Screening macroalgae for mitigation of enteric methane in vitro.

This study investigated the effects of 67 species of macroalgae on methanogenesis and rumen fermentation in vitro. Specimens were analyzed for their effect on ruminal fermentation and microbial community profiles. Incubations were carried out in an automated gas production system for 24-h and macroalgae were tested at 2% (feed dry matter basis) inclusion rate. Methane yield was decreased 99% by Asparagopsis taxiformis (AT) when compared with the control. Colpomenia peregrina also decreased methane yield 14% compared with control; no other species influenced methane yield. Total gas production was decreased 14 and 10% by AT and Sargassum horneri compared with control, respectively. Total volatile fatty acid (VFA) concentration was decreased between 5 and 8% by 3 macroalgae, whereas AT reduced it by 10%. Molar proportion of acetate was decreased 9% by AT, along with an increase in propionate by 14%. Asparagopsis taxiformis also increased butyrate and valerate molar proportions by 7 and 24%, respectively, whereas 3 macroalgae species decreased molar proportion of butyrate 3 to 5%. Vertebrata lanosa increased ammonia concentration, whereas 3 other species decreased it. Inclusion of AT decreased relative abundance of Prevotella, Bacteroidales, Firmicutes and Methanobacteriaceae, whereas Clostridium, Anaerovibrio and Methanobrevibacter were increased. Specific gene activities for Methanosphaera stadtmane and Methanobrevibacter ruminantium were decreased by AT inclusion. In this in vitro study, Asparagopsis taxiformis was most effective in decreasing methane concentration and yield, but also decreased total gas production and VFA concentration which indicates overall inhibition of ruminal fermentation. No other macroalgae were identified as potential mitigants of enteric methane.

Effects of phytonutrients and yeast culture supplementation on lactational performance and nutrient use efficiency in dairy cows.

Yeast culture and phytonutrients are dietary supplements with distinct modes of action, and they may have additive effects on the performance of dairy cattle. The objective of this study was to investigate the effects of a preparation of phytonutrients and a yeast culture from Saccharomyces cerevisiae on lactational performance, total-tract digestibility of nutrients, urinary nitrogen losses, energy metabolism markers, and blood cells in dairy cows. Thirty-six mid-lactation Holstein cows (10 primiparous and 26 multiparous) were used in an 8-wk randomized complete block design experiment with a 2-wk covariate period, 2 wk for adaptation to the diets, and a 4-wk experimental period for data and samples collection. Following a 2-wk covariate period, cows were blocked by days in milk, parity, and milk yield and randomly assigned to 1 of 3 treatments (12 cows per treatment): basal diet supplemented with 14 g/cow per day yeast culture (YC; S. cerevisiae), basal diet supplemented with 1.0 g/cow per day phytonutrients (PN; 5.5% cinnamaldehyde, 9.5% eugenol, and 3.5% capsicum oleoresin), or basal diet supplemented with a combination of YC and PN (YCPN). Treatments were top-dressed once daily on the total mixed ration at time of feeding. Dry matter intake, milk yield, and feed efficiency were not affected by treatments. Milk composition and energy-corrected milk yield were also not affected by supplementation of YC, PN, and YCPN. There were no differences in intake or total-tract digestibility of dietary nutrients among treatments. Compared with YC, the PN and YCPN treatments tended to decrease the proportion of short-chain fatty acids in milk fat. There was an additive effect of YC and PN supplementation on urinary urea nitrogen (UUN) excretion relative to total nitrogen intake. Cows fed a diet supplemented with YCPN had lower UUN excretion than cows in YC and tended to have lower UUN excretion compared with PN. Blood monocytes count and percentage were decreased in cows fed PN and YCPN diets compared with YC. Treatments did not affect concentrations of blood ß-hydroxybutyrate and total fatty acids. Overall, lactational performance, digestibility of nutrients, energy metabolism markers, and blood cells were not affected by YC, PN, or YCPN supplementation. A combination of PN and YC had an additive effect on nitrogen excretion in dairy cows.

Effects of an exogenous enzyme preparation extracted from a mixed culture of Aspergillus spp. on lactational performance, metabolism, and digestibility in primiparous and multiparous cows.

The objective of this study was to investigate the effects of an exogenous enzyme preparation from Aspergillus oryzae and Aspergillus niger on lactational performance of dairy cows. Forty-eight Holstein cows (32 primiparous and 16 multiparous) averaging (± SD) 36.3 ± 8.7 kg/d milk yield and 141 ± 52 d in milk were enrolled in a 10-wk randomized complete block design experiment (total of 24 blocks) and assigned to 1 of 2 treatments: basal diet, no enzyme supplementation (CON) or the basal diet supplemented with 4.2 g/kg dry matter intake (DMI) of an exogenous enzyme preparation containing amylolytic and fibrolytic activities (ENZ). After a 2-wk covariate period, premixes with the enzyme preparation or control were top-dressed daily by mixing with approximately 500 g of total mixed ration. Production data were collected daily and averaged by week. Milk samples were collected every other week, and milk composition was averaged by week. Blood, fecal, and urine samples were collected over 2 consecutive days at 0, 4, 8, 12, and 36 h after feeding during the last week of the experiment. Compared with CON, cows fed ENZ tended to increase DMI and had increased milk concentrations of true protein, lactose, and other solids. Milk fat content tended to be higher in CON cows. A treatment × parity interaction was found for some of the production variables. Primiparous cows receiving ENZ had greater yields of milk, energy-corrected milk, milk true protein, and lactose compared with CON primiparous cows; these production variables did not differ between treatments for multiparous cows. Intake and total-tract digestibility of nutrients did not differ between treatments. Concentrations of blood glucose and total fatty acids were not affected by ENZ supplementation, but ß-hydroxybutyrate concentration tended to be greater in ENZ cows. Overall, the exogenous enzyme preparation used in this study increased milk protein and lactose concentrations in all cows, and milk production in primiparous but not multiparous cows. The differential production response between primiparous and multiparous cows was likely a result of a greater increase in DMI with ENZ supplementation in the younger animals.

Symposium review: Effective nutritional strategies to mitigate enteric methane in dairy cattle.

Intensive research in the past decade has resulted in a better understanding of factors driving enteric methane (CH4) emissions in ruminants. Meta-analyses of large databases, developed through the GLOBAL NETWORK project, have identified successful strategies for mitigation of CH4 emissions. Methane inhibitors, alternative electron sinks, vegetable oils and oilseeds, and tanniferous forages are among the recommended strategies for mitigating CH4 emissions from dairy and beef cattle and small ruminants. These strategies were also effective in decreasing CH4 emissions yield and intensity. However, a higher inclusion rate of oils may negatively affect feed intake, rumen function, and animal performance, specifically milk components in dairy cows. In the case of nitrates (electron sinks), concerns with animal health may be impeding their adoption in practice, and potential emission trade-offs have to be considered. Tannins and tanniferous forages may have a negative effect on nutrient digestibility, and more research is needed to confirm their effects on overall animal performance in long-term experiments with high-producing animals. A meta-analysis of studies with dairy cows fed the CH4 inhibitor 3-nitrooxypropanol (3-NOP) at the Pennsylvania State University showed (1) a consistent 28 to 32% decrease in daily CH4 emissions or emissions yield and intensity; (2) no effect on dry matter intake, milk production, body weight, or body weight change, and a slight increase in milk fat concentration and yield (0.19 percentage units and 90 g/d, respectively); 3-NOP also appears to increase milk urea nitrogen concentration; (3) an exponential decrease in the mitigation effect of the inhibitor with increasing its dose (from 40 to 200 mg/kg of feed dry matter, corresponding to 3-NOP intake of 1 to 4.8 g/cow per day); and (4) a potential decrease in the efficacy of 3-NOP over time, which needs to be further investigated in long-term, full-lactation or multiple-lactation studies. The red macroalga Asparagopsis taxiformis has a strong CH4 mitigation effect, but studies are needed to determine its feasibility, long-term efficacy, and effects on animal production and health. We concluded that widespread adoption of mitigation strategies with proven effectiveness by the livestock industries will depend on cost, government policies and incentives, and willingness of consumers to pay a higher price for animal products with decreased carbon footprint.

Performance of dairy cows fed normal- or reduced-starch diets supplemented with an exogenous enzyme preparation.

The objective of this study was to investigate the effects of supplementation of an exogenous enzyme preparation (EEP) on performance, total-tract digestibility of nutrients, plasma AA profile, and milk fatty acids composition in lactating dairy cows fed a reduced-starch diet compared with a normal-starch diet (i.e., positive control). Forty-eight Holstein cows (28 primiparous and 20 multiparous) were enrolled in a 10-wk randomized complete block design experiment with 16 cows per treatment. Treatments were as follows: (1) normal-starch diet (control) containing (% dry matter basis) 24.8% starch and 33.0% neutral detergent fiber (NDF), (2) reduced-starch diet (RSD) containing 18.4% starch and 39.1% NDF, or (3) RSD supplemented with 10 g/cow per day of an EEP (ENZ). The EEP contained amylolytic and fibrolytic activities and was top-dressed on the total mixed ration at the time of feeding. Compared with normal-starch diet, dry matter intake and milk and energy-corrected milk (ECM) yields were lower (on average by 7.1, 9.5, and 7.2%, respectively) for cows on the RSD treatments. Concentrations, but not yields, of milk fat and total solids were increased by RSD. Energy-corrected milk feed efficiency did not differ among treatments. Total-tract digestibility of NDF tended to increase by RSD treatments. Plasma AA concentrations were not affected by treatment, except that of 3-methylhistidine was increased by ENZ, compared with RSD. Blood glucose concentration tended to be lower in cows on the RSD treatments, but ENZ increased glucose and tended to increase insulin concentrations at 4 h after feeding when compared with RSD. Cows on the RSD treatments had decreased concentrations of de novo fatty acids and tended to have increased concentrations of preformed fatty acids in milk. Overall, decreasing dietary starch concentration by 26% decreased dry matter intake, milk, and ECM yields, but ECM feed efficiency was not different among treatments. The negative effects of reducing dietary starch on production were not attenuated by the EEP.

Lactational performance and plasma and muscle amino acid concentrations in dairy cows fed diets supplying 2 levels of digestible histidine and metabolizable protein.

The objective of this experiment was to investigate the effect of dietary levels of digestible histidine (dHis) and MP on lactational performance and plasma and muscle concentrations of free AA in dairy cows. A randomized block design experiment was conducted with 48 Holstein cows, including 20 primiparous, averaging (±SD) 103 ± 22 d in milk and 45 ± 9 kg/d milk yield at the beginning of the experiment. A 2-wk covariate period preceded 12 experimental wk, of which 10 wk were for data and sample collection. Experimental treatments were (1) MP-adequate (MPA) diet with 2.1% dHis of MP (MPA2.1), (2) MPA with 3.0% dHis (MPA3.0), (3) MP-deficient (MPD) diet with 2.1% dHis (MPD2.1), and (4) MPD with 3.0% dHis (MPD3.0). Actual dHis supply was estimated at 64, 97, 57, and 88 g/d, respectively. Diets supplied MP at 110% (MPA) and 96% (MPD) of NRC 2001 dairy model requirements calculated based on DMI and production data during the experiment. Dry matter intake and milk yield data were collected daily, milk samples for composition and blood samples for AA analysis were collected every other week, and muscle biopsies at the end of covariate period, and during wk 12 of the experiment. The overall DMI was not affected by dHis or MP level. Milk yield tended to be increased by 3.0% dHis compared with 2.1% dHis. Milk true protein concentration and yield were not affected by treatments, whereas milk urea nitrogen concentration was lower for MPD versus the MPA diet. Milk fat concentration was lower for MPD versus MPA. There was a MP × dHis interaction for milk fat yield and energy-corrected milk; milk fat was lower for MPD3.0 versus MPD2.1, but similar for cows fed the MPA diet regardless of dHis level whereas energy-corrected milk was greater for MPA3.0 versus MPA2.1 but tended to be lower for MPD3.0 versus MPD2.1. Plasma His concentration was greater for cows fed dHis3.0, and concentration of sum of essential AA was greater, whereas carnosine, 1-Methyl-His and 3-Methyl-His concentrations were lower for cows fed MPA versus MPD diet. Muscle concentration of His was greater for cows fed dHis3.0 treatment. The apparent efficiency of His utilization was increased at lower MP and His levels. Overall, cows fed a corn silage-based diet supplying MP at 110% of NRC (2001) requirements tended to have increased ECM yield and similar milk protein yield to cows fed a diet supplying MP at 96% of requirements. Supplying dHis at 3.0% of MP (or 86 and 96 g/d, for MPD3.0 and MPA3.0, respectively) tended to increase milk yield and increased plasma and muscle concentrations of His but had minor or no effects on other production variables in dairy cows.

Dietary supplementation with rumen-protected capsicum during the transition period improves the metabolic status of dairy cows.

In ruminants, it has been observed that capsicum oleoresin can alter insulin responses and that high-intensity artificial sweetener can increase glucose absorption from the small intestine. Because glucose metabolism and insulin responses are critical during early lactation, these supplements might have an effect on the metabolic status of dairy cows during the transition period. The objective of this experiment was to evaluate the effects of rumen-protected capsicum oleoresin fed alone or in combination with artificial sweetener during the transition period on lactational performance and susceptibility to subclinical ketosis in dairy cows. Fifteen primiparous and 30 multiparous Holstein cows (a total of 39 cows finished the study) were arranged in a randomized complete block design during d -21 to 60 relative to parturition. Cows within block were randomly assigned to one of the following treatments: no supplement (CON), supplementation with 100 mg of rumen-protected capsicum/cow per day (RPCap), or RPCap plus 2 g of high-intensity artificial sweetener/cow per day (RPCapS). For both the RPCap and RPCapS treatments, only rumen-protected capsicum was fed during the dry period. From d 8 to 11 of lactation, intake was limited to 70% of predicted dry matter intake to induce subclinical ketosis. Production variables were recorded daily, samples for milk composition were collected on wk 2, 4, 6, and 8, and blood samples were collected on wk -2, 1, 2, and 4 of the experiment for analysis of metabolic hormones and blood cell counts. Supplementation with rumen-protected capsicum increased serum insulin and decreased ß-hydroxybutyrate concentrations precalving, indicating a decrease in lipolysis. During the lactation period, RPCap was associated with a trend for increased milk production and feed efficiency following the ketosis challenge. Supplementation with RPCapS appeared to negate the response to rumen-protected capsicum. All cows developed subclinical ketosis during the challenge, and this was not affected by treatment. We conclude that treatments did not decrease susceptibility to subclinical ketosis; however, dietary supplementation with rumen-protected capsicum was effective at improving energy status precalving and tended to increased milk production and feed efficiency. The mechanism underlying these responses is unclear.

Lactational performance, rumen fermentation, and enteric methane emission of dairy cows fed an amylase-enabled corn silage.

This study investigated the effects of an amylase-enabled corn silage on lactational performance, enteric CH4 emission, and rumen fermentation of lactating dairy cows. Following a 2-wk covariate period, 48 Holstein cows were blocked based on parity, days in milk, milk yield (MY), and CH4 emission. Cows were randomly assigned to 1 of 2 treatments in an 8-wk randomized complete block design experiment: (1) control corn silage (CON) from an isogenic corn without α-amylase trait and (2) Enogen hybrid corn (Syngenta Seeds LLC) harvested as silage (ECS) containing a bacterial transgene expressing α-amylase (i.e., amylase-enabled) in the endosperm of the grain. The ECS and CON silages were included at 40% of the dietary dry matter (DM) and contained, on average, 43.3 and 41.8% DM and (% DM) 36.7 and 37.5% neutral detergent fiber, and 36.1 and 33.1% starch, respectively. Rumen samples were collected from a subset of 10 cows using the ororuminal sampling technique on wk 3 of the experimental period. Enteric CH4 emission was measured using the GreenFeed system (C-Lock Inc.). Dry matter intake (DMI) was similar between treatments. Compared with CON, MY (38.8 vs. 40.8 kg/d), feed efficiency (1.47 vs. 1.55 kg of MY/kg of DMI), and milk true protein (1.20 vs. 1.25 kg/d) and lactose yields (1.89 vs. 2.00 kg/d) were increased, whereas milk urea nitrogen (14.0 vs. 12.7 mg/dL) was decreased, with the ECS diet. No effect of treatment on energy-corrected MY (ECM) was observed, but a trend was detected for increased ECM feed efficiency (1.45 vs. 1.50 kg of ECM/kg of DMI) for cows fed ECS compared with CON-fed cows. Daily CH4 emission was not affected by treatment, but emission intensity was decreased with the ECS diet (11.1 vs. 10.3 g/kg of milk, CON and ECS, respectively); CH4 emission intensity on ECM basis was not different between treatments. Rumen fermentation, apart from a reduced molar proportion of butyrate in ECS-fed cows, was not affected by treatment. Apparent total-tract digestibility of nutrients and urinary and fecal nitrogen excretions, apart from a trend for increased DM digestibility by ECS-fed cows, were not affected by treatment. Overall, ECS inclusion at 40% of dietary DM increased milk, milk protein, and lactose yields and feed efficiency, and tended to increase ECM feed efficiency but had no effect on ECM yield in dairy cows. The increased MY with ECS led to a decrease in enteric CH4 emission intensity, compared with the control silage.

Histidine dose-response effects on lactational performance and plasma amino acid concentrations in lactating dairy cows: 1. Metabolizable protein-adequate diet.

The objective of this experiment was to determine the effect of increasing digestible His (dHis) doses on milk production, milk composition, and plasma AA concentrations in lactating dairy cows fed diets that meet or exceed their energy and metabolizable protein (MP) requirements. In a companion paper (Räisänen et al., 2021) results are presented on the effect of increasing dHis dose with an MP-deficient basal diet. In this experiment, 16 Holstein cows (72 ± 15 d in milk) were used in a replicated 4 × 4 Latin square design experiment with four 28-d periods. Treatments were as follows: (1) control, total mixed ration (TMR) with 1.8% dHis of MP (TMR1; dHis1.8); (2) a different TMR with 2.2% dHis (TMR2; dHis2.2); (3) TMR2 supplemented with rumen-protected His (RP-His) to supply 2.6% dHis (dHis2.6); and (4) TMR2 supplemented with RP-His to supply 3.0% dHis of MP (dHis3.0). Estimated dHis intakes calculated at the end of the experiment were 46, 58, 69, and 79 g/d for dHis1.8, dHis2.2, dHis2.6, and dHis3.0, respectively. Contrasts were used to compare TMR1 with TMR2 and to test the linear and quadratic effects of RP-His inclusion rate on TMR2. We detected no effects of TMR or dHis dose on dry matter intake or milk yield, whereas energy-corrected milk (ECM) yield was quadratically increased, being greatest for cows on treatment dHis2.6. Milk true protein and lactose concentrations and milk true protein yield were not affected by TMR or dHis dose. Milk fat concentration and yield increased quadratically, and lactose yield tended to increase quadratically with increasing dHis dose. Calculated apparent efficiency of His utilization decreased quadratically with increasing dHis supply. Further, plasma concentration of His was greater for cows on TMR2 compared with TMR1. When an MP-adequate diet was fed to dairy cows, milk true protein concentration and yield were not affected by dHis supply, but milk fat and ECM yields of dairy cows were optimized at dHis supply of 69 g/d or 2.65% of MP.

Histidine dose-response effects on lactational performance and plasma amino acid concentrations in lactating dairy cows: 2. Metabolizable protein-deficient diet.

The objective of this experiment was to determine the effect of increasing digestible His (dHis) levels with a rumen-protected (RP) His product on milk production, milk composition, and plasma AA concentrations in lactating dairy cows fed a metabolizable protein (MP)-deficient diet, according to the National Research Council dairy model from 2001. The companion paper presents results on the effect of increasing dHis dose with a MP-adequate basal diet. Twenty Holstein cows, of which 8 were rumen-cannulated, were used in a replicated 4 × 4 Latin square design experiment with four 28-d periods. Treatments were a control diet supplying 1.8% dHis of MP or 37 g/d (dHis1.8) and the control diet supplemented RP-His to provide 2.2, 2.6, or 3.0%, dHis of MP, or 53, 63, and 74 g/d (dHis2.2, dHis2.6, and dHis3.0, respectively). Histidine dose did not affect dry matter intake, but milk yield increased quadratically and energy-corrected milk yield increased linearly with increasing dHis dose. Histidine dose had a quadratic effect on milk fat concentration but did not affect milk fat yield. Lactose concentration decreased linearly, whereas lactose yield increased linearly with increasing dHis dose. There was a tendency for a linear increase in milk true protein concentration, and milk true protein yield increased linearly with dHis dose. Further, plasma His concentration increased linearly with increasing dHis dose and calculated apparent efficiency of His utilization decreased quadratically with increasing dHis supply. Histidine had minor or no effects on rumen fermentation. In the conditions of this experiment, RP-His supplementation of an MP-deficient corn silage-based diet increased milk yield linearly up to a dHis supply of 63 g/d (or 2.6% dHis of MP) and increased feed efficiency, energy-corrected milk yield and milk true protein yield linearly up to a dHis supply of 74 g/d (or 3.0% dHis of MP) in lactating dairy cows.

Temporal changes in total and metabolically active ruminal methanogens in dairy cows supplemented with 3-nitrooxypropanol.

The purpose of this study was to investigate the effect of 3-nitrooxypropanol (3-NOP), a potent methane inhibitor, on total and metabolically active methanogens in the rumen of dairy cows over the course of the day and over a 12-wk period. Rumen contents of 8 ruminally cannulated early-lactation dairy cows were sampled at 2, 6, and 10 h after feeding during wk 4, 8, and 12 of a randomized complete block design experiment in which 3-NOP was fed at 60 mg/kg of feed dry matter. Cows (4 fed the control and 4 fed the 3-NOP diet) were blocked based on their previous lactation milk yield or predicted milk yield. Rumen samples were extracted for microbial DNA (total) and microbial RNA (metabolically active), PCR amplified for the 16S rRNA gene of archaea, sequenced on an Illumina platform, and analyzed for archaea diversity. In addition, the 16S copy number and 3 ruminal methanogenic species were quantified using the real-time quantitative PCR assay. We detected a difference between DNA and RNA (cDNA)-based archaea communities, revealing that ruminal methanogens differ in their metabolic activities. Within DNA and cDNA components, methanogenic communities differed by sampling hour, week, and treatment. Overall, Methanobrevibacter was the dominant genus (94.3%) followed by Methanosphaera, with the latter genus having greater abundance in the cDNA component (14.5%) compared with total populations (5.5%). Methanosphaera was higher at 2 h after feeding, whereas Methanobrevibacter increased at 6 and 10 h in both groups, showing diurnal patterns among individual methanogenic lineages. Methanobrevibacter was reduced at wk 4, whereas Methanosphaera was reduced at wk 8 and 12 in cows supplemented with 3-NOP compared with control cows, suggesting differential responses among methanogens to 3-NOP. A reduction in Methanobrevibacter ruminantium in all 3-NOP samples from wk 8 was confirmed using real-time quantitative PCR. The relative abundance of individual methanogens was driven by a combination of dietary composition, dry matter intake, and hydrogen concentrations in the rumen. This study provides novel information on the effects of 3-NOP on individual methanogenic lineages, but further studies are needed to understand temporal dynamics and to validate the effects of 3-NOP on individual lineages of ruminal methanogens.

Lactational performance, enteric gas emissions, and plasma amino acid profile of dairy cows fed diets with soybean or canola meals included on an equal protein basis.

This study investigated the effects of feeding solvent-extracted canola meal (CM), extruded soybean meal (ESBM), or solvent-extracted soybean meal (SSBM) on an equivalent crude protein basis on performance, plasma AA profiles, enteric gas emissions, milk fatty acids, and nutrient digestibility in lactating dairy cows. Fifteen Holstein cows (95 ± 20 d in milk) were used in a replicated 3 × 3 Latin square design experiment with 3 periods of 28 d each. Treatments were 3 diets containing 17.1% CM, 14.2% ESBM, or 13.6% SSBM (dry matter basis). Vegetable oil was added (canola oil for CM or soybean oil for SSBM) to equalize the ether extract concentration of the diets. Rumen-protected Met was supplemented targeting digestible Met supply of 2.2% of metabolizable protein in all diets. Canola meal increased dry matter intake (DMI) by 5.9 and 8.9% in comparison with ESBM and SSBM, respectively. Milk urea nitrogen was lowest in CM, followed by SSBM, and was highest for ESBM. No differences were observed in feed efficiency, energy-corrected milk yield, and milk composition or component yields among treatments. Cows fed CM emitted less enteric CH4 per kg of DMI compared with both ESBM and SSBM, but CH4 emission intensity (CH4 per kg of energy-corrected milk) was similar among treatments. In summary, replacement of ESBM or SSBM with CM, on an equal crude protein basis, in the diet of lactating dairy cows enhanced DMI, but yields of energy-corrected milk and milk components and feed efficiency were similar among treatments.

Effects of the macroalga Asparagopsis taxiformis and oregano leaves on methane emission, rumen fermentation, and lactational performance of dairy cows.

Asparagopsis taxiformis (AT) is a source of multiple halogenated compounds and, in a limited number of studies, has been shown to decrease enteric CH4 emission in vitro and in vivo. Similarly, oregano has been suggested as a potential CH4 mitigating agent. This study consisted of 2 in vitro and 2 in vivo experiments. Experiment (Exp.) 1 was aimed at establishing the effect of AT on CH4 emission in vitro. Two experiments (Exp. 2 and 3) with lactating dairy cows were conducted to determine the antimethanogenic effect of AT and oregano (Exp. 3) in vivo. Another experiment (Exp. 4) was designed to investigate stability of bromoform (CHBr3) in AT over time. In Exp. 3, 20 Holstein cows were used in a replicated 4 × 4 Latin square design with four 28-d periods. Treatments were basal diet (control) or basal diet supplemented with (dry matter basis) 0.25% AT (LowAT), 0.50% AT (HighAT), or 1.77% oregano (Origanum vulgare L.) leaves. Enteric gas emissions were measured using the GreenFeed system (C-Lock Inc., Rapid City, SD), and rumen samples were collected for fermentation analysis using the ororuminal technique. In Exp.1 (in vitro), relative to the control, AT (at 1% dry matter basis, inclusion rate) decreased CH4 yield by 98%. In Exp. 3, HighAT decreased average daily CH4 emission and CH4 yield by 65% and 55%, respectively, in experimental periods 1 and 2, but had no effect in periods 3 and 4. The differential response to AT among experimental periods was likely a result of a decrease in CHBr3 concentration in AT over time, as observed in Exp. 4 (up to 84% decrease in 4 mo of storage). In Exp. 3, H2 emission was increased by AT and, as expected, the proportion of acetate in the total volatile fatty acids in the rumen was decreased and those of propionate and butyrate were increased by HighAT compared with the control. Compared with the control, HighAT decreased dry matter intake, milk yield, and energy-corrected milk yield in Exp. 3. Milk composition was not affected by treatment, except lactose percentage and yield were decreased by HighAT. Concentrations of iodine and bromide in milk were increased by HighAT compared with the control. Milk CHBr3 concentration and its organoleptic characteristics were not different between control and HighAT. Oregano had no effect on CH4 emission or lactational performance of the cows in Exp. 3. Overall, AT included at 0.50% in the ration of dairy cows can have a large mitigation effect on enteric CH4 emission, but dry matter intake and milk production may also decrease. There was a marked decrease in the CH4 mitigation potential of AT in the second half of Exp. 3, likely resulting from CHBr3 decay over time.

Comparing noninvasive sampling techniques with standard cannula sampling method for ruminal microbial analysis.

Rumen microbes play an important role in the conversion of indigestible plant material to energy and protein in dairy cows. Sampling for ruminal contents via cannula is considered the gold standard technique for microbial analysis, but the technique requires ruminally cannulated animals and specialized animal facilities. The purpose of this study was to determine whether other sampling methods and locations along the digestive tract may serve as noninvasive proxies to the cannula method for microbial analysis. Six ruminally cannulated lactating Holstein dairy cows were adapted to a standard total mixed ration for 2 wk and sampled during the third week. Sampling locations and methods included salivary content, rumination bolus (regurgitated digesta collected from the cow's mouth), feces, and rumen contents via stomach tube and cannula. Stomach tube and cannula samples differ in proportions of solid and liquid material and were therefore separated into whole (as collected), liquid, and solid fractions. Samples were collected at 0 (before feeding), 2, 4, 6, 8, and 12 h after feeding over 2 d. All samples were extracted for total genomic DNA and selected samples for metabolically active DNA (RNA), PCR-amplified for the V1-V2 region of the 16S rRNA bacterial gene, and analyzed for bacterial diversity using the QIIME2 pipeline followed by statistical analysis in R (https://www.R-project.org/). In DNA-based analysis, at the community level, saliva, rumination bolus, and fecal samples clustered in separate groups, whereas all fractions of stomach tube and cannula samples clustered together, indicating that microbial communities of stomach tube and cannula samples were homogeneous. Rumination bolus samples at 6, 8, and 12 h after feeding clustered with stomach tube and cannula samples, indicating that rumination bolus samples may be an alternative for cannula samples; however, time of sampling is critical for sampling of bolus digesta. Results of the RNA-based analysis of rumination bolus samples and solid samples from cannula and stomach tube at 0 and 6 h after feeding were similar. We concluded that the solid fraction of samples obtained via the stomach tube method may serve as a proxy for the solid fraction of whole ruminal contents obtained via cannula for DNA-based microbial investigations. Both rumination bolus and stomach tube solid samples may serve as proxies for cannula solid samples for RNA-based microbial analysis.

Enteric methane emission, milk production, and composition of dairy cows fed 3-nitrooxypropanol.

This study examined the effect of 3-nitrooxypropanol (3-NOP), an investigational substance, on enteric methane emission, milk production, and composition in Holstein dairy cows. Following a 3-wk covariate period, 48 multi- and primiparous cows averaging (± standard deviation) 118 ± 28 d in milk, 43.4 ± 8 kg/d milk yield, and 594 ± 57 kg of body weight were blocked based on days in milk, milk yield, and enteric methane emission and randomly assigned to 1 of 2 treatment groups: (1) control, no 3-NOP, and (2) 3-NOP applied at 60 mg/kg feed dry matter. Inclusion of 3-NOP was through the total mixed ration and fed for 15 consecutive weeks. Cows were housed in a freestall barn equipped with a Calan Broadbent Feeding System (American Calan Inc., Northwood, NH) for monitoring individual dry matter intake and fed ad libitum once daily. Enteric gaseous emissions (methane, carbon dioxide, and hydrogen) were measured using 3 GreenFeed (C-Lock Inc., Rapid City, SD) units. Dry matter intake, cow body weight, and body weight change were not affected by 3-NOP. Compared with the control group, 3-NOP applied at 60 mg/kg feed dry matter decreased daily methane emission, emission yield, and emission intensity by 26, 27, and 29%, respectively. Enteric emission of carbon dioxide was not affected, and hydrogen emission was increased 6-fold by 3-NOP. Administration of 3-NOP had no effect on milk and energy-corrected milk yields and feed efficiency, increased milk fat and milk urea nitrogen concentrations, and increased milk fat yield but had no other effects on milk components. Concentration of C6:0 and C8:0 and the sum of saturated fatty acids in milk fat were increased by 3-NOP. Total trans fatty acids and the sum of polyunsaturated fatty acids were decreased by 3-NOP. In this experiment, 3-NOP decreased enteric methane daily emission, yield, and intensity without affecting dry matter intake and milk yield, but increased milk fat in high-producing dairy cows.

Short communication: Short-term effect of 3-nitrooxypropanol on feed dry matter intake in lactating dairy cows.

The objective of this study was to investigate the effect of 3-nitrooxypropanol (3-NOP), an enteric methane inhibitor under investigation, on short-term dry matter intake (DMI) in lactating dairy cows. Following a 1-wk adaptation period, 12 multiparous Holstein cows were fed a basal total mixed ration (TMR) containing increasing levels of 3-NOP during 5 consecutive, 6-d periods. The experiment was conducted in a tiestall barn. Feed bins were split in half by a solid divider, and cows simultaneously received the basal TMR supplemented with the following: (1) a placebo without 3-NOP or (2) 3-NOP included in the TMR at 30, 60, 90, or 120 mg/kg of feed dry matter (experimental periods 2, 3, 4, and 5, respectively). Cows received the control diet (basal TMR plus placebo premix) during experimental period 1. A premix containing ground corn grain, soybean oil, and dry molasses was used to incorporate 3-NOP in the ration. Cows were fed twice daily as follows: 60% of the daily feed allowance at 0800 h and 40% at 1800 h. Feed offered and refused was recorded at each feeding. During the morning feedings, each cow was offered either control or 3-NOP-treated TMR at 150% of her average intake during the previous 3 d. After collection of the evening refusals, cows received only the basal TMR without the premix until the next morning feeding. The test period for the short-term DMI data collection was defined from morning feeding to afternoon refusals collection during each day of each experimental period. Location (left or right) of the control and 3-NOP diets within a feed bin was switched every day during each period to avoid feed location bias. Dry matter intake of TMR during the test period was quadratically increased by 3-NOP compared with the control. Inclusion of 3-NOP at 120 mg/kg of feed dry matter resulted in decreased 10-h DMI compared with the lower 3-NOP doses, but was similar to the control. There was no effect of feed location (left or right) within feed bin on DMI. Data from this short-term study suggests that 3-NOP does not have a negative effect on DMI in lactating dairy cows.

Short communication: Relationship of dry matter intake with enteric methane emission measured with the GreenFeed system in dairy cows receiving a diet without or with 3-nitrooxypropanol.

The relationship between DM intake (DMI) and enteric methane emission is well established in ruminant animals but may depend on measurement technique (e.g. spot v. continuous gas sampling) and rumen environment (e.g. use of fermentation modifiers). A previous meta-analysis has shown a poor overall (i.e. 24 h) relationship of DMI with enteric methane emission in lactating dairy cows when measured using the GreenFeed system (GF; Symposium review: uncertainties in enteric methane inventories, measurement techniques, and prediction models. Journal of Dairy Science 101, 6655 to 6674). Therefore, we examined this relationship in a 15-week experiment with lactating dairy cows receiving a control diet or a diet containing the investigational product 3-nitrooxypropanol (3-NOP), an enteric methane inhibitor, applied at 60 mg/kg feed DM. Daily methane emission, measured using GF, and DMI were clustered into 12 feed-intake timeslots of 2 h each. Methane emission and DMI were the lowest 2 h before feeding and the highest within 6 h after feed provision. The overall (24 h) relationship between methane emission and DMI was poor (R2 = 0.01). The relationship for the control (but not 3-NOP) cows was improved (R2 = 0.31; P < 0.001) when DMI was allocated to timeslots and was strongest (R2 = 0.51; P < 0.001) 8 to 10 h after feed provision. Analysis of the 3-NOP emission data showed marked differences in the mitigation effect over time. There was a lack of effect in the 2-h timeslot before feeding, the mitigation effect was highest (45%) immediately after feed provision, persisted at around 32% to 39% within 10 h after feed provision, and decreased to 13%, 4 h before feeding. These trends were clearly related to DMI (i.e. 3-NOP intake) by the cows. The current analysis showed that the relationship of enteric methane emission, as measured using GF, and DMI in dairy cows depends on the time of measurement relative to time of feeding. The implication of this finding is that a sufficient number of observations, covering the entire 24-h feeding cycle, have to be collected to have representative emission estimates using the GF system. This analysis also revealed that the methane mitigation effect of 3-NOP is highest immediately after feed provision and lowest before feeding.

Dose-response effect of 3-nitrooxypropanol on enteric methane emissions in dairy cows.

This experiment was designed to test the effect of inclusion rate of 3-nitrooxypropanol (3-NOP), a methane inhibitor, on enteric methane emissions in dairy cows. The study was conducted with 49 multiparous Holstein cows in a randomized complete block design in 2 phases; phase 1 was with 28 cows, and phase 2 with 21 cows. Cows were fed a basal total mixed ration ad libitum and were blocked based on days in milk, milk yield, and enteric methane emissions during a 14-d covariate period. Treatments were control (no 3-NOP) and 40, 60, 80, 100, 150, and 200 mg of 3-NOP/kg of feed dry matter. Following a 14-d adaptation period, enteric gaseous emissions (methane, carbon dioxide, and hydrogen) were measured using the GreenFeed system (C-Lock Inc., Rapid City, SD) over a 3-d period. Compared with the control, inclusion rate of 3-NOP quadratically decreased daily enteric methane emissions from 22 to 40%. Maximum mitigation effect was achieved with the 3 highest 3-NOP doses (with no statistical difference among 100, 150, and 200 mg/kg). The decrease in methane emission yield and emission intensity ranged from 16 to 36% and from 25 to 45%, respectively. Emissions of hydrogen quadratically increased 6- to 10-fold, compared with the control; the maximum increase was with 150 mg/kg 3-NOP. Treatment did not affect daily emissions of carbon dioxide, but a linear increase in carbon dioxide emission yield was observed with increasing 3-NOP doses. Dry matter intake and milk yield of the cows was not affected by 3-NOP. Milk fat concentration and yield were increased by 3-NOP due to increased concentration of de novo synthetized short-chain fatty acids in milk. Inclusion of 3-NOP also tended to increase milk urea nitrogen but had no other effects on milk components. In this short-term experiment, 3-NOP decreased enteric methane emissions without affecting dry matter intake or milk yield and increased milk fat in dairy cows. Maximum mitigation effect was achieved at 100 to 200 mg/kg of feed dry matter.