. Sci. 96, 250–264. Keunen, J.E., Plaizier, J.C., Kyriazakis, L., Duffield, T.F., Widowski, T.M., Lindinger, M.I., McBride, B.W., 2002. Effects of a subacute ruminal acidosis model on the diet selection of dairy cows. J. Dairy Sci. 85, 3304–3313. Klieve, A.V., Hennessy, D., Ouwerkerk, D., Forster, R.J., Mackie, R.I., Attwood, G.T., 2003. Establishing populations of Megasphaera elsdenii YE 34 and Butyrivibrio fibrisolvens YE 44 in the rumen of cattle fed high grain diets. J. Appl. Microbiol. 95, 621–630. Klieve, A.V., Hudman, J.F., Bauchop, T., 1989. Inducible bacteriophages from ruminal bacteria. Appl. Environ. Microbiol. 55, 1630–1634. Koch, A.L., 1994. Growth measurement. In: Gerhardt, P., Murray, R.G.E., Wood, W.A., Krieg, N.R. (Eds.), Methods for General and Molecular Bacteriology. ASM, Washington, DC, USA, pp. 249–277. Krause, K.M., Combs, D.K., 2003. Effects of forage particle size, forage source and grain fermentability on performance and ruminal pH in midlactation cows. J. Dairy Sci. 86, 1382–1397. Krause, K.M., Oetzel, G.R., 2005. Inducing subacute ruminal acidosis in lactating dairy cows. J. Dairy Sci. 88, 3633–3639. Krause, K.M., Oetzel, G.R., 2006. Understanding and preventing subacute ruminal acidosis in dairy herds: a review. Anim. Feed Sci. Technol. 126, 215–236. Marden, J.P., Julien, C., Monteils, V., Auclair, E., Moncoulon, R., Bayourthe, C., 2008. How does live yeast differ from sodium bicarbonate to stabilize ruminal pH in high-yielding dairy cows? J. Dairy Sci. 91, 3528–3535. Martin, C., Brossard, L., Doreau, M., 2006. Mécanismes d’apparition de l’acidose ruminale latente et conséquences physiopathologiques et zootechniques [mechanisms of appearance of ruminal acidosis and consequences on physiopathology and performances]. (In French, with English abstract). INRA Prod. Anim. 19, 93–108. Mutsvangwa, T., Walton, J.D., Plaizier, J.C., Duffield, T.F., Bagg, R., Dick, P., Vessie, G., McBride, B.W., 2002. Effects of a monensin controlled-release capsule or premix on attenuation of subacute ruminal acidosis in dairy cows. J. Dairy Sci. 85, 3454–3461. National Research Council (NRC), 1989. Nutrient Requirements of Dairy Cattle, 6th rev. ed. National Academy of Science, Washington, DC, USA. Nocek, J.E., Kautz, W.P., Leedle, J.A.Z., Allman, J.G., 2002. Ruminal supplementation of direct-fed microbials on diurnal pH variation and in situ digestion in dairy cattle. J. Dairy Sci. 85, 429–433. Nolan, T., Hands, R.E., Bustin, S.A., 2006. Quantification of mRNA using real-time RT-PCR. Nat. Protocols 1, 1559–1582. Norris, K.P., Powell, E.O., 1961. Improvements in determining total counts of bacteria. J. R. Micros. Soc. 80, 107–119. Oellermann, S.O., Arambel, M.J., Kent, B.A., Walters, J.L., 1990. Effect of graded amounts of Aspergillus oryzae fermentation extract on ruminal characteristics and nutrient digestibility in cattle. J. Dairy. Sci. 73, 2412–2416. Osborne, J.K., Mutsvangwa, T., Alzahal, O., Duffield, T.F., Bagg, R., Dick, P., Vessie, G., McBride, B.W., 2004. Effects of monensin on ruminal forage digestibility and total tract diet digestibility in lactating dairy cows during grain-induced subacute ruminal acidosis. J. Dairy Sci. 87, 1840–1847.

J. Chiquette / Animal Feed Science and Technology 153 (2009) 278–291

291

Ouwerkerk, D., Klieve, A.V., Forster, R.J., 2002. Enumeration of Megasphaera elsdenii in rumen contents by real-time Taq nuclease assay. J. Appl. Microbiol. 92, 753–758. Programme d’analyse des troupeaux laitiers du Québec, 1999. Rapport de production du PATLQ (PATLQ production report). (In French.) In: Le producteur de lait québécois, June 2000, pp. 48–50. Reilly, K., Attwood, G.T., 1998. Detection of Clostridium proteoclasticum and closely related strains in the rumen by competitive PCR. Appl. Environ. Microbiol. 64, 907–913. SAS Statistical Analysis System, Release 9.1, 2002. SAS Institute, Inc., Cary, NC, USA. Sievert, S.J., Shaver, R.D., 1993. Carbohydrate and Aspergillus oryzae effect on intake, digestion and milk production by dairy cows. J. Dairy Sci. 76, 2454–3254. Stewart, C.S., 1977. Factors affecting the cellulolytic activity of rumen contents. Appl. Environ. Microbiol. 33, 497–502. Stone, W.C., 1999. The effect of subclinical rumen acidosis on milk components. In: Proceedings of the 61st Cornell Nutr. Conference for Feed Manuf. Cornell University Press, Ithaca, NY, USA, pp. 40–46. Tajima, K., Aminov, R.I., Nagamine, T., Matsui, H., Nakamura, M., Benno, Y., 2001. Diet-dependent shifts in the bacterial population of the rumen revealed with real-time PCR. Appl. Environ. Microbiol. 67, 2766–2774. Taylor, K.A.C.C., 1996. A simple colorimetric assay for muramic acid, lactic acid, glyceraldehyde, acetaldehyde and formaldehyde. Appl. Biochem. Biotechnol. 56, 49–58. Van Soest, P.J., Robertson, J.B., Lewis, B.A., 1991. Symposium: carbohydrate methodology, metabolism and nutritional implications in dairy cattle. Methods for dietary fiber, neutral detergent fiber, and nonstarch polysaccharides in relation to animal nutrition. J. Dairy Sci. 74, 3583–3597. Varel, V.H., Kreikemeier, K.K., Jung, H.J.G., Hatfield, R.D., 1993. In vitro stimulation of forage fiber degradation by ruminal microorganisms with Aspergillus oryzae fermentation extract. Appl. Environ. Microbiol. 59, 3171–3176. Williams, P.E.V., Newbold, C.J., 1990. Rumen probiosis: the effects of novel microorganisms on rumen fermentation and ruminant productivity. In: Haresign, W., Cole, D.J.A. (Eds.), Recent Advances in Animal Nutrition. Butterworths, Toronto, ON, Canada, pp. 211–227.