Low emission feed : opportunities to mitigate enteric methane production of dairy cows

As global demand for high-quality food originating from animal production is expected to rise due to an increasing human population and consumer income level, the expected role of ruminants in meeting this demand brings multiple challenges. Ruminant production needs to adapt to environmental changes and, at the same time, reduce its impact on the environment. Ruminants production systems have a major impact on the environment through the emission of greenhouse gases such as methane (CH4), nitrous oxide and carbon dioxide. Microbial fermentation of feeds in the gastrointestinal tract, known as enteric fermentation, is the main source of CH4 emissions from dairy production. Enteric CH4 emission is strongly related to the amount of feed fermented in the rumen, which depends on feed intake, feed composition and rumen fermentation conditions associated to the intrinsic characteristics of these feeds and the characteristics of the whole diet. Important gaps in knowledge remain however. The prime aim of this thesis was to investigate the effects of various feeding strategies to mitigate enteric CH4 emissions of dairy cows. First experiment was conducted to investigate the effects of type and level of starch in the concentrate. Inclusion of a high level (53%) of starch in the concentrate that accounted for 40% of the total mixed ration dry matter (DM) produced lower CH4 per unit of estimated rumen fermentable organic matter (eRFOM) than a low level (27% of DM) of starch (43.1 vs. 46.9 g/kg of eRFOM). Methane production per kg of eRFOM also was lower for diets based on rapidly fermentable starch (gelatinized maize grain) compared to diets based on slowly fermentable starch (native maize grain) (42.6 vs. 47.4 g/kg of eRFOM). However, inclusion of 53% of starch in the concentrate from both types of starch did not affect CH4 emission intensity (CH4 Ei) (CH4 emission per kg of fat- and protein-corrected milk; FPCM). In a subsequent experiment, maize silage was prepared from whole-plant maize harvested at a very early (25% DM), early (28% DM), medium (32% DM) and late (40% DM) stage of maturity and fed to dairy cows as an alternative to concentrate as starch source. Diet consisted of (on DM basis) 75% maize silage, 20% concentrate and 5% wheat straw. Increasing harvest maturity of maize silage linearly decreased CH4 yield (21.7, 23.0, 21.0 and 20.1 g/kg of DM intake) and CH4 emission as a fraction of gross energy intake (6.3, 6.7, 6.3 and 6.0%). Methane Ei tended to decrease linearly with maturity (13.0, 13.4, 13.2 and 12.1 g/kg FPCM). In another experiment grass silage as roughage source was tested. This experiment was designed to investigate the effects of N fertilisation of grassland and maturity of grass at cutting on CH4 emission in dairy cows. Two N fertilisation rates (65 vs. 150 kg of N/ha) were examined in combination with three stages of grass maturity (early, 28 days of regrowth; mid, 41 days of regrowth; and late, 62 days of regrowth). Diet contained 80:20 ratio (on DM basis) of grass silage (mainly ryegrass) and concentrate. Dry matter intake decreased with N fertilisation and maturity, and FPCM decreased with maturity but was unaffected by N fertilisation. Methane Ei (mean 15.0 g/kg of FPCM) increased by 31% and CH4 per unit digestible OM intake (mean 33.1 g/kg of DOMI) increased by 15% with increasing maturity. Methane yield (mean 23.5 g/kg of DM intake) and CH4 as a fraction of gross energy intake (mean 7%) increased by 7 and 9% with maturity, respectively, which implies an increased loss of dietary energy with progressing grass maturity. Rate of N fertilisation had no effect on CH4 Ei and CH4 yield. Despite the importance of in vitro gas production technique for evaluating feeds, in vitro study as a stand-alone approach was considered inadequate to fully evaluate the potential effect of feeds and rumen fermentation modifiers on CH4 production, because in vitro studies are frequently performed separately rather than in parallel with in vivo studies. To test this hypothesis, both in vitro and in vivo CH4 measurements were measured simultaneously using cows in the first experiment that were fed (and adapted to) the same dietary material used as a substrate for in vitro incubation, as donor for microbial inoculum. It was found that 24-h in vitro CH4 (mL/g of incubated organic matter) correlated well with in vivo CH4 when expressed per unit of eRFOM (R2 = 0.54), but not when expressed per unit of organic matter ingested (R2 = 0.04). In the same experiment, results showed that incubation of the same substrate with rumen inocula obtained from donor cows adapted to different diets produced a variable amount of CH4 suggesting that it is important to consider the diet of the donor animal when collecting rumen inocula for in vitro incubation. Even though the in vitro technique has limitations to represent in vivo conditions, it is useful for screening of large sets of animal feeds or feed additives to be used as a CH4 mitigation strategy. In this thesis, two in vitro experiments were conducted to examine the effects of variation in structural composition of condensed tannins (CT) in sainfoin accessions collected from across the world on CH4 production, and CT extracts obtained from a selected sainfoin accessions on CH4 production. Results revealed substantial variation among CT in their effect on in vitro CH4 production and this variation was attributed to differences in chemical structure of CT. Condensed tannins evaluated in this thesis showed to have potential to reduce in vitro CH4 production, but require further investigations to fully evaluate their in vivo effects. In conclusion, results from the research work conducted in this thesis show that changes in the basal diet of dairy cows and in roughage production management can substantially reduce the amount of enteric CH4 produced and thereby influence the impact of dairy production on the environment

Antagonistic intestinal microflora produces antimicrobial substance inhibitory to pseudomonas species and other spoilage organisms

Chicken intestine harbors a vast number of bacterial strains. In the present study, antimicrobial substance produced by lactic acid bacteria (LAB) isolated from the gastrointestinal tract of healthy chicken was detected, characterized, and purified. Based on 16S rRNA sequencing, the bacteria were identified as Lactobacillus plantarum vN. The antimicrobial substance produced by this bacterium was designated vN-1 and exhibited a broad-spectrum of activity against many important pathogenic and spoilage microorganisms, including Pseudomonas aeruginosa, Staphylococcus aureus, Micrococcus luteus, Salmonella Typhimurium, and Erwinia amylovova. vN-1 was determined to be thermostable, insensitive to pH values ranging from 2.0 to 8.0, resistant to various organic solvents and to enzymatic inactivation. The inhibition kinetics displayed a bactericidal mode of action. This study revealed an antimicrobial substance with low molecular mass of less than 1 kDa as determined by ultrafiltration and having features not previously reported for LAB isolated from chicken intestines. The detection of this antimicrobial substance addresses an important aspect of biotechnological control agents of spoilage caused by Pseudomonas spp. and promises the possibility for preservation of refrigerated poultry meat

Feeding nitrate and docosahexaenoic acid affects enteric methane production and milk fatty acid composition in lactating dairy cows

An experiment was conducted to study potential interaction between the effects of feeding nitrate and docosahexaenoic acid (DHA; C22:6 n-3) on enteric CH4 production and performance of lactating dairy cows. Twenty-eight lactating Holstein dairy cows were grouped into 7 blocks of 4 cows. Within blocks, cows were randomly assigned to 1 of 4 treatments: control (CON; urea as alternative nonprotein N source to nitrate), NO3 [21 g of nitrate/kg of dry matter (DM)], DHA (3 g of DHA/kg of DM and urea as alternative nonprotein N source to nitrate), or NO3 + DHA (21 g of nitrate/kg of DM and 3 g of DHA/kg of DM, respectively). Cows were fed a total mixed ration consisting of 21% grass silage, 49% corn silage, and 30% concentrates on a DM basis. Feed additives were included in the concentrates. Cows assigned to a treatment including nitrate were gradually adapted to the treatment dose of nitrate over a period of 21 d during which no DHA was fed. The experimental period lasted 17 d, and CH4 production was measured during the last 5 d in climate respiration chambers. Cows produced on average 363, 263, 369, and 298 g of CH4/d on CON, NO3, DHA, and NO3 + DHA treatments, respectively, and a tendency for a nitrate × DHA interaction effect was found where the CH4-mitigating effect of nitrate decreased when combined with DHA. This tendency was not obtained for CH4 production relative to dry matter intake (DMI) or to fat- and protein corrected milk (FPCM). The NO3 treatment decreased CH4 production irrespective of the unit in which it was expressed, whereas DHA did not affect CH4 production per kilogram of DMI, but resulted in a higher CH4 production per kilogram of fat- and protein-corrected milk (FPCM) production. The FPCM production (27.9, 24.7, 24.2, and 23.8 kg/d for CON, NO3, DHA, and NO3 + DHA, respectively) was lower for DHA-fed cows because of decreased milk fat concentration. The proportion of saturated fatty acids in milk fat was decreased by DHA, and the proportion of polyunsaturated fatty acids was increased by both nitrate and DHA. Milk protein concentration was lower for nitrate-fed cows. In conclusion, nitrate but not DHA decreased enteric CH4 production and no interaction effects were found on CH4 production per kilogram of DMI or per kilogram of FPCM

Effects of grass silage quality and level of feed intake on enteric methane production in lactating dairy cows

The objective of this study was to determine the effect of level of feed intake and quality of ryegrass silage as well as their interaction on enteric methane (CH4) emission from dairy cows. In a randomized block design, 56 lactating dairy cows received a diet of grass silage, corn silage, and a compound feed meal (70:10:20 on DM basis). Treatments consisted of 4 grass silage qualities prepared from grass harvested from leafy through late heading stage, and offered to dairy cows at 96 ± 2.4 (mean ± SEM) days in milk (namely, high intake) and 217 ± 2.4 d in milk (namely, low intake). Grass silage CP content varied between 124 and 286 g/kg of DM, and NDF content between 365 and 546 g/kg of DM. After 12 d of adaptation, enteric CH4 production of cows was measured in open-circuit climate-controlled respiration chambers for 5 d. No interaction between DMI and grass quality on CH4 emission, or on milk production, diet digestibility, and energy, and N retention was found (P ≥ 0.17). Cows had a greater DMI (16.6 vs. 15.5 kg/d; SEM 0.46) and greater fat- and protein-corrected milk (FPCM) yield (29.9 vs. 25.4 kg/d; SEM 1.24) at high than low intake (both P ≤ 0.001). Apparent total-tract nutrient digestibility was not affected (P ≥ 0.08) by DMI level. Total enteric CH4 production (346 ± 10.9 g/d) was not affected (P = 0.15) by DMI level. A small, significant (P = 0.025) decrease at high compared with low intake occurred for CH4 yield (21.8 ± 0.59 g/kg of DMI; −4%). Methane emission intensity (12.8 ± 0.56 g/kg of FPCM; −12%) was considerably smaller (P ≤ 0.001) at high intake as a result of greater milk yields realized in early lactation. As grass quality decreased from leafy through late heading stage, FPCM yield and apparent total-tract OM digestibility declined (−12%; P ≤ 0.015), whereas total CH4 production (+13%), CH4 yield (+21%), and CH4 emission intensity (+28%) increased (P ≤ 0.001). Our results suggest that improving grass silage quality by cutting grass at an earlier stage considerably reduces enteric CH4 emissions from dairy cows, independent of DMI. In contrast, losses of N in manure increased for the earlier cut grass silage treatments. The small increase in DMI at high intake was associated with a small to moderate reduction in CH4 emission per unit of DMI and GE intake. This study confirmed that enteric CH4 emissions from dairy cows at distinct levels of feed intake depend on the nutritive value and chemical composition of the grass silage

Increasing harvest maturity of whole-plant corn silage reduces methane

The objective of this study was to investigate the effects of increasing maturity of whole-plant corn at harvest on CH4 emissions by dairy cows consuming corn silage (CS) based diets. Whole-plant corn was harvested at a very early [25% dry matter (DM); CS25], early (28% DM; CS28), medium (32% DM; CS32), and late (40% DM; CS40) stage of maturity. In a randomized block design, 28 lactating Holstein-Friesian dairy cows, of which 8 were fitted with rumen cannula, received 1 of 4 dietary treatments designated as T25, T28, T32, and T40 to reflect the DM contents at harvest. Treatments consisted of (DM basis) 75% CS, 20% concentrate, and 5% wheat straw. Feed intake, digestibility, milk production and composition, energy and N balance, and CH4 production were measured during a 5-d period in climate respiration chambers after an adaptation to the diet for 12 d. Corn silage starch content varied between 275 (CS25) and 385 (CS40) g/kg of DM. Treatments did not affect DM intake (DMI), milk yield, or milk contents. In situ ruminal fractional degradation rate of starch decreased linearly from 0.098 to 0.059/h as maturity increased from CS25 to CS40. Apparent total-tract digestibility of DM, organic matter, crude protein, neutral detergent fiber, crude fat, starch, and gross energy (GE) decreased linearly with maturity. Treatments did not affect ruminal pH, volatile fatty acids, and ammonia concentrations, and volatile fatty acids molar proportions. The concentration of C18:3n-3 in milk fat decreased linearly, and the concentration of C18:2n-6 and the n-6:n-3 ratio increased linearly with maturity. A quadratic response occurred for the total saturated fatty acid concentration and total monounsaturated fatty acid concentration in milk fat. Methane production relative to DMI (21.7, 23.0, 21.0, and 20.1 g/kg) and relative to GE intake (0.063, 0.067, 0.063, and 0.060 MJ/MJ; values for T25, T28, T32, and T40, respectively) decreased linearly with maturity. Also, CH4 emission relative to fat- and protein-corrected milk tended to decrease linearly with maturity (13.0, 13.4, 13.2, and 12.1 g/kg of fat- and protein-corrected milk, for T25, T28, T32, and T40, respectively). Intake of GE and metabolizable energy, and energy retained, all expressed per unit of metabolic body weight, did not differ among treatments. Nitrogen intake, N use efficiency (milk N/N intake), and N balance were not influenced by treatments. Increasing maturity of whole-plant corn at harvest may offer an effective strategy to decrease CH4 losses with feeding CS without negatively affecting cow performance

Relationship between in vitro and in vivo methane production measured simultaneously with different dietary starch sources and starch levels in dairy cattle

To investigate the relationship between in vitro and in vivo methane (CH4) production measured simultaneously using the same rumen-fistulated cows in both experiments, four dietary treatments based on concentrate that accounted for 400 g/kg of the mixed diet DM, were formulated to contain starch varying in rate of fermentation (slowly (S) vs. rapidly (R): native vs. gelatinized maize grain) and level of inclusion (low (L) vs. high (H): 270 vs. 530 g/kg of concentrate DM). Sixteen rumen-fistulated lactating dairy cows were used in a complete randomized block design with these treatments replicated in four periods of 17 d each. In experiment 1, after 12 d of adaptation, the cows were housed in respiration chambers for 5 d to measure CH4 production. In experiment 2, in each period in vitro gas and CH4 production were measured (in duplicate per period) for mixed diet samples from the same diet as fed to the donor cows using rumen inocula adapted to the respective diets for an average of 16 d. In addition, samples of two concentrate ingredients, viz. grass silage and beet pulp, were incubated with four different inocula obtained from individual donor cows. Gas production (GP) was measured using automated GP system with CH4 measured at distinct time points. In vitro (24-h) CH4 production of mixed diet was lower with R than S (42.9 vs. 49.5 ml/g of incubated organic matter (OM); P=0.004), and higher with L than H (49.8 vs. 42.6 ml/g of incubated OM; P=0.002). A significant interaction effect between source and level of starch (P=0.015) was also found, indicating the CH4 production of the RH diet decreased in particular. In vivo, an increased rate of starch fermentation resulted in a lower CH4 per unit of estimated rumen-fermentable OM (eRFOM; 55.6 vs. 61.2 ml/g of eRFOM; P=0.007), and higher level of starch tended (P=0.089) to reduce CH4 per unit of eRFOM, but dietary starch level and source did not affect CH4 per unit of OM consumed. Across the diets tested, 24-h in vitro CH4 (ml/g of incubated OM) correlated well with in vivo CH4 expressed per unit of eRFOM (R2 = 0.54; P=0.040), but not when expressed per unit of OM ingested (R2 = 0.04; P=0.878). For grass silage (the same trend for beet pulp), inocula adapted to R- and H-based diets compared with S- and L-based diets resulted in a lower CH4 production (36.1 vs. 44.8 ml/g of incubated OM, R vs. S; and 37.4 vs. 43.4 ml/g of incubated OM, H vs. L;

Effects of dietary starch content and rate of fermentation on methane production in lactating dairy cows

The objective of this study was to investigate the effects of starch varying in rate of fermentation and level of inclusion in the diet in exchange for fiber on methane (CH4) production of dairy cows. Forty Holstein-Friesian lactating dairy cows of which 16 were rumen cannulated were grouped in 10 blocks of 4 cows each. Cows received diets consisting of 60% grass silage and 40% concentrate (dry matter basis). Cows within block were randomly assigned to 1 of 4 different diets composed of concentrates that varied in rate of starch fermentation [slowly (S) vs. rapidly (R) rumen fermentable; native vs. gelatinized corn grain] and level of starch (low vs. high; 270 vs. 530 g/kg of concentrate dry matter). Results of rumen in situ incubations confirmed that the fractional rate of degradation of starch was higher for R than S starch. Effective rumen degradability of organic matter was higher for high than low starch and also higher for R than S starch. Increased level of starch, but not starch fermentability, decreased dry matter intake and daily CH4 production. Milk yield (mean 24.0 ± 1.02 kg/d), milk fat content (mean 5.05 ± 0.16%), and milk protein content (mean 3.64 ± 0.05%) did not differ between diets. Methane expressed per kilogram of fat- and protein-corrected milk, per kilogram of dry matter intake, or as a fraction of gross energy intake did not differ between diets. Methane expressed per kilogram of estimated rumen-fermentable organic matter (eRFOM) was higher for S than R starch–based diets (47.4 vs. 42.6 g/kg of eRFOM) and for low than high starch–based diets (46.9 vs. 43.1 g/kg of eRFOM). Apparent total-tract digestibility of neutral detergent fiber and crude protein were not affected by diets, but starch digestibility was higher for diets based on R starch (97.2%) compared with S starch (95.5%). Both total volatile fatty acid concentration (109.2 vs. 97.5 mM) and propionate proportion (16.5 vs. 15.8 mol/100 mol) were higher for R starch– compared with S starch–based diets but unaffected by the level of starch. Total N excretion in feces plus urine and N retained were unaffected by dietary treatments, and similarly energy intake and output of energy in milk expressed per unit of metabolic body weight were not affected by treatments. In conclusion, an increased rate of starch fermentation and increased level of starch in the diet of dairy cattle reduced CH4 produced per unit of eRFOM but did not affect CH4 production per unit of feed dry matter intake or per unit of milk produced

Effect of nitrogen fertilization rate and regrowth interval of grass herbage on methane emission of zero-grazing lactating dairy cows

Dairy cattle farming in temperate regions often relies on grass herbage (GH)-based diets but the effect of several grass management options on enteric CH4 emission has not been fully investigated yet. We investigated the combined effect of N fertilization rate and length of regrowth period of GH (predominantly ryegrass) on CH4 emission from lactating dairy cows. In a randomized block design, 28 lactating Holstein-Friesian dairy cows received a basal diet of GH and compound feed [85:15; dry matter (DM) basis]. Treatments consisted of GH cut after 3 or 5 weeks of regrowth, after receiving either a low (20 kg of N/ha) or a high (90 kg of N/ha) fertilization rate after initial cut. Feed intake, digestibility, milk production and composition, N and energy balance, and CH4 emission were measured during a 5-d period in climate respiration chambers after an adaptation to the diet for 12 d. Cows were restricted-fed during measurements and mean DM intake was 15.0 ± 0.16 kg/d. Herbage crude protein content varied between 76 and 161 g/kg of DM, and sugar content between 186 and 303 g/kg of DM. Fat- and protein-corrected milk (FPCM) and feed digestibility increased with increased N fertilization rates and a shorter regrowth interval. Increasing the N fertilization rate increased daily CH4 emission per cow (+10%) and per unit of DM intake (+9%), tended to increase the fraction of gross energy intake emitted as CH4 (+7%), and (partly because of the low crude protein content for the low fertilized GH) only numerically reduced CH4 per unit of FPCM. The longer regrowth interval increased CH4 emission per unit of FPCM (+14%) compared with the shorter regrowth interval, but did not affect CH4 emission expressed in any other unit. With increasing N fertilization CH4 emission decreased per unit of digestible neutral detergent fiber intake (-13%) but not per unit of digestible organic matter intake. There was no interaction of the effect of N fertilization rate and regrowth interval on CH4 emission, but effects of N fertilization were generally most distinct with GH of 5 wk regrowth. The present results suggest that altering grass quality through an increase of N fertilization and a shorter regrowth interval can reduce CH4 emission in zero-grazing dairy cows, depending on the unit in which it is expressed. The larger amount of CH4 produced per day and cow with the more intensively managed GH is compensated by a higher feed digestibility and FPCM yield