Abstract
The potential of dietary supplements of 2 live yeast strains (Saccharomyces cerevisiae) or camelina oil to lower ruminal methane (CH4) and carbon dioxide (CO2) production and the associated effects on animal performance, rumen fermentation, rumen microbial populations, nutrient metabolism, and milk fatty acid (FA) composition of cows fed grass silage-based diets were examined. Four Finnish Ayrshire cows (53 ± 7 d in milk) fitted with rumen cannula were used in a 4 × 4 Latin square with four 42-d periods. Cows received a basal total mixed ration (control treatment) with a 50:50 forage-to-concentrate ratio [on a dry matter (DM) basis] containing grass silage, the same basal total mixed ration supplemented with 1 of 2 live yeasts, A or B, administered directly in the rumen at 1010 cfu/d (treatments A and B), or supplements of 60 g of camelina oil/kg of diet DM that replaced concentrate ingredients in the basal total mixed ration (treatment CO). Relative to the control, treatments A and B had no effects on DM intake, rumen fermentation, ruminal gas production, or apparent total-tract nutrient digestibility. In contrast, treatment CO lowered DM intake and ruminal CH4 and CO2 production, responses associated with numerical nonsignificant decreases in total-tract organic matter digestibility, but no alterations in rumen fermentation characteristics or changes in the total numbers of rumen bacteria, methanogens, protozoa, and fungi. Compared with the control, treatment CO decreased the yields of milk, milk fat, lactose, and protein. Relative to treatment B, treatment CO improved nitrogen utilization due to a lower crude protein intake. Treatment A had no influence on milk FA composition, whereas treatment B increased cis-9 10:1 and decreased 11-cyclohexyl 11:0 and 24:0 concentrations. Treatment CO decreased milk fat 8:0 to 16:0 and total saturated FA, and increased 18:0, 18:1, 18:2, conjugated linoleic acid, 18:3n-3, and trans FA concentrations. Decreases in ruminal CH4 production to treatment CO were related, at least in part to lowered DM intake, whereas treatments had no effect on ruminal CH4 emission intensity (g/kg of digestible organic matter intake or milk yield). Results indicated that live yeasts A and B had no influence on animal performance, ruminal gas production, rumen fermentation, or nutrient utilization in cows fed grass silage-based diets. Dietary supplements of camelina oil decreased ruminal CH4 and CO2 production, but also lowered the yields of milk and milk constituents due to an adverse effect on intake.
Key words
Introduction
Ruminal methane (CH4) production occurs due to the metabolic activity of methanogenic archaea capable of utilizing hydrogen (H2) and carbon dioxide (CO2) as substrates (
Martin et al., 2010
). Formation of CH4 contributes to the efficiency of ruminal OM degradation by preventing the accumulation of H2, which in high concentrations may inhibit microbial enzyme activity and ruminal fermentation (Morgavi et al., 2010
). Nutritional strategies to mitigate ruminal CH4 to lower greenhouse gas emissions from ruminant livestock production have sought to identify and investigate various feed additives and dietary ingredients to promote alternative pathways to CH4 formation for the dissipation of metabolic H2 in the rumen. Dietary supplements of the medium-chain SFA, 12:0 and 14:0 (Jordan et al., 2006
; Machmüller, 2006
; Hristov et al., 2009
), plant oils, or oilseeds (- Hristov A.N.
- Vander Pol M.
- Agle M.
- Zaman S.
- Schneider C.
- Ndegwa P.
- Vaddella V.K.
- Johnson K.
- Shingfield K.J.
- Karnati S.K.R.
Effect of lauric acid and coconut oil on ruminal fermentation, digestion, ammonia losses from manure, and milk fatty acid composition in lactating cows.
J. Dairy Sci. 2009; 92: 5561-5582
McGinn et al., 2004
; Beauchemin and McGinn, 2006
; Martin et al., 2008
) are known to lower ruminal CH4 production in lactating or growing cattle. However, the decreases in ruminal CH4 formation to relatively high amounts of lipid (≥50 g/kg of DM) from plant oils or oilseeds in the diet are often accompanied by decreases in DMI, nutrient digestion, and animal performance (Beauchemin and McGinn, 2006
; Jordan et al., 2006
; Martin et al., 2008
).Camelina (Camelina sativa L.), an ancient summer annual oilseed crop, is tolerant to drought and can adapt to different climatic and soil conditions (
Hurtaud and Peyraud, 2007
). Cultivation of camelina has received renewed attention due to the relatively high 18:3n-3 content of camelina oil (Hurtaud and Peyraud, 2007
). In lactating cows, dietary supplements of camelina seeds or camelina oil increased cis-9 18:1, cis-9,trans-11 CLA, and 18:3 n-3 and lowered medium-chain SFA concentrations in milk (Hurtaud and Peyraud, 2007
; Halmemies-Beauchet-Filleau et al., 2011
), changes considered beneficial to human health (Shingfield et al., 2013
). However, the effects of camelina oil on ruminal fermentation, CH4 and CO2 production, and microbial communities have not been determined.Recent evaluations have concluded that dietary lipid supplements resulted in the most consistent decrease in CH4 output when compared with alterations in forage-to-concentrate ratio, direct-fed microbials, or exogenous enzymes (
Martin et al., 2010
; Grainger and Beauchemin, 2011
). Furthermore, simply replacing forages with concentrate ingredients also ignores the important role of ruminants in utilizing fibrous feeds not suitable for human consumption (Grainger and Beauchemin, 2011
), and that when fed in excess, concentrates increase the risk of ruminal acidosis (Fonty and Chaucheyras-Durand, 2006
).Live yeast products are used as feed additives for ruminants to improve feed efficiency and prevent ruminal acidosis through the scavenging of oxygen within the rumen, provision of microbial growth factors, or direct competition with autochthonous species in the rumen (
Newbold et al., 1996
; Fonty and Chaucheyras-Durand, 2006
). Selection of Saccharomyces cerevisiae strains for specifically lowering ruminal CH4 production has been postulated (McGinn et al., 2004
; Chaucheyras-Durand et al., 2008
; Chung et al., 2011
), whereas other strains may indirectly decrease CH4 production per unit of milk or meat through improvements in ruminal fiber degradation and overall feed conversion efficiency.The present experiment examined the potential of 2 strains of live yeasts or camelina oil enriched in PUFA to lower ruminal CH4 and CO2 production, as well as the associated effects on rumen fermentation, rumen microbial populations, nutrient digestibility, energy and nitrogen (N) metabolism, milk production, and milk FA composition of lactating cows fed grass silage-based diets. Live yeast strains were selected on the basis of in vitro studies demonstrating positive effects on fiber degradation during incubations with mixed rumen populations or Fibrobacter succinogenes.
Materials and Methods
Animals, Experimental Design, and Diets
All experimental procedures were approved by the National Ethics Committee (Hämeenlinna, Finland) in accordance with the guidelines established by the European Community Council Directive 86/609/EEC (
European Union, 1986
). Four multiparous Finnish Ayrshire dairy cows fitted with rumen cannulas (#1C, i.d. 100 mm, Bar Diamond Inc., Parma, ID) of (mean ± SD) similar parity (3.8 ± 0.63), DIM (53 ± 7), and BW (711 ± 23 kg) producing 33.8 ± 3.4 kg of milk/d were allocated at random to experimental diets according to a 4 × 4 Latin square with four 42-d periods comprising 23 d of adaptation, 5 d of sample collection, and 14 d of washout.Cows were housed in individual tiestalls in a dedicated metabolism unit, had free access to water and salt block, and were milked in situ twice daily at 0700 and 1645 h. Treatments comprised a basal TMR (designated as the control treatment; forage-to-concentrate ratio 50:50 on a DM basis) or a TMR containing camelina oil (CO) based on grass silage with an in vitro OM digestibility of 686 g/kg of DM. Experimental silage was prepared from primary growths of tall fescue (Festuca arundinacea) grown at Jokioinen (60°49′N, 23°28′E), cut with a mower conditioner, and wilted for 5 h. Cut grass was ensiled with a mixture of Pediococcus acidilactici strain MA18/5M, Lactobacillus buchneri strain NCIMB 40788, cellulase, and hemicellulase applied at 5.0 L/tonne of fresh matter (Lalsil Dry, Lallemand Animal Nutrition).
Cows on treatments A and B received the basal TMR (Table 1) supplemented with 1 of 2 live yeast (Saccharomyces cerevisiae) strains, A or B, supplied as 0.5 g/d of a highly concentrated dried product (Lallemand Animal Nutrition) at 1010 cfu/d. Live yeasts were selected based on in vitro studies demonstrating positive effects on fiber degradation by mixed rumen populations (strain A), or the ability to promote the growth and degradation of fiber by Fibrobacter succinogenes [strain B, a rumen bacterium that does not produce H2; F. Chaucheyras-Durand, A. Ameilbonne (Lallemand Animal Nutrition, Blagnac, France), and E. Forano, unpublished data]. Viable yeast counts were checked before the start of the experiment and both dried products conformed to expectations. Yeast supplements were stored in the dark at 4°C, away from humidity, throughout the experiment. Previous experiments in vitro have demonstrated that each yeast strain survived in the rumen for at least 12 h [F. Chaucheyras-Durand, A. Ameilbonne (Lallemand Animal Nutrition), and E. Forano, unpublished data]. Live yeast supplements were placed directly into the rumen via the cannula at 0600 h, and mixed with rumen solid contents to ensure that all viable cells for treatments A and B were administered. For treatment CO, 60 g of camelina oil/kg of diet DM (Raisio Feed Ltd., Raisio, Finland) replaced concentrate ingredients, which resulted in a higher gross energy (GE) and FA and lower CP and carbohydrate concentration compared with the basal TMR (Table 1). The FA composition of grass silage, concentrates, and camelina oil is presented in Table 2. Diets were offered 4 times daily at 0600, 1000, 1630, and 1900 h as a TMR to avoid selection of dietary components and to maintain the desired forage-to-concentrate ratio. Experimental diets were offered ad libitum to result in 10% refusals and formulated to meet or exceed ME and protein requirements of lactating cows producing 30 kg of milk/d (
MTT Agrifood Research Finland, 2006
).MTT Agrifood Research Finland. 2006. Finnish feed tables and feeding recommendations: 2006. Accessed Nov. 13, 2010. http://www.mtt.fi/mtts/pdf/mtts106.pdf
Table 1Formulation and chemical composition of the experimental diets
| Item | Treatment | |||
|---|---|---|---|---|
| Control | A | B | CO | |
| Ingredient (g/kg of DM) | ||||
| Grass silage | 500 | 500 | 500 | 500 |
| Barley, rolled | 225 | 225 | 225 | 197 |
| Sugar beet pulp, molasses | 120 | 120 | 120 | 105 |
| Rapeseed meal, solvent extracted | 140 | 140 | 140 | 123 |
| Camelina oil | — | — | — | 60 |
| Vitamin and mineral premix 4 Vitamin and mineral supplement (Onni-Kivennäinen, Melica Finland Ltd., Vaasa, Finland) declared as containing calcium (205g/kg), magnesium (72g/kg), sodium (85g/kg), phosphorus (27g/kg), zinc (1.46g/kg), manganese (0.35g/kg), copper (0.27g/kg), iodine (39mg/kg), cobalt (27mg/kg), selenium (20mg/kg), retinyl acetate (120 IU/g), cholecalciferol (25 IU/g), and dl-α tocopheryl acetate (0.34 IU/g). | 15.0 | 15.0 | 15.0 | 15.0 |
| Chemical composition (g/kg of DM, unless otherwise stated) | ||||
| DM (g/kg as fed) | 327 | 327 | 327 | 328 |
| OM | 913 | 913 | 913 | 917 |
| CP | 163 | 163 | 163 | 152 |
| NDF | 401 | 401 | 401 | 387 |
| Potentially digestible NDF | 325 | 325 | 325 | 313 |
| ADF | 228 | 228 | 228 | 221 |
| Water-soluble carbohydrate | 32.7 | 32.7 | 32.7 | 29.0 |
| Starch | 120 | 120 | 120 | 106 |
| FA | 21.9 | 21.9 | 21.9 | 77.3 |
| Gross energy (MJ/kg of DM) | 18.7 | 18.7 | 18.7 | 20.0 |
1 Refers to grass silage-based diets containing no additional supplement (control diet) or supplemented with 0.5 g/d of 1 of 2 strains of probiotic live yeast strains A or B or 60 g/kg DM of camelina oil (CO).
2 Mean fermentation characteristics of experimental silage: pH 4.55; in DM lactic acid, 37.8 g/kg; acetic acid, 84.1 g/kg; propionic acid, 16.1 g/kg; butyric acid, 0.6 g/kg; soluble N, 693 g/kg of total N, ammonia-N, 127 g/kg of total N; GE, 19.0 MJ/kg of DM.
3 Gross energy content 40.2 MJ/kg of DM.
4 Vitamin and mineral supplement (Onni-Kivennäinen, Melica Finland Ltd., Vaasa, Finland) declared as containing calcium (205 g/kg), magnesium (72 g/kg), sodium (85 g/kg), phosphorus (27 g/kg), zinc (1.46 g/kg), manganese (0.35 g/kg), copper (0.27 g/kg), iodine (39 mg/kg), cobalt (27 mg/kg), selenium (20 mg/kg), retinyl acetate (120 IU/g), cholecalciferol (25 IU/g), and dl-α tocopheryl acetate (0.34 IU/g).
5 Calculated as NDF – indigestible NDF.
Table 2Fatty acid composition and content of grass silage, concentrate supplements, and camelina oil
| FA (g/100 g of FA, unless otherwise noted) | Ingredient | ||
|---|---|---|---|
| Silage | Concentrate | Camelina oil | |
| 12:0 | 0.30 | 0.03 | 0.01 |
| 14:0 | 0.64 | 0.28 | 0.06 |
| 16:0 | 23.3 | 14.9 | 5.62 |
| trans-3 16:1 | 2.18 | — | — |
| cis-9 16:1 | 0.18 | 0.62 | 0.08 |
| 18:0 | 1.62 | 1.40 | 2.39 |
| cis-9 18:1 | 4.23 | 26.9 | 11.6 |
| cis-11 18:1 | 0.50 | 5.46 | 0.71 |
| 18:2n-6 | 18.0 | 39.4 | 15.7 |
| 18:3n-3 | 35.6 | 6.54 | 37.0 |
| 20:0 | 0.70 | 0.36 | 1.51 |
| cis-11 20:1 | 0.33 | 0.76 | 15.1 |
| 20:2n-6 | 0.00 | 0.09 | 2.15 |
| 22:0 | 1.10 | 0.32 | 0.32 |
| cis-13 22:1 | 0.21 | 0.18 | 5.25 |
| 22:2n-6 | 0.00 | 0.00 | 0.16 |
| 24:0 | 0.97 | 0.35 | 0.17 |
| 26:0 | 1.14 | 0.07 | — |
| 28:0 | 0.91 | 0.01 | — |
| 30:0 | 0.61 | 0.01 | — |
| Unidentified | 4.49 | 0.19 | — |
| Other 1 Includes the sum of 15:0 anteiso, 15:0, 16:1 (n=5 isomers), 16:2 (n=2), 17:0, 17:1 (double bond position and geometry indeterminant), 18:1 (n=9), 18:0 iso, 19:0, 18:2 (n=6), 18:3 (n=2), 20:1 (n=4), 21:0, 18:4n-3, cis-15 22:1, 20:4n-6, 23:0, cis-14 23:1, cis-15 24:1, 25:0, cis-17 26:1, 29:0, and 10-oxo-18:0. | 2.92 | 2.16 | 2.21 |
| Σ SFA | 32.8 | 18.3 | 10.2 |
| Σ MUFA | 6.98 | 35.1 | 34.6 |
| Σ PUFA | 55.7 | 46.5 | 55.2 |
| Total FA (g/kg of DM) | 13.8 | 30.0 | 954 |
1 Includes the sum of 15:0 anteiso, 15:0, 16:1 (n = 5 isomers), 16:2 (n = 2), 17:0, 17:1 (double bond position and geometry indeterminant), 18:1 (n = 9), 18:0 iso, 19:0, 18:2 (n = 6), 18:3 (n = 2), 20:1 (n = 4), 21:0, 18:4n-3, cis-15 22:1, 20:4n-6, 23:0, cis-14 23:1, cis-15 24:1, 25:0, cis-17 26:1, 29:0, and 10-oxo-18:0.
Measurements and Chemical Analysis
Daily feed intake was determined as the amount of TMR offered minus refusals and milk yield were recorded throughout the experiment. Measurements of intake between d 24 and 28 were used for statistical analysis. During this period, representative samples of silage and concentrates were collected daily, composited by period, and submitted for chemical composition determinations (
Shingfield et al., 2002
). The chemical composition of TMR was calculated based on the chemical composition of silage, concentrates, and camelina oil, assuming no selection of dietary ingredients as indicated by negligible differences in the DM content of experimental TMR offered and feed refusals determined for each cow in all experimental periods. Concentrations of GE in samples of silage, concentrates, camelina oil, feces, and urine were determined by bomb calorimetry (1108 Oxygen bomb, Parr Instrument, Moline, IL). Indigestible NDF concentration of silage, concentrates, and feces was determined in duplicate by incubation of 1.0 g of sample DM in nylon bags (60 × 120 mm, pore size 0.017 mm) within the rumen of 2 cows fed a grass silage-based diet (forage-to-concentrate ratio = 70:30 on a DM basis) for 12 d. Once removed from the rumen, bags were rinsed in cold water for 25 min using a household washing machine, incubated for 1 h in boiling neutral detergent solution, rinsed, and dried to a constant weight at 60°C. Potentially digestible NDF (pdNDF) concentration was calculated as NDF – indigestible NDF. Samples of rumen fluid (n = 8) were collected on d 28 of each period from each cow at 1.5-h intervals from 0600 to 1630 h. Following the removal of rumen fluid, pH was measured and samples were filtered through 2 layers of cheesecloth and stored at −20°C until analyzed for VFA and ammonia-N (Shingfield et al., 2002
).For microbial assessments, 100-g samples of digesta were collected from each region of the rumen (anterior dorsal, anterior ventral, posterior dorsal, and posterior ventral) immediately after the collection of rumen fluid and just before morning feeding on d 28 of each experimental period. Samples of ruminal digesta were combined and mixed thoroughly. Subsamples (50 g) of composite digesta were mixed with 100 mL of RNAlater solution (Fisher Scientific, Illkirch, France), split into smaller aliquots, and stored at −80°C until quantitative PCR (qPCR) analysis.
Ruminal gas production was estimated using the sulfur hexafluoride (SF6) tracer gas technique (
Boadi et al., 2002
) with modifications. Measurements of CO2 and CH4 were made over a 4-d interval starting at 0600 h on d 24 of each experimental period. Thin wafer permeation tubes containing SF6 (i.d. 16.4 × 46.5 mm, Fine Permeation Tubes, Spadafora, Italy) with a declared rate of release of 1.0 mg/d were placed into the rumen of each cow on d 16 of the experiment. Actual SF6 release rates (1.12 ± 0.17 mg/d) for each tube \ determined by weight difference over the course of the experiment, from d 16 through 168, were used in the calculations. Gases in the rumen headspace were withdrawn continuously (1.7 mL/min) over 4 consecutive 24-h periods into evacuated 5.5-L air-tight canisters via a 100-mm length of capillary tubing (PEEK 1.6 × 0.13 mm i.d., VICI Valcro Instruments Co, Houston, TX). Gas collection tubes were fitted with a water lock (50 mL) and a filter (0.2-µm pore size) to prevent rumen liquor entering the collection tube and gas canisters. The tubes were anchored securely to the neck of the rumen cannula allowing gas to be collected at approximately 5 cm above the rumen mat. The end of each tube was covered with nylon (17-µm pore size) that prevented the entrance of rumen particulates. Each canister was replaced after 24 h, pressurized with N2 gas to 110 kPa, and left for 2 h to ensure a thorough mixing of N2 with the collected gases. Gases were released slowly from the canister, subsampled in triplicate, transferred into evacuated 10-mL glass tubes equipped with a rubber stopper, and analyzed for CH4, CO2, and SF6 concentrations.Apparent total-tract digestibility coefficients were determined by total fecal collection over a 96-h interval starting at 1800 h on d 24 of each experimental period. Feces excreted was weighed, thoroughly mixed, subsampled (5% wt/wt), and stored at –20°C before chemical analysis. Urine was separated from feces by means of a light harness and flexible tubing attached to the vulva and collected in plastic canisters containing 500 mL of 5 mol/L sulfuric acid. Collection vessels were changed at 12-h intervals.
Samples of milk were collected over 4 consecutive milking starting at 1645 h on d 24. Milk samples treated with preservative (Bronopol, Valio Ltd., Helsinki, Finland) and analyzed for milk fat, CP, and lactose by infrared analysis (MilkoScan FT6000, Foss Electric, Hillerød, Denmark). Milk composition was calculated based on weighted average milk yield. Unpreserved milk samples were also collected, composited according to yield, and stored at −20°C until analyzed for FA composition.
Gas Analysis
Samples of gases collected from the rumen were analyzed in triplicate for CH4, CO2, and SF6 concentrations using a gas chromatograph (Agilent 6890N, Agilent Technologies, Santa Clara, CA) equipped with flame ionization (FID) and electron-capture detectors, autosampler, and a nickel catalyst for converting CO2 to CH4 (
Regina and Alakukku, 2010
). Concentration of gases was determined based on calibration curves constructed using authentic standards (AGA Ltd., Espoo, Finland) over a range of 2.5 to 25% for CO2 and CH4 and 0.00625 to 0.125 µL/L for SF6. Peaks were identified by retention time comparisons with authentic standards. Daily CH4 and CO2 production was calculated based on known amount of SF6 released in the rumen. No correction was made for background CH4 and CO2 concentrations because cows were housed in a well-ventilated facility (72 m3/min) and fitted with custom-made sponges placed between the outer edge of the cannula flange and the abdominal wall to minimize the exchange of surrounding air with ruminal contents.Enumeration of Rumen Microbial Populations by qPCR
Microbial DNA was extracted from 250 mg of centrifuged rumen contents in quadruplicate with the Nucleospin for soil kit (Macherey-Nagel, Hoerdt, France). Samples were homogenized by bead beating in the presence of 700 µL of lysis buffer SL1 and 15 µL of Enhancer SX buffer using the Fast Prep-24 instrument (MP Biomedicals, Illkirch, France). All steps were performed according to the instructions provided by the manufacturer. The DNA extracts were assessed for purity and quantity using a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific, Illkirch, France), and stored at −20°C until molecular analysis.
Rumen microbes were quantified by qPCR targeting of the 16S rRNA gene for bacteria and methanogenic archaea, the 18S rRNA gene for protozoa, and the ITS1 region for fungi (Table 3). The qPCR was performed using SYBR-green chemistry on a MasterCycler ep Realplex thermal cycler (Eppendorf, Le Pecq, France). Primer sets and PCR conditions used were the same as reported in the literature for methanogenic archaea (
Ohene-Adjei et al., 2007
), protozoa (Sylvester et al., 2005
), Ruminococcus flavefaciens, Fibrobacter succinogenes, and fungi (Denman and McSweeney, 2006
). Total bacteria were quantified using 520F and 799R2 primers (Edwards et al., 2007
). The qPCR was carried out in a total volume of 20 μL containing 2× Takara SYBR Premix Ex Taq kit (Lonza, Levallois-Perret, France), 40 ng of DNA template, and 0.5 μM of each forward and reverse primer. Each reaction was run in triplicate in 96-well plates (BioRad, Marnes-la-Coquette, France). Standards were used to determine the absolute abundance of microbial groups, expressed as the log number of DNA copies per microgram of DNA. For total bacteria, cellulolytic bacteria, and methanogenic archaea, the standard curves were prepared according to Mosoni et al. (2011)
). For protozoa, the standard curve was developed using pSC-A-amp/kan plasmids (Strataclone PCR cloning kit, Agilent Technologies) containing the near full 18S rRNA gene from Polyplastron multivesiculatum, Eudiplodinium maggii, and Ostracodinium dentatum mixed in equal amounts. For fungi, the ITS1 fragment from Piromyces spp. was amplified using qPCR primers (Lwin et al., 2011
) and cloned using the pCR2.1 Topo TA Cloning kit (Invitrogen, Life Technologies, Saint Aubin, France). The number of gene copies present in each plasmid was calculated using the plasmid DNA concentration and the molecular mass of the vector and the insert. For each target, a standard curve was prepared from 102 to 109 copies by serial dilution. Efficiency of the qPCR for each target varied between 97 and 102% with a slope from −3.0 to −3.4 and a regression coefficient above 0.95 in accordance with the MIQE guidelines (Bustin et al., 2009
).Table 3Oligonucleotide primers used for real-time quantitative PCR
| Microbial group | Primer set | Fragment length (bp) | Reference |
|---|---|---|---|
| Total bacteria | 5′-AGCAGCCGCGGTAAT-3′ 5′-CAGGGTATCTAATCCTGTT-3′ | 280 | Edwards et al., 2007 |
| Fibrobacter succinogenes | 5′-GTTCGGAATTACTGGGCGTAAA-3′ 5′-CGCCTGCCCCTGAACTATC-3′ | 121 | Denman and McSweeney, 2006 |
| Ruminococcus flavefaciens | 5′-CGAACGGAGATAATTTGAGTTTACTTAGG-3′ 5′-CGGTCTCTGTATGTTATGAGGTATTACC-3′ | 132 | Denman and McSweeney, 2006 |
| Methanogenic archaea | 5′-GAGGAAGGAGTGGACGACGGTA-3′ 5′-ACGGGCGGTGTGTGCAAG-3′ | 233 | Ohene-Adjei et al., 2007 |
| Fungi | 5′-GAGGAAGTAAAAGTCGTAACAAGGTTTC-3′ 5′-CAAATTCACAAAGGGTAGGATGATT-3′ | 120 | Denman and McSweeney, 2006 |
| Protozoa | 5′-GCTTTCGWTGGTAGTGTATT-3′ 5′-CTTGCCCTCYAATCGTWCT-3′ | 223 | Sylvester et al., 2005 |
Lipid Analysis
Fatty acid methyl esters in freeze-dried feed samples were prepared in a one-step extraction-transesterification procedure using chloroform and 2% (vol/vol) sulfuric acid in methanol (
Halmemies-Beauchet-Filleau et al., 2011
). Lipid in a 1-mL milk sample was extracted in triplicate using a mixture of ammonia, ethanol, diethylether, and hexane (0.2:1.0:2.5:2.5 by vol). Extracts were combined and evaporated to dryness at 40°C under oxygen-free N2. Samples were dissolved in hexane and methyl acetate and transesterified to FAME using freshly prepared methanolic sodium methoxide (Halmemies-Beauchet-Filleau et al., 2011
).The FAME prepared from samples of feeds and milk fat were quantified using a gas chromatograph (model 6890N, Agilent Technologies) equipped with an FID, automatic injector, split injection port, and a 100-m fused silica capillary column (i.d. = 0.25 mm) coated with a 0.2-μm film of cyanopropyl polysiloxane (CP-Sil 88, Agilent Technologies;
Halmemies-Beauchet-Filleau et al., 2011
). For methyl esters not available as commercial standards, peaks were identified based on GC-MS analysis of 4,4-dimethyloxoline derivatives prepared from FAME (Halmemies-Beauchet-Filleau et al., 2011
). The distribution of CLA isomers in milk fat FAME was determined by Ag+ HPLC (Model 1090, Agilent Technologies; Halmemies-Beauchet-Filleau et al., 2011
).Milk FA composition was expressed as a weight percentage of total FA using theoretical relative response factors to account for the carbon deficiency in the FID response for FAME containing 4- to 10-carbon atoms (
Halmemies-Beauchet-Filleau et al., 2011
). Concentrations of CLA isomers were calculated based on proportionate peak area responses determined by HPLC and the sum of trans-7,cis-9 CLA, trans-8,cis-10 CLA, and cis-9,trans-11 CLA weight percentage determined by GC analysis.Calculations
Digestible and metabolizable energy (MJ/d) were calculated as
Daily ECM yield (kg/d) was calculated according to
Daily ECM yield (kg/d) was calculated according to
Sjaunja et al. (1990)
):Statistical Analysis
Experimental data were analyzed by ANOVA for a 4 × 4 Latin square using the Mixed procedure of SAS (version 9.2, SAS institute, Cary, NC) with a model that included the fixed effects of period and treatment and random effect of cow. Measurements of rumen fermentation characteristics were analyzed by ANOVA for repeated measures using a model that included the fixed effects of period, treatment, time, and their interaction and random effect of cow assuming an autoregressive order-one covariance structure fitted on the basis of Akaike information and Schwarz Bayesian model-fit criteria. Least squares means ± SEM are reported. Treatment effects were declared significant at P ≤ 0.05, and a trend was assumed for probabilities <0.1 and >0.05. When the overall effect of treatment was significant, differences among means were further evaluated using the Fisher’s least significant difference test.
Results
Nutrient Intake
Treatment CO lowered (P = 0.05) DMI and tended (P = 0.06) to lower OM intake compared with the control and treatment B (Table 4). Relative to the control and treatments A and B, treatment CO decreased (P < 0.05) the intake of CP, NDF, pdNDF, ADF, water-soluble carbohydrate, and starch, but increased (P < 0.05) the intake of all FA other than 12:0, 26:0, 28:0, and 30:0 (Table 4).
Table 4Effect of dietary supplements of 2 live yeast strains or camelina oil on nutrient intake of lactating cows
| Intake (kg/d, unless otherwise stated) | Treatment | SEM | P-value | |||
|---|---|---|---|---|---|---|
| Control | A | B | CO | |||
| DM | 19.0 | 18.4ab | 19.2 | 16.7 | 0.91 | 0.05 |
| OM | 17.2 | 16.7 | 17.4 | 15.2 | 0.83 | 0.06 |
| CP | 3.09 | 3.01 | 3.13 | 2.53 | 0.143 | 0.004 |
| NDF | 7.61 | 7.39 | 7.68 | 6.45 | 0.359 | 0.02 |
| Potentially digestible NDF | 6.16 | 5.99 | 6.22 | 5.23 | 0.291 | 0.02 |
| ADF | 4.32 | 4.20 | 4.37 | 3.68 | 0.205 | 0.02 |
| Water-soluble carbohydrate | 0.62 | 0.60 | 0.63 | 0.49 | 0.003 | 0.002 |
| Starch | 2.28 | 2.22 | 2.30 | 1.77 | 0.105 | 0.002 |
| Gross energy (MJ/d) | 355 | 347 | 360 | 330 | 17.6 | 0.35 |
| FA (g/d) | ||||||
| 12:0 | 0.50 | 0.49 | 0.50 | 0.48 | 0.024 | 0.52 |
| 14:0 | 1.61 | 1.57 | 1.63 | 1.88 | 0.092 | 0.04 |
| 16:0 | 71.7 | 69.8 | 72.6 | 109 | 4.90 | <0.001 |
| trans-3 16:1 | 3.05 | 2.97ab | 3.09 | 2.68 | 0.147 | 0.05 |
| cis-9 16:1 | 1.87 | 1.82 | 1.89 | 2.19 | 0.107 | 0.04 |
| 18:0 | 5.94 | 5.791 | 6.02 | 26.0 | 1.03 | <0.001 |
| cis-9 18:1 | 76.5 | 74.5 | 77.6 | 163 | 6.82 | <0.001 |
| cis-11 18:1 | 15.1 | 14.7 | 15.3 | 18.0 | 0.88 | 0.02 |
| 18:2n-6 | 129 | 126 | 131 | 242 | 10.3 | <0.001 |
| 18:3n-3 | 67.1 | 65.3 | 67.9 | 385 | 14.9 | <0.001 |
| 20:0 | 1.93 | 1.88 | 1.95 | 15.0 | 0.58 | <0.001 |
| cis-11 20:1 | 2.48 | 2.41 | 2.51 | 135 | 5.14 | <0.001 |
| 20:2n-6 | 0.25 | 0.24 | 0.25 | 19.2 | 0.73 | <0.001 |
| 22:0 | 2.39 | 2.32 | 2.42 | 4.82 | 0.202 | <0.001 |
| cis-13 22:1 | 0.77 | 0.74 | 0.77 | 47.2 | 1.81 | <0.001 |
| 22:2n-6 | 0.00 | 0.00 | 0.00 | 1.43 | 0.053 | <0.001 |
| 24:0 | 2.26 | 2.21 | 2.30 | 3.42 | 0.152 | <0.001 |
| 26:0 | 1.78 | 1.73 | 1.79 | 1.61 | 0.088 | 0.14 |
| 28:0 | 1.31 | 1.27 | 1.32 | 1.14 | 0.065 | 0.06 |
| 30:0 | 0.88 | 0.86 | 0.89 | 0.77 | 0.044 | 0.06 |
| Other | 16.6 | 16.1 | 16.7 | 33.6 | 1.38 | <0.001 |
| Σ SFA | 93.9 | 91.5 | 95.1 | 168 | 7.29 | <0.001 |
| Σ MUFA | 102 | 99.3 | 103 | 387 | 15.2 | <0.001 |
| Σ PUFA | 200 | 195 | 203 | 653 | 26.0 | <0.001 |
| Total | 403 | 392 | 408 | 1,213 | 48.7 | <0.001 |
a,b Within a row, means without a common superscript differ (P < 0.05).
1 Refers to grass silage-based diets containing no additional supplement (control diet) or supplemented with 0.5 g/d of 1 of 2 strains of live yeasts A or B or 60 g/kg DM of camelina oil (CO).
2 Calculated as NDF – indigestible NDF.
Rumen Fermentation and Microbial Populations
Relative to the control and treatment A, treatment CO tended (P = 0.06) to decrease rumen pH, but treatments had no effect (P > 0.05) on ruminal ammonia-N and total VFA concentrations (Table 5). Treatments had no influence (P > 0.05) on the molar proportions of major VFA or on the molar acetate-to-propionate ratio. However, treatment CO lowered (P < 0.001) the molar proportion of isobutyrate and tended (P = 0.06) to decrease molar proportions of valerate and caproate, and treatment B decreased (P < 0.001) the molar proportion of isobutyrate compared with the control (Table 5). Abundance of F. succinogenes, R. flavefaciens, total bacteria, methanogenic archaea, protozoa, and fungi were not affected (P > 0.05) by experimental treatments (Table 5).
Table 5Effect of dietary supplements of 2 live yeast strains or camelina oil on rumen fermentation characteristics and rumen microbial populations of lactating cows
| Item | Treatment | SEM | P-value | |||
|---|---|---|---|---|---|---|
| Control | A | B | CO | |||
| pH | 6.65 | 6.65 | 6.52 | 6.40 | 0.065 | 0.06 |
| Ammonia-N (mmol/L) | 6.43 | 6.42 | 6.28 | 5.53 | 0.621 | 0.31 |
| Total VFA (mmol/L) | 98.6 | 97.2 | 103 | 100 | 3.60 | 0.67 |
| Molar proportion (mmol/mol) | ||||||
| Acetate | 672 | 670 | 668 | 675 | 4.2 | 0.67 |
| Propionate | 189 | 192 | 192 | 198 | 4.0 | 0.22 |
| Butyrate | 86.9 | 88.6 | 89.4 | 80.7 | 3.45 | 0.21 |
| Isobutyrate | 10.0 | 9.70 | 9.16 | 7.73 | 0.225 | <0.001 |
| Valerate | 15.9 | 15.0 | 15.7 | 14.2 | 0.41 | 0.06 |
| Isovalerate | 17.0 | 17.1 | 16.6 | 14.5 | 0.91 | 0.17 |
| Caproate | 9.59 | 8.94 | 9.25 | 7.92 | 0.392 | 0.06 |
| Molar ratio | ||||||
| Acetate:propionate | 3.58 | 3.52 | 3.47 | 3.41 | 0.081 | 0.45 |
| Microbial numbers (log gene copies/µg of DNA) | ||||||
| Total bacteria | 10.08 | 10.13 | 10.16 | 10.13 | 0.032 | 0.44 |
| Methanogens | 7.27 | 7.35 | 7.30 | 7.30 | 0.034 | 0.26 |
| Protozoa | 7.63 | 7.59 | 7.60 | 7.42 | 0.083 | 0.23 |
| Fungi | 6.10 | 6.07 | 5.98 | 6.02 | 0.056 | 0.43 |
| Fibrobacter succinogenes | 7.64 | 7.58 | 7.77 | 7.61 | 0.071 | 0.31 |
| Ruminococcus flavefaciens | 6.50 | 6.34 | 6.47 | 6.75 | 0.100 | 0.12 |
a–c Within a row, means without a common superscript differ (P < 0.05).
1 Refers to grass silage-based diets containing no additional supplement (control diet) or supplemented with 0.5 g/d of 1 of 2 strains of live yeasts A or B or 60 g/kg DM of camelina oil (CO).
Nutrient Utilization
Apparent whole-tract nutrient digestibility, other than that of starch and GE, were unaffected (P > 0.05) by treatments (Table 6). Digestibility of starch (P = 0.05) and GE tended (P = 0.08) to be lower for treatment CO compared with the control and treatment B. Relative to the control, treatment CO decreased (P < 0.05) N intake, urinary N excretion, and milk N secretion, and tended (P = 0.07) to lower fecal N output. Compared with treatment B, treatment CO increased (P < 0.05) the efficiency of N utilization. However, treatments had no effect (P > 0.05) on milk energy secretion or energy excreted in feces, whereas treatment CO tended (P = 0.06) to lower energy losses as CH4 and in urine.
Table 6Effect of dietary supplements of 2 live yeast strains or camelina oil on apparent total-tract digestibility of nutrients and energy and nitrogen metabolism of lactating cows
| Item | Treatment | SEM | P-value | |||
|---|---|---|---|---|---|---|
| Control | A | B | CO | |||
| Apparent digestibility (g/kg, unless otherwise stated) | ||||||
| OM | 724 | 716 | 724 | 693 | 8.5 | 0.13 |
| CP | 711 | 710 | 712 | 688 | 10.5 | 0.31 |
| NDF | 585 | 570 | 589 | 545 | 13.0 | 0.18 |
| Potentially digestible NDF | 691 | 680 | 693 | 655 | 11.4 | 0.17 |
| ADF | 595 | 582 | 600 | 557 | 13.9 | 0.22 |
| Starch | 982 | 981 | 983 | 977 | 1.1 | 0.05 |
| Gross energy (KJ/MJ) | 709 | 700 | 709 | 675 | 8.3 | 0.08 |
| Energy (MJ/d) | ||||||
| Intake | 355 | 346 | 359 | 333 | 17.7 | 0.36 |
| Feces | 104 | 104 | 105 | 107 | 5.2 | 0.70 |
| Urine | 12.8 | 11.6 | 12.2 | 10.9 | 0.77 | 0.06 |
| Methane | 21.2 | 19.3 | 20.2 | 15.1 | 1.38 | 0.06 |
| Milk | 83.3 | 79.9 | 78.8 | 74.7 | 7.46 | 0.19 |
| Digestible energy | 252 | 242 | 254 | 225 | 13.5 | 0.20 |
| ME | 218 | 211 | 222 | 199 | 12.8 | 0.43 |
| Nitrogen (g/d) | ||||||
| Intake | 494 | 481 | 500 | 405 | 22.9 | 0.006 |
| Feces | 143 | 139 | 144 | 126 | 5.7 | 0.07 |
| Urine | 184 | 179 | 181 | 153 | 11.5 | 0.01 |
| Milk | 138 | 132 | 129 | 120 | 10.0 | 0.04 |
| Retained N | 32.1 | 33.5 | 49.0 | 9.57 | 10.62 | 0.14 |
| Milk N/intake N | 0.274 | 0.270 | 0.254 | 0.290 | 0.0203 | 0.04 |
a,b Within a row, means without a common superscript differ (P < 0.05).
1 Refers to grass silage-based diets containing no additional supplement (control diet) or supplemented with 0.5 g/d of 1 of 2 strains of live yeasts A or B 60 g/kg of DM of camelina oil (CO).
2 Calculated as NDF – indigestible NDF.
3 Digestible energy = energy intake – fecal energy.
4 Metabolizable energy = digestible energy – urinary energy – methane energy.
5 Retained N = N intake – fecal N – urinary N – milk N.
Ruminal CH4 and CO2 Production
Treatments A and B had no effect (P > 0.05) on ruminal CH4 production, whereas treatment CO decreased (P < 0.05) ruminal CH4 output (Table 7). However, treatments had no effect (P > 0.05) on ruminal CH4 production intensity (g/kg of OM or NDF digested in total digestive tract or g/kg of milk) or the proportion of GE lost as CH4. Compared with the control, treatment CO decreased (P < 0.05) ruminal CO2 production. Ruminal CO2 output per kilogram of milk tended (P = 0.09) to be lower for treatment CO compared with the control and treatment B.
Table 7Effect of dietary supplements of 2 live yeast strains or camelina oil on ruminal gas production of lactating cows
| Item | Treatment | SEM | P-value | |||
|---|---|---|---|---|---|---|
| Control | A | B | CO | |||
| Ruminal methane | ||||||
| g/d | 407 | 365 | 372 | 287 | 22.3 | 0.03 |
| g/kg of DMI | 21.4 | 19.7 | 19.4 | 17.6 | 1.27 | 0.30 |
| g/kg of OMD | 32.7 | 30.4 | 29.6 | 27.9 | 2.12 | 0.51 |
| g/kg of NDFD | 57.8 | 53.3 | 52.5 | 49.7 | 3.60 | 0.51 |
| g/kg of milk | 15.6 | 13.8 | 15.0 | 13.1 | 1.33 | 0.27 |
| % of GEI | 6.31 | 5.82 | 5.73 | 4.86 | 0.368 | 0.14 |
| Ruminal carbon dioxide | ||||||
| g/d | 3,493 | 3,006 | 3,172 | 2,295 | 226.4 | 0.02 |
| g/kg of DMI | 184 | 163 | 166 | 138 | 10.5 | 0.11 |
| g/kg of OMD | 281 | 251 | 252 | 220 | 16.8 | 0.18 |
| g/kg of NDFD | 497 | 440 | 447 | 392 | 28.5 | 0.18 |
| g/kg of milk | 133 | 114 | 127 | 104 | 10.4 | 0.09 |
a,b Within a row, means without a common superscript differ (P < 0.05).
1 Refers to grass silage-based diets containing no additional supplement (control diet) or supplemented with 0.5 g/d of 1 of 2 strains of live yeasts A or B or 60 g/kg DM of camelina oil (CO).
2 OM apparently digested in total digestive tract.
3 NDF apparently digested in total digestive tract.
4 Gross energy intake.
Milk Production and Composition
Compared with treatment A and the control, treatment CO decreased (P < 0.05) the yields of milk and milk protein (Table 8). Furthermore, treatment CO decreased (P < 0.05) yields of ECM and milk lactose compared with the control and treatments A and B, and lowered (P < 0.05) milk fat yield relative to the control and treatment B. Treatments had no effect (P > 0.05) on milk fat and lactose concentrations, whereas milk protein content tended (P = 0.08) to be lower for treatment A and CO.
Table 8Effect of dietary supplements of 2 live yeast strains or camelina oil on milk yield and milk composition of lactating cows
| Item | Treatment | SEM | P-value | |||
|---|---|---|---|---|---|---|
| Control | A | B | CO | |||
| Yield | ||||||
| Milk (kg/d) | 27.0 | 26.5 | 25.6 | 22.4 | 2.30 | 0.05 |
| ECM (kg/d) | 26.4 | 25.5 | 25.7 | 22.1 | 2.18 | 0.04 |
| Fat (g/d) | 1,070 | 1,020 | 1,062 | 920 | 96.4 | 0.05 |
| Protein (g/d) | 859 | 827 | 824 | 696 | 57.5 | 0.01 |
| Lactose (g/d) | 1,240 | 1,228 | 1,183 | 1,017 | 115.7 | 0.03 |
| Composition (g/kg) | ||||||
| Fat | 39.9 | 38.3 | 41.6 | 40.8 | 1.31 | 0.24 |
| Protein | 32.4 | 31.2 | 32.4 | 31.3 | 1.08 | 0.08 |
| Lactose | 45.8 | 46.3 | 46.2 | 44.6 | 1.11 | 0.45 |
a,b Within a row, means without a common superscript differ (P < 0.05).
1 Refers to grass silage-based diets containing no additional supplement (control diet) or supplemented with 0.5 g/d of 1 of 2 strains of live yeasts A or B or 60 g/kg DM of camelina oil (CO).
2 Energy-corrected milk = milk (kg/d) × [38.3 × fat (g/kg) + 24.2 × protein (g/kg) + 16.54 × lactose (g/kg) + 20.7]/3,140 (
Sjaunja et al. (1990)
).Milk FA Composition
Administration of live yeasts into the rumen had little influence on milk FA composition (Table 9). In contrast, treatment CO resulted in substantial changes in milk FA composition that were characterized by decreases (P < 0.05) in 6:0, 8:0, 10:0, 12:0, 14:0, 16:0, and total SFA and increases (P < 0.05) in 18:0, total 18:1, 18:2, CLA, 18:3n-3, total 20- and 22-carbon, MUFA, PUFA, and trans FA concentrations (Table 9).
Table 9Effect of dietary supplements of 2 live yeast strains or camelina oil on milk FA composition of lactating cows
| FA (g/100 g of FA) | Treatment | SEM | P-value | |||
|---|---|---|---|---|---|---|
| Control | A | B | CO | |||
| 4:0 | 3.10 | 3.18 | 3.11 | 3.18 | 0.225 | 0.98 |
| 6:0 | 1.90 | 1.93 | 1.88 | 1.56 | 0.116 | 0.03 |
| 8:0 | 1.12 | 1.11 | 1.11 | 0.79 | 0.065 | 0.001 |
| 10:0 | 2.66 | 2.62 | 2.60 | 1.55 | 0.132 | <0.001 |
| cis-9 10:1 | 0.296 | 0.276 | 0.303 | 0.193 | 0.0131 | <0.001 |
| 12:0 | 3.24 | 3.13 | 3.17 | 1.84 | 0.126 | <0.001 |
| cis-9 12:1 | 0.082 | 0.075 | 0.083 | 0.043 | 0.0041 | <0.001 |
| trans-9 12:1 | 0.081 | 0.073 | 0.082 | 0.046 | 0.0042 | <0.001 |
| 14:0 | 12.1 | 11.8 | 11.9 | 8.10 | 0.32 | <0.001 |
| cis-9 14:1 | 1.12 | 1.02 | 1.15 | 0.77 | 0.073 | <0.001 |
| trans-9 14:1 | 0.013 | 0.011 | 0.013 | 0.009 | 0.0007 | 0.01 |
| Σ 15 | 2.36 | 2.31 | 2.22 | 1.55 | 0.126 | 0.004 |
| 16:0 | 34.4 | 35.8 | 34.6 | 21.3 | 1.32 | <0.001 |
| Σ cis 16:1 | 2.20 | 2.09 | 2.23 | 1.64 | 0.153 | 0.02 |
| Σ trans 16:1 | 0.230 | 0.227 | 0.236 | 0.456 | 0.0148 | <0.001 |
| Σ 16:1 | 2.43 | 2.32 | 2.46 | 2.10 | 0.152 | 0.14 |
| Σ 17 | 1.18 | 1.16 | 1.16 | 0.86 | 0.031 | <0.001 |
| 18:0 | 8.78 | 9.41 | 8.33 | 12.9 | 0.778 | 0.003 |
| 10-oxo-18:0 | 0.465 | 0.457 | 0.456 | 0.247 | 0.0430 | <0.001 |
| 13-oxo-18:0 | 0.022 | 0.023 | 0.023 | 0.017 | 0.0019 | 0.12 |
| Σ cis 18:1 | 17.5 | 16.4 | 18.4 | 25.3 | 1.54 | 0.02 |
| Σ trans 18:1 | 2.31 | 2.23 | 2.28 | 6.71 | 0.280 | <0.001 |
| Σ 18:1 | 19.8 | 18.6 | 20.6 | 32.0 | 1.34 | 0.001 |
| Σ 18:2 | 1.71 | 1.63 | 1.69 | 2.65 | 0.095 | <0.001 |
| Σ CLA | 0.38 | 0.36 | 0.39 | 0.95 | 0.047 | <0.001 |
| 18:3n-3 | 0.454 | 0.437 | 0.437 | 0.489 | 0.0224 | 0.007 |
| 18:3n-6 | 0.015 | 0.016 | 0.016 | 0.007 | 0.0012 | 0.005 |
| cis-9,trans-11,cis-15 18:3 | 0.036 | 0.031 | 0.032 | 0.056 | 0.0050 | 0.02 |
| 20:0 | 0.178 | 0.169 | 0.146 | 1.69 | 0.056 | <0.001 |
| Σ cis 20:1 | 0.247 | 0.211 | 0.218 | 2.48 | 0.089 | <0.001 |
| Σ trans 20:1 | 0.040 | 0.031 | 0.031 | 0.585 | 0.0293 | <0.001 |
| Σ 20:1 | 0.287 | 0.242 | 0.250 | 3.07 | 0.112 | <0.001 |
| 20:2n-6 | 0.024 | 0.023 | 0.022 | 0.063 | 0.0044 | <0.001 |
| 20:3n-3 | 0.008 | 0.008 | 0.007 | 0.046 | 0.0033 | <0.001 |
| 20:3n-6 | 0.047 | 0.049 | 0.047 | 0.033 | 0.0068 | 0.01 |
| 20:4n-3 | 0.034 | 0.035 | 0.036 | 0.026 | 0.0034 | 0.03 |
| 20:4n-6 | 0.066 | 0.065 | 0.059 | 0.046 | 0.0068 | 0.04 |
| 20:5n-3 | 0.049 | 0.052 | 0.048 | 0.032 | 0.0038 | 0.01 |
| 22:0 | 0.054 | 0.057 | 0.050 | 0.157 | 0.0078 | <0.001 |
| cis-9 22:1 | 0.011 | 0.011 | 0.011 | 0.054 | 0.0038 | <0.001 |
| cis-13 22:1 | 0.014 | 0.012 | 0.012 | 0.232 | 0.0139 | <0.001 |
| Σ 22:1 | 0.025 | 0.023 | 0.023 | 0.286 | 0.0155 | <0.001 |
| 22:4n-6 | 0.018 | 0.018 | 0.020 | 0.013 | 0.0033 | 0.33 |
| 22:5n-3 | 0.060 | 0.055 | 0.055 | 0.038 | 0.0064 | 0.001 |
| Σ 22 | 0.157 | 0.152 | 0.147 | 0.494 | 0.0248 | <0.001 |
| 24:0 | 0.039 | 0.037 | 0.035 | 0.031 | 0.0038 | 0.002 |
| cis-15 24:1 | 0.010 | 0.010 | 0.010 | 0.028 | 0.0017 | <0.001 |
| 26:0 | 0.030 | 0.029 | 0.029 | 0.013 | 0.0025 | <0.001 |
| Other | 0.161 | 0.153 | 0.155 | 0.234 | 0.0088 | <0.001 |
| Summary | ||||||
| Σ trans FA | 3.37 | 3.21 | 3.30 | 11.8 | 0.412 | <0.001 |
| Σ SFA | 72.1 | 73.7 | 71.3 | 56.0 | 1.50 | <0.001 |
| Σ MUFA | 24.7 | 23.3 | 25.6 | 39.1 | 1.47 | <0.001 |
| Σ PUFA | 2.89 | 2.78 | 2.86 | 4.46 | 0.164 | <0.001 |
| Total FA (g/100 g of fat) | 94.6 | 94.3 | 94.3 | 94.3 | 0.13 | 0.47 |
a–c Within a row, means without a common superscript differ (P < 0.05).
1 Refers to grass silage-based diets containing no additional supplement (control diet) or supplemented with 0.5 g/d of 1 of 2 strains of live yeasts A or B or 60 g/kg DM of camelina oil (CO).
2 Sum of nonmethylene interrupted 18:2 isomers.
3 Sum of 8-oxo-16:0, 10-oxo-16:0, 16:2n-4, 9-oxo-18:0, 15-oxo-18:0, cis-9,trans-11,trans-15 18:3, 18:4n-3, 22:2n-6, 22:3n-3, 22:6n-3, and 28:0.
Relative to the control, treatment A lowered (P < 0.05) milk cis-10 16:1 concentration and treatment CO increased (P < 0.05) cis-10 16:1, cis-12 16:1 and trans-9 to -13 16:1 content and decreased (P < 0.05) cis-9 16:1 and cis-13 16:1 concentrations (Supplemental Table S1; http://dx.doi.org/10.3168/jds.2014-7976). Treatments A and B had no effect (P > 0.05) on the distribution of milk fat 18:1 isomers, whereas treatment CO increased (P < 0.05) cis-9, -12, -15, and -16 18:1 and trans (Δ4–16) 18:1 concentrations. Furthermore, treatment CO enriched (P < 0.05) milk fat cis (Δ9–15) and trans (Δ9–13) 20:1 concentrations.
Treatments A and B had no influence (P > 0.05) on milk fat 18:2 or CLA isomer concentrations (Supplemental Table S2; http://dx.doi.org/10.3168/jds.2014-7976). In contrast, treatment CO increased (P < 0.05) the abundance of nonmethylene interrupted 18:2 isomers, including cis-12,cis-15 18:2, cis-9,trans-13 18:2, and trans-11,cis-15 18:2, but decreased (P < 0.05) the concentration of 18:2n-6. Relative to other treatments, treatment CO increased (P < 0.01) milk fat cis-9,trans-11 CLA, cis,trans CLA (12,14), trans,cis CLA (7,9; 9,11; 10,12; 11,13; and 12,14), and trans,trans CLA (11,13 and 12,14) concentrations.
Treatments A and B had no influence (P > 0.05) on the concentrations of the majority of odd- and branched-chain FA (OBCFA) in milk fat, with the exception of a decrease (P < 0.001) in 11-cyclohexyl 11:0 concentration for treatment B compared with the control (Supplemental Table S3; http://dx.doi.org/10.3168/jds.2014-7976). Compared with other treatments, treatment CO lowered (P < 0.05) the abundance of most OBCFA in milk fat and increased (P < 0.001) 21:0 and cis-12 21:1 concentrations.
Discussion
Intake and Nutrient Digestion
Treatments A and B had no effect on DMI, but given the relatively small numbers of cows used in our experiment, these findings require confirmation in a larger-scale experiment. However, an extensive evaluation of data from multiple studies concluded that a single live strain of Saccharomyces cerevisiae had no effect on intake of lactating cows (
De Ondarza et al., 2010
). Nevertheless, some indications were found that the influence of live yeasts on fiber digestion are dependent on the inherent digestibility of dietary forages, with the largest improvements reported in cows fed low-quality forages (Guedes et al., 2008
). Furthermore, strains of live yeasts have been shown to be effective in the prevention of ruminal acidosis (Fonty and Chaucheyras-Durand, 2006
), a condition not prevalent in this experiment as indicated from measurements of rumen pH.At relatively high levels of supplementation (≥50 g of oil/kg of DM) plant oils and oilseeds typically lower DMI, a response often attributed to various mechanisms including the adverse effect of unsaturated FA on ruminal microbial communities, lower OM and NDF digestion in the rumen, a tendency to shift the site of nutrient digestion from the rumen to the intestines, and elevate plasma gut peptide concentrations (
Allen, 2000
; Lock and Shingfield, 2004
). Compared with the control diet (fat content 24.3 g/kg of DM), treatment CO containing 85.9 g of fat/kg of DM resulted in a 12.1% decrease in DMI. An earlier study also reported that camelina lipid supplements might lower intake in lactating cows. Supplementing maize silage-based diets with camelina seeds (29 g/kg of DM) or camelina meal (95 g/kg of DM), both providing 10.3 g of additional oil/kg of DM, equivalent to 39 and 35% of total dietary lipid content was associated with decreases in DMI of −1.9 and −5.7%, respectively (Hurtaud and Peyraud, 2007
). In cows fed red clover silage-based diets, supplements of camelina oil (14.9 g/kg of DM) or camelina expeller (106 g/kg of DM), providing an additional 17.6 g of FA/kg of DM, were found to have no effect on DMI (Halmemies-Beauchet-Filleau et al., 2011
). However, the effects of plant oils on DMI are known to vary depending on the composition and lipid content of the basal diet and the source and inclusion rate of lipid supplements (Chilliard, 1993
; Benchaar et al., 2012
; - Benchaar C.
- Romero-Pérez G.A.
- Chouinard P.Y.
- Hassanat F.
- Eugene M.
- Petit H.V.
- Côrtes C.
Supplementation of increasing amounts of linseed oil to dairy cows fed total mixed rations: Effects on digestion, ruminal fermentation characteristics, protozoal populations, and milk fatty acid composition.
J. Dairy Sci. 2012; 95: 4578-4590
Rabiee et al., 2012
).Decreases in DMI on the treatment CO were accompanied by numerical decreases in total-tract OM, NDF, pdNDF, starch, or GE digestibility coefficients (−4.3, −6.8, −5.2, −0.5, and −4.8%, respectively), differences that may well be determined significant if the current study was repeated with more animals. Plant oils and oilseeds have variable effects on diet digestibility in lactating cows depending on the amount and source of lipid added and composition of the basal diet, with decreases often being attributed to the inhibitory adverse effect of unsaturated FA on the growth of microbial populations in the rumen (
Palmquist and Jenkins, 1980
; Jenkins, 1993
). In cows fed alfalfa and barley silage, including 88 g of rapeseed oil/kg of diet, DM in the ration had no influence on ruminal or total-tract OM and NDF digestion (Chelikani et al., 2004
). Sunflower oil offered up to 50 g/kg of DM was shown to progressively decrease ruminal pdNDF and total NDF digestibility in cows fed grass silage-based diets (Shingfield et al., 2008
). When fed as part of a TMR based on grass hay, linseed oil (40 g/kg of diet DM) had no negative effects on nutrient digestion (Benchaar et al., 2012
), whereas linseed oil or whole or processed linseeds supplying 57 g of lipid/kg of diet DM lowered total-tract OM and NDF digestibility in cows fed maize silage-based diets (- Benchaar C.
- Romero-Pérez G.A.
- Chouinard P.Y.
- Hassanat F.
- Eugene M.
- Petit H.V.
- Côrtes C.
Supplementation of increasing amounts of linseed oil to dairy cows fed total mixed rations: Effects on digestion, ruminal fermentation characteristics, protozoal populations, and milk fatty acid composition.
J. Dairy Sci. 2012; 95: 4578-4590
Martin et al., 2008
). In cows fed a diet based on grass and maize silage, supplemental lipid from linseed oil (400 g/d) and linseed hulls (1.8 kg/d containing 29.4 g of fat/kg of DM) had more adverse effects on intake and total-tract fiber digestibility when administered in the rumen than the abomasum (Kazama et al., 2010
). Direct comparisons are limited, but at low levels of inclusion (10 g/kg of DM), no differences in total-tract nutrient digestion have been reported for camelina oil compared with rapeseed or sunflower oil in cows fed red clover silage (Halmemies-Beauchet-Filleau et al., 2011
).Regulation of the intake of lactating cows involves both physical and metabolic feedback mechanisms (
Allen, 2000
). No measurements of rumen pool sizes and digestion kinetics were made to explore physical constraints on intake. Nevertheless, the numerical decreases in total-tract digestibility do not appear to fully explain the decrease in DMI on the treatment CO, suggesting that metabolic factors may also have contributed to the decrease in intake in response to camelina oil. Decreases in DMI to high amounts of lipid supplements have often been explained as a consequence of alterations in ruminoreticular motility, the release of gut hormones, including cholecystokinin-octapeptide, and oxidation of fat in the liver (Chilliard, 1993
; Allen, 2000
). Abomasal infusion of unsaturated FA from soybean (178–534 g/d; Litherland et al., 2005
) or linseed oil (250 and 500 g/d; Côrtes et al., 2011
) have been shown to lower DMI without altering diet digestibility. These studies also demonstrated that free FA induce more pronounced decreases in DMI compared with esterified FA (Litherland et al., 2005
), and that the decreases in intake to infusions of linseed oil were greater in cows fed diets containing 24.7 than 73.2 g of fat/kg of DM (Côrtes et al., 2011
). Even though increases in FA at the small intestine have been shown to stimulate enterocyte secretion of glucagon-like peptide-1 and cholecystokinin-octapeptide (Litherland et al., 2005
), the mechanisms involved in the regulation of intake in cows fed lipid supplements merit further investigation.Across all diets, the efficiency of N utilization, defined as milk N/N intake, averaged 0.272, which is consistent with a mean of 0.277 for cows fed grass silage-based diets containing on average 165 g of CP/kg of DM (
Huhtanen and Hristov, 2009
). Camelina oil improved N utilization compared with treatment B, which can, for the most part, be explained by a lower N intake. It is well established that dietary N intake is a major determinant of the efficiency of N utilization for milk production in lactating cows (Huhtanen and Hristov, 2009
), whereas partitioning into urine increases exponentially when N intake exceeds 400 g/d (Castillo et al., 2000
).Rumen Fermentation
Treatments A and B had no significant influence on ruminal fermentation, which is in agreement with the findings of an earlier experiment in growing cattle (
McGinn et al., 2004
). In dry cows, 1 strain of Saccharomyces cerevisiae was shown to increase propionate and decrease acetate whereas another had no influence on ruminal molar VFA proportions, highlighting the variability in responses to yeast supplements in vivo (Chung et al., 2011
).Camelina oil had limited influence on rumen fermentation, other than a tendency to lower rumen pH and decrease the molar proportion of isobutyrate. In cows fed maize silage, camelina seeds or meal have been shown to lower molar proportions of acetate and increase those of propionate and valerate in rumen VFA (
Hurtaud and Peyraud, 2007
). Differences between experiments may relate to the form in which camelina lipid is included in the diet and the composition of other feed ingredients. Dietary plant oil supplements may influence molar VFA proportions depending on the composition of the basal diet, amount of added lipid, and the amount of lipid in the basal diet (Chelikani et al., 2004
; Shingfield et al., 2008
; Benchaar et al., 2012
). A recent meta-analysis of 106 treatment means derived from experiments in cattle fed grass silage-based diets indicated that differences in silage lactate concentrations have a greater influence on rumen fermentation patterns compared with dietary fat content (- Benchaar C.
- Romero-Pérez G.A.
- Chouinard P.Y.
- Hassanat F.
- Eugene M.
- Petit H.V.
- Côrtes C.
Supplementation of increasing amounts of linseed oil to dairy cows fed total mixed rations: Effects on digestion, ruminal fermentation characteristics, protozoal populations, and milk fatty acid composition.
J. Dairy Sci. 2012; 95: 4578-4590
Huhtanen et al., 2013
). Plant oils and oilseeds typically have adverse effects on rumen fermentation when included in rations to provide 60 to 70 g of fat/kg of diet DM (Jenkins, 1993
; Grainger and Beauchemin, 2011
), but little evidence exists from this or earlier studies (Shingfield et al., 2008
) to suggest that high amounts of plant oils (≥50 g/kg of DM) result in substantial changes in rumen pH or molar VFA proportions in cows fed high-forage diets based on silage prepared from mixed timothy and meadow fescue.Ruminal Gas Production and Microbial Ecology
Administration of live yeast strains was associated with numerical decreases in ruminal CO2 and CH4 production compared with the control, but because of the limited number of observations and inherent sensitivity of the SF6 tracer technique it did not establish these differences as significant. Further evaluation of live yeast strains A and B using a larger number of cows would be required to confirm possible efficacy. In growing cattle, probiotic yeasts have been shown to result in variable and often nonsignificant differences in ruminal CH4 production of between −3 and 6% (
McGinn et al., 2004
). Consistent with minimal changes in rumen fermentation patterns and gas production, treatments A and B had no effect on the abundance of microbial groups or targeted ruminal cellulolytic bacterial species. Although it has been suggested that change-over designs may not be the most appropriate for assessing the effects of live yeast effects on rumen function due to possible treatment carry-over effects on the rumen microbiota (Chaucheyras-Durand et al., 2012
), a 14-d washout between treatments was used in the present study to minimize these effects.Dietary supplements of camelina oil resulted in a 4.9% decrease in CH4 production per 10 g/kg of diet DM increase in diet oil content, a response similar to that of 4.8% reported for cows fed diets containing 58 g of linseed oil/kg of diet DM (
Martin et al., 2008
). The decrease in both ruminal CH4 and CO2 production on treatment CO (−29.5 and −34.3%, respectively) can be explained, at least to some extent, by the lower DMI on treatment CO (−12.1%). Supplementing the diet of growing cattle with 46 g of rapeseed oil/kg of diet DM was also reported to lower absolute CH4 emissions (Beauchemin and McGinn, 2006
). However, energy losses of CH4 as a percentage of digestible energy intake did not differ among treatments, indicating that the changes in ruminal CH4 formation to rapeseed oil reported by Beauchemin and McGinn, 2006
) could be explained by a lower DMI.Both 18:2n-6 and 18:3n-3 contained in camelina oil exert toxic effects on ciliate protozoa and bacteriostatic effects on cellulolytic bacteria (
Doreau and Ferlay, 1995
; Jenkins et al., 2008
). Both protozoal and bacterial populations contribute to fiber digestion and H2 production in the rumen, indicating that PUFA may potentially inhibit ruminal CH4 production (Martin et al., 2010
). Based on the analysis of samples collected prefeeding, camelina oil had no effect on the abundance of protozoa in the rumen, or on the populations of fiber-degrading bacteria F. succinogenes and R. flavefaciens. There are no reports on the effects of camelina oil on rumen microbial populations. However, the FA composition of camelina oil is more similar to linseed oil than other plant oils typically fed to ruminants (Halmemies-Beauchet-Filleau et al., 2011
), and therefore the effects of linseed and camelina oils on rumen microbial ecology could be expected to be rather similar.Dietary linseed oil supplements were reported to have no effect on rumen microbial populations in samples collected from sheep before feeding, but decreased protozoal numbers with no influence on fibrolytic bacteria in samples collected 3 h postfeeding (
Mosoni et al., 2008
). In contrast, linseed oil was demonstrated to have no effect on the total number or the genera distribution of protozoa in cattle collected 2 h postfeeding (Benchaar et al., 2012
). Consistent with earlier findings (- Benchaar C.
- Romero-Pérez G.A.
- Chouinard P.Y.
- Hassanat F.
- Eugene M.
- Petit H.V.
- Côrtes C.
Supplementation of increasing amounts of linseed oil to dairy cows fed total mixed rations: Effects on digestion, ruminal fermentation characteristics, protozoal populations, and milk fatty acid composition.
J. Dairy Sci. 2012; 95: 4578-4590
Mosoni et al., 2008
), the lack of treatment effects on rumen protozoal numbers in our study may also be related to the collection of ruminal digesta immediately before morning feeding. An in vitro study reported that crushed linseeds and sunflower seeds decrease CH4 production without changes in protozoal numbers, suggesting that other mechanisms than an inhibition of protozoa may also be involved, including changes in molar VFA proportions or changes in the diversity or activity of archaea (Popova et al., 2011
). Even though treatment CO decreased daily CH4 production, the abundance of ruminal methanogens was not affected, suggesting that rather than influencing numbers, camelina oil may modulate the composition and functioning of this microbial population (Popova et al., 2011
).Milk Production
Treatments A and B had no influence on milk yield, composition, or energy secretion in milk, consistent with no changes in intake or digestibility compared with the control. However, the number of cows used to assess the effects of live yeasts on animal performance in our study was limited, with the corollary that a larger experiment with more animals would be required to establish fully the effects of treatments A and B on intake and milk production. A recent meta-analysis based on data from 14 experiments including measurements of more than 1,600 cows fed a range of diets indicated that live yeasts can be expected to improve FCM yield and stimulate an increase in milk protein and fat secretion (
De Ondarza et al., 2010
).Compared with the control, treatment CO lowered the yields of milk, milk fat, protein, and lactose. In cows fed diets based on red clover silage, camelina oil (14.9 g/kg of DM) or expeller (106 g/kg of DM) had no adverse effects on milk yield and composition (
Halmemies-Beauchet-Filleau et al., 2011
). However, camelina meal (95 g/kg of DM), but not camelina seeds (29 g/kg of DM), was shown to decrease milk protein yield in cows fed maize silage-based diets (Hurtaud and Peyraud, 2007
). It is generally accepted that energy intake is the major nutritional factor influencing milk protein synthesis, but the relation between increases in ME and milk protein secretion only holds true for dietary protein and carbohydrate components (Lock and Shingfield, 2004
). Depending on inclusion rate, dietary lipid supplements including animal fats and plant oils typically result in a 1- to 4-g/kg decrease in milk protein concentration (Wu and Huber, 1994
). Often the decrease has been attributed to increases in milk yield rather than lowered milk protein output, but there is evidence to indicate that the decrease in milk protein percentage is not simply due to dilution, but reflects true physiological responses to fat supplements (Wu and Huber, 1994
). Compared with the control, treatment CO lowered milk fat output that was related to the overall decrease in milk yield, rather than a decrease in milk fat content.Milk FA Composition
Live yeasts do not contain substantial amounts of lipid (
Ratledge and Evans, 1989
), and therefore the possible effects on milk fat composition could be anticipated to involve changes in the relative abundance of OBCFA and specific biohydrogenation intermediates originating from the rumen. The similarities in milk FA composition between the control and treatments A and B would tend to suggest that the live yeast strains tested had no major influence on ruminal lipolysis, biohydrogenation, or microbial lipid synthesis.Compared with the control, treatment CO resulted in a substantial decrease in milk fat SFA due to lower 6- to 16-carbon FA concentrations, which represents a typical response to relatively high amounts of dietary plant oil supplements (
Chilliard et al., 2007
; Glasser et al., 2008
; Shingfield et al., 2013
). Much of the decrease can be attributed to an increase in the availability of 18-carbon and longer FA, which inhibits acyl-CoA carboxylase activity and decreases the synthesis of 6:0 to 16:0 de novo in the mammary glands (Chilliard et al., 2007
).Treatment CO elevated milk fat cis-MUFA concentrations compared with other treatments, principally due to increases in cis-9 18:1 that can be attributed to both higher intake and ruminal escape of cis-9 18:1, as well as increased availability of 18:0 for desaturation in the mammary glands. Studies in cows indicate that between 44 and 78% of 18:0 extracted from the blood is desaturated in the mammary gland of lactating cows (
Shingfield et al., 2013
).For all treatments, cis-9 16:1 represented the second-most abundant cis-MUFA in milk fat, more than 50% of which is synthesized endogenously via the action of stearoyl CoA desaturase on 16:0 in the mammary gland (
Shingfield et al., 2013
). Milk fat also contained 14 cis and trans 16:1 isomers that may originate from the isomerization of dietary cis-9 16:1 and trans-3 16:1, or from the oxidation of 18:1 biohydrogenation intermediates in the rumen (Destaillats et al., 2000
).Compared with the control, treatment CO increased total trans FA content as a result of elevated trans 16:1, trans 18:1, and trans 18:2 concentrations. Much of the increase in milk trans FA was associated with trans-11 18:1, that represents a common intermediate formed during the penultimate step of PUFA biohydrogenation in the rumen (
Harfoot and Hazlewood, 1988
). Enrichment of trans FA in milk by treatment CO can be attributed to the incomplete ruminal biohydrogenation of unsaturated FA (Chilliard et al., 2007
) in camelina oil. Despite the differences in milk trans 18:1 concentrations, the relative abundance of individual 18:1 isomers did not differ substantially among treatments, indicating that camelina oil did not induce substantial alterations in the major biohydrogenation pathways in the rumen (Jenkins et al., 2008
; Shingfield et al., 2013
). Compared with an earlier report (Halmemies-Beauchet-Filleau et al., 2011
), the increases in milk trans-11 18:1 concentrations per gram of camelina oil per kilogram of diet DM on the treatment CO were lower than expected, with the implication that unsaturated 18-carbon FA were extensively biohydrogenated to 18:0 in the rumen. Camelina oil also enriched milk fat cis-9,trans-11 CLA concentrations, responses that are in the range that could be expected when plant oils rich in 18:2n-6 and 18:3n-3 are used as supplements for diets of similar composition to the experimental TMR fed in our study (Chilliard et al., 2007
; Shingfield et al., 2013
).Even though cows fed treatment CO had a higher intake of 18:2n-6 and 18:3n-3, the enrichment of 18:3n-3 in milk was 7.7% higher compared with the control, whereas 18:2n-6 abundance was decreased, changes that are in line with earlier reports on milk FA composition responses to camelina oil (
Halmemies-Beauchet-Filleau et al., 2011
), seeds, or expeller (Hurtaud and Peyraud, 2007
). Lower concentrations of most OBCFA in milk from cows receiving treatment CO are consistent with milk fat composition responses to plant oils in lactating cows (Glasser et al., 2008
). Although the appearance of OBCFA in milk fat originate principally from microbial lipids synthesized de novo in the rumen, changes in the relative concentrations in milk may at least in part be related to a decrease in microbial synthesis or alterations in the relative abundance of specific populations of bacteria and protozoa in the rumen (Vlaeminck et al., 2006
).Conclusions
Ruminal administration of live yeast strains had no influence on ruminal CH4 and CO2 production, rumen fermentation, or animal performance in cows fed diets based on grass silage. Supplements of camelina oil decreased ruminal CH4 and CO2 production, responses that were accompanied by lowered intake, yield of milk and milk constituents, and altered milk FA composition without a substantial change in nutrient digestion, rumen fermentation, or ruminal microbial populations. Live yeast strains had no influence on the proportions of the major FA in milk. Dietary supplements of camelina oil decreased 12:0, 14:0, 16:0, and 18:2n-6 and increased cis-9 18:1, trans-11 18:1, cis-9,trans-11 CLA, 18:3n-3, and total trans FA concentrations. The effect of these changes in milk fat composition on human health merits further investigation.
Acknowledgments
The authors thank staff in the metabolism unit at Natural Resources Institute (Jokioinen, Finland) for technical support, care of experimental animals, and assistance in sample collection. Competitive funding from the Finnish Ministry of Agriculture (Helsinki) as part of the GreenDairy project is gratefully acknowledged. Plasmids used for protozoa quantification were a generous donation from F. Ossa (Institut Rosell/Lallemand, Montréal, Canada).
Supplementary Table
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Article info
Publication history
Published online: February 25, 2015
Accepted:
January 14,
2015
Received:
January 23,
2014
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© 2015 American Dairy Science Association. Published by Elsevier Inc.
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