Effects of Feed Additives on Ruminal Methane Emissions, Fermentation Patterns, and Microbial Populations in Cattle

87 ISSN Online: 3007-1941 ISSN Print: 3007-1933 EFFECTS OF FEED ADDITIVES ON RUMINAL METHANE EMISSIONS, FERMENTATION PATTERNS, AND MICROBIAL POPULATIONS IN CATTLE Article Details A B S T R A C T Keywords: Methane Mitigation, Asparagopsis Taxiformis, Nitrate, Monensin, Rumen Microbiota, Fermentation, Cattle Ayesha Khan Department of Zoology, Wildlife and Fisheries, PMASArid Agriculture University, Rawalpindi, Punjab, Pakistan Muhammad Yousaf* Department of Animal Nutrition, The University of Agriculture, Peshawar, KPK, Pakistan Email: [email protected] Waseem Ahmed Department of Dairy and Livestock, Holstein Research Management (HRM), Pakistan Ammar Faiz Department of Meat Science and Technology, University of Veterinary and Animal Sciences, Lahore, Punjab, Pakistan Sadaf Ilyas Department of Zoology, University of Sialkot, Punjab, Pakistan Zaheer Ahmad Department of Livestock Production and Management, The University of Veterinary and Animal Sciences (UVAS), Swat, Khyber Pakhtunkhwa, Pakistan Atta Ur Rehman Department of Animal Nutrition, The University of Agriculture, Peshawar, KPK, Pakistan Muhammad Nasir Hayat Department of Animal Nutrition, University of Agriculture Faisalabad, Punjab, Pakistan Tauqeer Ahsan Livestock and Dairy Development Extension, Peshawar, Khyber Pakhtunkhwa, Pakistan Muhammad Abdul Wahab Department of Animal Nutrition, Cholistan University of Veterinary and Animal Sciences, Bahawalpur, Punjab, Pakistan This study assessed the impact of three feed additives such as monensin, nitrate, and Asparagopsis taxiformis on ruminal methane emissions, fermentation properties, and microbial population in cattle. Forty bull steers (Bos taurus; 350 ± 22 kg) were assigned in a randomized block design to four treatments: control, monensin (24 mg/kg DM), nitrate (1.5% NO₃⁻ of DM), and Asparagopsis (0.25% DM). The trial was conducted over 10 weeks, encompassing both an adaptation phase and a measurement period. Methane emissions were measured using open-circuit respiration chambers, and rumen fluid was examined for volatile fatty acids (VFA) and microbial gene abundances by qPCR. The results indicated substantial decreases (p < 0.001) in methane generation and yield for all the additions relative to the control group. Asparagopsis achieved the highest mitigation at 49%, followed by nitrate at 25% and monensin at 10%, all without adversely affecting the dry matter intake or average daily gain. The methane reduction was closely linked to diminished mcrA gene abundance and protozoal numbers, coupled with an elevated proportion of Prevotella spp. and decreased acetate-to-propionate ratios, signifying a metabolic transition towards propionate production. The findings indicate that Asparagopsis taxiformis is a highly effective natural feed additive for methane reduction, whereas nitrate and monensin provide moderate but complementary effects. This study provides quantitative and mechanistic data supporting the incorporation of feed-based interventions as sustainable methods for mitigating enteric methane emissions in ruminant production systems. Ayesha Khan1, Muhammad Yousaf2*, Waseem Ahmed3, Ammar Faiz4, Sadaf Ilyas5, Zaheer Ahmad6, Atta Ur Rehman7, Muhammad Nasir Hayat8, Tauqeer Ahsan9, Muhammad Abdul Wahab10 https://msra.online/index.php/Journal/about https://msra.online/index.php/Journal/about Volume 3, Issue 4 (2025) 88 https://msra.online/index.php/Journal/about Volume 3, Issue 4 (2025) INTRODUCTION: Methane (CH₄) produced by ruminant livestock is a significant source of anthropogenic greenhouse gases, responsible for as much as one-third of the agricultural methane emissions worldwide. These emissions not only contribute to climate change but also represent a significant loss of dietary energy that could be used to support animal productivity. According to Gerber et al. (2013), livestock are responsible for up to 14.5% of the world's greenhouse gas emissions, predominantly through enteric methane emissions. This issue has increasingly become an area of scientific research, focusing on sustainable strategies for mitigation in the livestock sector. Rumen harbors a diverse microbial community that ferments feed and is the leading site of methanogenesis. Morgavi et al. (2010) characterized the rumen as a highly integrated microbial community, in which methanogenic archaea convert hydrogen during carbohydrate fermentation into methane, rather than carbon dioxide. Accordingly, the mitigation of methane has focused on manipulating the microbial ecosystem and fermentation pathways to redirect hydrogen away from methanogenesis. In ruminants, methanogenesis is a microbial process carried out by hydrogenotrophic archaea via the methyl-coenzyme M reductase pathway (Thauer et al., 2008). The mcrA gene encodes an essential enzyme in the last step of methane production. Inhibition of methanogenic archaea and their syntrophic relationships is an effective way to mitigate methane production. Ungerfeld (2020) highlighted the potential to redirect metabolic H 2 towards alternative sinks, such as propionate formation or nitrate reduction, leading to a lower methane yield. Feed additives are emerging as promising candidates for achieving this redirection without detrimental impacts on animal health or productivity. The ionophore antibiotic monensin has a selective inhibitory effect on gram-positive hydrogen-producing bacteria and therefore reduces methane production (Hook et al., 2010). Nitrate competes as an electron acceptor for hydrogen, reducing reducing equivalents that would otherwise be used in methane formation, and is concomitantly converted into ammonia (Van Zijderveld et al., 2010). The interventions have had a moderate effect, generally decreasing methane by 10–25% depending on the inclusion rate and ad libitum diet. Recently, macroalgae (Asparagopsis taxiformis) have been characterized as strong natural feed additives with profound antimethanogenic activity. Kinley et al. (2016) showed that adding Asparagopsis to cow feed could decrease methane emissions by more than 80% due to bromoform and other halogenated compounds, which suppress methyl-coenzyme M reductase activity. Parallel microbial analyses by Pandey et al. (2021) also observed the ability of Asparagopsis and nitrate to reduce not only methanogen numbers but also shift the rumen bacterial community more broadly towards propionate-producing organisms such as Prevotella. However, there are few comparative data characterizing these additives in the same experimental system. Thus, this study was established to elucidate the effects of monensin, nitrate, and Asparagopsis taxiformis on ruminal methane production, fermentation parameters, and microbial populations in cattle, with an emphasis on providing mechanistic evidence for their relative efficacy and microbial interactions. Methodology Experimental Design A randomized controlled feeding trial was performed at University of Agriculture, Faisalabad, to investigate the effects of feed additives on ruminal methane production and the microbial community in cattle. Forty clinically healthy beef steers (Bos taurus) aged 12–14 months with an average body weight of 350 ± 22 kg were used in the current study. Animals were blocked based on baseline variation into four quartiles and then randomly allocated within each block to one of the four treatment groups, with 10 animals per treatment. The four treatments were a diet without an additive (control), a similar diet with monensin, a diet containing nitrate, and another diet containing Asparagopsis taxiformis. The entire experimental period was ten weeks, consisting of a two-week adaptation to the diet and eight weeks used for measurements. The animals were kept in ventilated battery cages and had libitum access to clean drinking water, with the experimental conditions remaining constant throughout the experiment. 89 https://msra.online/index.php/Journal/about Volume 3, Issue 4 (2025) Diets and Feed Additives All animals were fed a total mixed ration (TMR) formulated to meet the nutrient requirements of growing beef cattle. The basal diet consisted of 40% grass silage, 30% corn silage, 25% ground corn, and 5% soybean meal on a dry matter basis, and was enriched with a mineral and vitamin premix. The monensin group and the control group were fed the basal diet with or without 24 mg/kg of monensin in dry matter, respectively. The nitrate treatment was administered as calcium nitrate via dietary inclusion to achieve 1.5% nitrate in the diet dry matter. In comparison, the Asparagopsis group received the basal diet supplemented with dried Asparagopsis taxiformis at a 0.25% inclusion level on a dry matter basis. Feed was provided daily at 08:00 h, and refusals were weighed based on the amount refused before subsequent feed was dispensing to measure the daily dry matter intake. Feed Intake and Growth Performance For all animals, feed intake was recorded, and the amount of feed offered versus refused was monitored daily. DMI was expressed in kilograms per day. Body weight was measured on days 0, 14, 42, and 70 after overnight fasting. Bodyweight ADG was calculated based on the slope of bodyweight over time using linear regression during the measurement period. Feed efficiency was calculated as weight gain divided by feed intake. Methane Measurement Enteric Methane was determined using open-circuit respiration chambers. Individual steers were housed in a chamber for over 6 hours, twice per week, from week 3 to week 10 of the trial. Gas concentrations were continuously written to data files and background-corrected during post-processing. The methane emission rate (grams per day) was determined as the product of the methane concentration over the airflow and the duration of the measurements. The methane yield (g/kg of DMI) was calculated as the total daily methane production divided by the daily feed intake. Rumen Sampling and Fermentation Parameters Rumen fluid was collected using an ororuminal tube for three h after feeding every 4th, seventh, and 10th weeks. The collected liquid was filtered through four layers of cheesecloth, and the pH was determined immediately using a standard calibration pH meter. Another subsample was collected and preserved at −20°C for volatile fatty acid (VFA) analysis, which was performed by gas chromatography postdeproteinization with 25% metaphosphoric acid. The concentration of VFA was presented as millimoles per liter, and the ratio of A:P was determined to describe the ruminal fermentation profile. Microbial DNA Extraction and Quantitative PCR A bead-beating and silica-column procedure was used for genomic DNA extraction from 1 ml of rumen fluid. Quantitative PCR (qPCR) with SYBR Green detection chemistry was conducted to estimate the abundance of total bacteria (16S rRNA), methanogens (mcrA gene), Ruminococcus albus (cellulolytic bacterium), and Prevotella spp. (amylolytic and proteolytic bacteria) and total rumen protozoa (18S rRNA). The amplification efficiency was 90%-105% and standard curves were prepared using serially diluted plasmid DNA carrying the fragments encoding the target genes. Microbial populations are presented as log₁₀ gene copies/mL of rumen fluid. Outcome Variables The primary outcome variable was methane yield, expressed as grams of CH₄ per kilogram of dry matter intake. Secondary variables included total methane production (g/day), dry matter intake (kg/day), average 90 https://msra.online/index.php/Journal/about Volume 3, Issue 4 (2025) daily gain (kg/day), and the relative abundance of microbial groups in log₁₀ copies per milliliter. Ruminal fermentation parameters such as total VFA concentration and the acetate-to-propionate ratio were also considered secondary outcomes. Statistical Analysis Statistical analyses were conducted using IBM SPSS Statistics (version 27). Normality and outliers were tested using descriptive statistics (boxplots and the Shapiro–Wilk test). The variance homogeneity of the treated groups was tested using Levene's test. For each response variable, the effects of treatment were analyzed using a general linear model (GLM), with the treatments as a fixed factor and block as a covariate to adjust for body weight differences. For post hoc multiple comparisons, each feed additive group was compared with the control group using Dunnett's test. Data are presented as mean ± standard error of the mean (SEM). The correlations between methane yield and microbial quantity or fermentation factors were analyzed using Pearson's correlation, and the significant parameters were subsequently used for multiple linear regression models. Differences were considered statistically significant at p < 0.05, and trends were discussed at p < 0.10. Ethical Considerations All animal procedures were performed in accordance with institutional and national regulations on animal use. The Institutional Animal Ethics Committee approved experimental design. Results Feed Intake and Growth Performance Dry matter intake (DMI) and growth performance data are summarized in Table 1. Feed additive supplementation caused slight but significant variations in feed intake among the treatments. The mean DMI decreased gradually from 10.5 ± 0.5 kg/day in the control group to 9.9 ± 0.5 kg/day in cattle receiving Asparagopsis taxiformis (p = 0.032). Average daily gain (ADG) ranged between 1.19 and 1.27 kg/day and did not differ significantly among treatments (p > 0.05). Feed efficiency improved slightly in the monensin and Asparagopsis groups, indicating enhanced nutrient utilization efficiency without compromising growth performance. These observations are illustrated in Figure 1, which depicts a modest decline in DMI across treatments with no adverse impact on growth rate. The visual trend demonstrates that methane-mitigating feed additives, particularly Asparagopsis, did not negatively affect cattle performance. Table 1: Effect of Feed Additives on Feed Intake and Growth Performance in Cattle Treatment DMI (kg/day) ADG (kg/day) Feed Efficiency (ADG/DMI) Control 10.50 ± 0.50 1.25 ± 0.07 0.119 Monensin 10.30 ± 0.50 1.27 ± 0.07 0.123 Nitrate 10.10 ± 0.50 1.19 ± 0.07 0.118 Asparagopsis 9.90 ± 0.50 1.22 ± 0.07 0.123 p-value 0.032 0.218 0.064 91 https://msra.online/index.php/Journal/about Volume 3, Issue 4 (2025) Figure 1: Effect of feed additives on dry matter intake (DMI) and average daily gain (ADG) in cattle. Each bar represents mean ± standard deviation (n = 10). Feed additives did not adversely affect growth performance despite slight differences in feed intake. Methane Emissions Feed additives exerted a marked effect on enteric methane production and yield (Table 2). Mean methane output declined significantly (p < 0.001) from 230 ± 15 g/day in the control group to 118 ± 15 g/day in the Asparagopsis group, representing nearly a 49% reduction. Monensin and nitrate supplementation also decreased emissions by 10% and 25%, respectively, relative to the control. Methane yield (g/kg DMI) exhibited a similar pattern, with Asparagopsis-fed cattle showing the lowest values (12.0 ± 1.6 g/kg DMI) compared with 22.0 ± 1.7 g/kg DMI in controls. These trends are depicted in Figure 2, showing a consistent reduction in both absolute methane production and methane yield across treatments. The visual gradient emphasizes the superior methane-suppressing efficacy of Asparagopsis taxiformis, followed by nitrate and monensin. Table 2: Effect ff Feed Additives on Methane Production and Yield in Cattle Treatment CH₄ (g/day) CH₄ Yield (g/kg DMI) Control 230 ± 15 22.0 ± 1.7 Monensin 207 ± 15 20.1 ± 1.6 Nitrate 173 ± 15 17.3 ± 1.6 Asparagopsis 118 ± 15 12.0 ± 1.6 p-value <0.001 <0.001 92 https://msra.online/index.php/Journal/about Volume 3, Issue 4 (2025) Figure 2: Effect of feed additives on methane production and methane yield (g/kg DMI). Bars represent mean ± standard deviation (n = 10). All additives significantly reduced methane emissions compared to the control, with the greatest reduction observed in the Asparagopsis group. Rumen Fermentation Characteristics Feed additive supplementation did not significantly alter total VFA concentrations but modified the pattern of ruminal fermentation (Table 3). Total VFA concentration ranged between 115 ± 6 and 118 ± 6 mmol/L across treatments (p = 0.186). However, the acetate-to-propionate (A:P) ratio was significantly reduced (p = 0.007) in the Asparagopsis and monensin groups, indicating a shift toward greater propionate production and improved hydrogen utilization efficiency. As shown in Figure 3, the A:P ratio declined progressively from 3.10 in the control group to 2.50 in the Asparagopsis group. This fermentation shift aligns with the observed reductions in methane output, suggesting that feed additives redirected ruminal fermentation pathways away from methanogenesis. Table 3: Ruminal Fermentation Parameters as Affected by Feed Additives Treatment Total VFA (mmol/L) A:P Ratio Control 115 ± 6 3.10 ± 0.20 Monensin 118 ± 6 2.70 ± 0.20 Nitrate 116 ± 6 2.85 ± 0.20 Asparagopsis 117 ± 6 2.50 ± 0.20 p-value 0.186 0.007 93 https://msra.online/index.php/Journal/about Volume 3, Issue 4 (2025) Figure 3: Changes in ruminal fermentation (acetate-to-propionate ratio) in response to feed additive supplementation. Bars show mean ± SD (n = 10). Both Asparagopsis and monensin significantly lowered the A:P ratio compared to the control, indicating a shift toward propionate production. Ruminal Microbial Populations Feed additives significantly influenced the abundance of rumen microbial populations involved in methanogenesis and carbohydrate fermentation (Table 4). The mcrA gene copy number, representing methanogenic archaea, was highest in the control group (8.20 ± 0.20 log₁₀ copies/mL) and lowest in the Asparagopsis group (7.10 ± 0.20 log₁₀ copies/mL), showing a strong treatment effect (p < 0.001). Similar reductions were noted in protozoal abundance, with both nitrate and Asparagopsis treatments reducing counts relative to the control (p = 0.018). Ruminococcus albus populations declined modestly under nitrate and Asparagopsis supplementation, while Prevotella spp. increased slightly, indicating a microbial shift toward propionate-producing bacteria. These dynamics are illustrated in Figure 4, which highlights the marked suppression of methanogenic archaea (mcrA) across treatments, particularly with Asparagopsis. Table 4: Effect of Feed Additives on Ruminal Microbial Populations (log₁₀ copies/mL) Treatment mcrA Total Bacteria R. albus Prevotella spp. Protozoa Control 8.20 ± 0.20 9.20 ± 0.18 7.60 ± 0.18 8.00 ± 0.16 7.10 ± 0.15 Monensin 8.00 ± 0.20 9.25 ± 0.18 7.45 ± 0.18 8.10 ± 0.16 6.95 ± 0.15 Nitrate 7.70 ± 0.20 9.15 ± 0.18 7.40 ± 0.18 8.05 ± 0.16 6.90 ± 0.15 Asparagopsis 7.10 ± 0.20 9.10 ± 0.18 7.20 ± 0.18 8.15 ± 0.16 6.70 ± 0.15 p-value <0.001 0.248 0.041 0.032 0.018 94 https://msra.online/index.php/Journal/about Volume 3, Issue 4 (2025) Figure 4: Effect of feed additives on methanogenic archaea (mcrA) abundance (log₁₀ copies/mL). Bars show mean ± SD (n = 10). A significant reduction in methanogen abundance was observed in all additive groups, with the lowest counts in Asparagopsis-fed cattle. Relationship Between Methane Yield and Microbial Markers Pearson correlation analysis revealed strong positive relationships between methane yield and mcrA gene abundance (r = 0.88, p < 0.001) as well as the acetate-to-propionate ratio (r = 0.71, p < 0.01). Conversely, methane yield was negatively correlated with Prevotella spp. abundance (r = −0.52, p < 0.05). Multiple regression analysis identified mcrA abundance and A:P ratio as the primary predictors of methane yield (R² = 0.83, p < 0.001). These associations are graphically presented in Figure 5, which demonstrates a clear linear relationship between mcrA abundance and methane yield across treatments. The gradient indicates that a decline in methanogen population density corresponded strongly with methane mitigation efficiency. 95 https://msra.online/index.php/Journal/about Volume 3, Issue 4 (2025) Figure 5: Correlation between methanogen abundance (mcrA) and methane yield in cattle fed different feed additives. Each point represents treatment mean (n = 10). The strong positive correlation indicates that methane reduction is closely associated with decreased methanogenic activity. Summary of Findings Overall, the results demonstrate that supplementation with Asparagopsis taxiformis produced the most pronounced methane mitigation effect without compromising animal performance or ruminal fermentation stability. Both monensin and nitrate also significantly reduced methane emissions but to a lesser extent. The reduction in methane emissions was mechanistically linked to lower methanogen abundance and a shift in fermentation pathways favoring propionate production. The graphical evidence (Figures 1–5) collectively supports the conclusion that strategic use of specific feed additives can substantially mitigate methane emissions in ruminant systems while maintaining productive efficiency. Discussion This study showed that adding feed additives, such as monensin, nitrate, and Asparagopsis taxiformis, significantly affected methane emissions and rumen microbial populations in the present experiment without negatively impacting growth. The extent to which methane production is inhibited in cattle supplemented with Asparagopsis is the most conspicuous, showing a nearly 50% decrease compared to control animals. These results are consistent with those of Kinley et al. (2016), who observed a more than 80% reduction in methane emissions from Aparagopsis-fed cattle and verified its vigorous antimethanogenic activity. Monensin and nitrate also displayed modest methane inhibition, which was equivalent to the reductions previously pooled in meta-analyses by Palangi and Lackner (2022), which is a normal decrease between 7