Whole Cottonseed as an Effective Strategy to Mitigate Enteric Methane Emissions in Cattle Fed Low-Quality Forages

1. Introduction

Human-induced activities are responsible for approximately 60% of global methane (CH₄) emissions, with enteric CH₄ production and manure management contributing about 32% of global anthropogenic emissions ( [1]. Combined with non-anthropogenic sources, these activities account for 19.2% of total global CH₄ emissions [2]. Methane is a potent greenhouse gas (GHG) with a global warming potential (GWP) 21 to 26 times greater than carbon dioxide (CO₂), making it a significant contributor to climate change. Furthermore, CH₄ production in the rumen represents an energy loss for ruminants, compounding its environmental and economic implications [3, 4].

Multiple studies (e.g., Wilkerson et al., [5] have established a positive correlation between CH₄ production and the intake of digestible cellulose, hemicellulose, and non-fiber carbohydrates, while fat intake shows a negative correlation. Lipid supplementation has emerged as a promising strategy for mitigating enteric CH₄ emissions across various ruminant species [6, 7, 8]. This reduction is achieved through three primary mechanisms. First, lipids serve as an alternative hydrogen sink, as unsaturated fatty acids undergo biohydrogenation in the rumen [9]. Second, supplemental lipids often reduce dry matter intake (DMI), indirectly decreasing CH₄ emissions [10, 11, 12]. Lastly, specific lipids, particularly medium-chain fatty acids, can alter the rumen microbiome, suppressing protozoa and archaea populations [13, 14].

Industrial by-products such as whole cottonseed (WCS), corn dried distillers grains with solubles (DDGS), and soybean meal are rich in fat, especially polyunsaturated fatty acids (PUFAs). For example, WCS contains 77.68% PUFA [15], DDGS contains 82% PUFA [16], and soybean meal contains 82.3% PUFA [17]. These by-products are widely used as supplements to enhance the average daily gain (ADG) of beef cattle grazing on low-quality forages [18], primarily due to their high concentrations of rumen-degradable protein (RDP) and metabolizable energy (ME) [19, 20, 21]. The fat content in these feedstuffs also positions them as potential mitigators of enteric CH₄ emissions. However, their effects on CH₄ emissions in forage-based diets, particularly under tropical and subtropical conditions, remain poorly understood [6, 9].

In Argentina, the provinces of Santiago del Estero and Chaco account for 79% of the country’s cotton production. Whole cottonseed, a by-product of the textile industry, is widely used in ruminant diets for beef cattle [22], dairy cattle [23], and sheep [24] due to its high protein content and metabolizable energy, primarily derived from lipids. Given its composition, WCS has the potential to reduce enteric CH₄ emissions and improve animal performance, particularly in diets based on low-quality forages.

We hypothesize that supplementing beef cattle diets with WCS will reduce enteric CH₄ production while enhancing animal performance in cattle consuming low-quality forages. Therefore, the objective of this study was to quantitatively evaluate the effects of WCS supplementation on intake, digestion, animal performance, and enteric CH₄ emissions in Braford crossbred heifers fed low-quality forage diets.

2. Materials and Methods

The experiment was conducted over 85 days (June 30, 2022, to September 23, 2022) during the dry, cold season at the INTA Santiago del Estero Research Station (28° 01´ 32´´ S, 64° 13´ 58´´ W; 170 m elevation). All experimental procedures were approved by the INTA Tucumán-Santiago Institutional Animal Care and Use Committee (Approval No. 03/22).

2.1. Animals, experimental design and diets

Twenty-four crossbred Braford beef heifers (318.21 ± 31.18 kg BW) were randomly assigned to four pens (three heifers per pen) for each treatment across two measurement periods. Each pen measured 240 m² (12 × 20 m). The experimental design was completely randomized, with each pen considered an experimental unit.

Two dietary treatments were evaluated: 0WCS: Guinea grass hay (GGH; Megathyrsus maximus cv. Gatton panic) with no supplementation (forage-to-concentrate ratio: 100:0); and 0.5WCS: Guinea grass hay supplemented with whole cottonseed (WCS) at 0.5% of BW (as-fed basis), resulting in a forage-to-concentrate ratio of 74:26. Animals were fed once daily at 7:00 AM. Guinea grass hay was offered in a 6 m-long canvas feed bunk, while WCS was provided in individual 1.5 m plastic feed bunks. Water was available ad libitum.

Feed intake was determined by weighing feed refusals separately for hay and supplement. The WCS used was untreated, consisting of small seeds surrounded by lint. The chemical composition of GGH and WCS is presented in Table 1.

The experiment was divided into 2 measurement periods, Period 1 (18 to 40 d) and Period 2 (59 to 81 d) both consisted of 14 d for treatment adaptation, 5 d for feed intake measurement, 4 d for enteric CH₄ emission monitoring, and 3 d for digestion evaluation.

2.2. Feed Intake and Digestibility

Feed intake was calculated as the difference between feed offered (kg DM) and feed refusals (kg DM). Nutrient intake was corrected for nutrient concentration in both offered feeds and refusals.

Total tract digestibility was estimated using acid detergent insoluble ash as an internal marker, following Cochran and Galyean's method [25]. Fecal grab samples were collected every 6 hours during the final 3 days of each evaluation period (days 38 to 40 for Period 1 and days 79 to 81 for Period 2). Sampling times were rotated by 3 hours daily to cover a 24-hour cycle and minimize diurnal variation in marker excretion.

2.3. Enteric methane emission

The enteric CH₄ emission (g CH₄/day) was measured using the sulfur hexafluoride (SF₆) tracer technique, as proposed by Johnson et al. [26] and adapted for extended periods [27, 28]. Samples were collected over 4 days, specifically on days 33 to 37 for Period 1 and days 74 to 78 for Period 2.

Permeation tubes containing 1.83 ± 0.17 g of SF₆ were dosed orally using a custom dispenser. Tubes were pre-weighed weekly for 8 weeks while stored at 39°C to estimate permeation rates (average: 11.18 ± 2.14 mg/d).

The sample collection system included two 0.5 L steel containers, while the flow regulator consisted of a 10 cm metal capillary, with a 5 mm segment compressed to achieve a target flow rate of 0.05 mL/min (the restrictor was calibrated to maintain a pressure between 0.4 - 0.6 bars at the end of the sampling period). Air samples were collected continuously over 4 days using 0.5 L stainless steel cylinders placed near the nostrils of each animal (Figure 1). Before sampling, the cylinders were cleaned with nitrogen gas (99.9% purity) and evacuated to a pressure of -0.99 bar relative to atmospheric pressure. The restrictor and the sampling line were housed in 5 cm polyethylene tubing (12 mm inner diameter), with a polyester fabric cover to prevent blockage from water or dust. Background air samples were collected near the pens for CH₄ and SF₆ concentration corrections.

The CH₄ and SF₆ concentrations were analyzed via gas chromatography (Perkin Elmer 600, USA) at the Pathobiology Veterinary Institute (CICVyA, INTA, Argentina) according to the methodology described by Gere et al. [29]. Methane production was calculated using the following equation:

$${CH}_4\left(g/d\right)=\mathrm{P}\mathrm{R}{SF}_6\left(\mathrm{g}/\mathrm{d}\right)\times \left({\displaystyle \frac{\left[{CH}_4-{CH}_{4B}\right]}{\left[{SF}_6-{SF}_{6B}\right]}}\right)\times {\displaystyle \frac{\mathrm{M}\mathrm{W}\mathrm{C}\mathrm{H}₄}{\mathrm{M}\mathrm{W}\mathrm{S}\mathrm{F}₆}}$$

Where:

CH₄: CH₄ emission rate (g/d); CH₄, SF₆: gas concentrations from exhaled air; CH_4B, SF_6B: background gas concentrations; PRSF₆: SF₆ permeation rate (g/d); MWCH₄, MWSF₆: Molecular weights of CH₄ and SF₆.

2.4. Statistical analyses

The experimental design was completely randomized. Data were analyzed using Software INFOSTAT 2020 [30] with an interface with R through mixed linear models. Each pen was considered an experimental unit. WCS supplementation levels were considered fixed effects in each period; the pen was a random effect. Multiple comparisons between means were performed using the LSD Fisher test (p < 0.05). The following model was fitted to the data set for all variables:

Yij = µ + T_i + P_j + A_k + (TxP)_ij + ε_ijk

Where Y_ij is the response to treatment, µ is the overall mean, Ti is the fixed effect of treatment i, Pj is the fixed effect of period, Ak is the random effect of Pen k, (TxP)ij is the interaction between treatment and period, and ɛ_ijk is the experimental error.

3. Results and discussion

3.1. Feed Intake

As shown in Table 2, dry matter intake (DMI) decreased by 19.5% in Period 1 and 12.65% in Period 2 with WCS supplementation (p < 0.01). This reduction indicates a substitution effect, with a marked depression in DMI. A similar decline was observed in forage dry matter intake (FDMI), which decreased by 39.96% and 30.34% in Periods 1 and 2, respectively (p < 0.01). Additionally, total organic matter intake (TOMI), neutral detergent fiber intake (NDFI), and acid detergent fiber intake (ADFI) were also lower in supplemented animals (p < 0.01).

In contrast, crude protein intake (CPI) and ether extract intake (EEI) increased significantly (p < 0.01) with supplementation, as expected due to the higher protein and fat content of WCS. While gross energy intake (GEI) showed no difference in Period 1, it was significantly higher in supplemented animals during Period 2 (p < 0.02). The period effect (p < 0.05) could be explained by variations in animal weight across periods. The treatment–period interaction did not reach statistical significance (p > 0.10).

Previous research suggests that WCS supplementation should be around 0.5% of body weight (BW), corresponding to approximately 2.3 to 3.2 kg WCS per cow per day [31, 32]. In this study, WCS supplementation reduced total dry matter intake (TDMI), forage dry matter intake (FDMI), and total organic matter intake (TOMI), consistent with the substitution effect caused by the high lipid content of WCS (18.71% ether extract). This observation aligns with the well-established finding that lipid supplementation generally reduces DMI in various types of diets [10, 11, 12].

Studies by Hill et al. [32] in beef cows fed bermudagrass hay and Chuntrakort et al. [32] in Zebu cattle fed rice straw-based diets reported reductions in DMI only at higher levels of WCS supplementation (1% BW) than those used in this study. Conversely, Beck et al. [8] observed a decrease in FDMI but an increase in TDMI with WCS supplementation at 0.5% BW, reflecting a balance between substitution and addition effects. Other studies [14, 34, 35] found no differences in DMI or forage intake, likely due to variations in experimental conditions.

Despite the reduced intake of dry matter and forage, CPI and EEI were higher in the supplemented group, reflecting the increased availability of protein and fat in WCS. This finding aligns with Beck et al. [8], who reported similar increases in protein and energy intake with WCS supplementation.

3.2. Digestion

As shown in Table 3, WCS supplementation improved crude protein (CP) digestibility (CPD) and ether extract digestibility (EED) (p < 0.01). However, no significant differences were observed in dry matter digestibility (DMD), neutral detergent fiber digestibility (NDFD), or acid detergent fiber digestibility (ADFD).

The lack of effect on DMD is consistent with previous studies on dairy cows fed total mixed rations (TMR) [36] and beef cattle on high-forage diets [32]. In contrast, Chuntrakort et al. [33] observed a reduction in DMD with higher WCS supplementation levels (1% BW). Similarly, Hill et al. [32]reported negative effects on digestibility when WCS was offered free choice.

The increased CP digestibility in the 0.5WCS group aligns with some studies, although others found no significant differences between the control and supplemented groups [33, 36, 37]. The unaffected NDFD is consistent with findings by Hill et al. [32], Nogueira [36], and Beck et al. [8], although Chuntrakort et al. [33]) reported reductions with WCS supplementation. Similarly, ADFD showed no differences between treatments, corroborating earlier reports [33, 36, 38], although Beck et al. [8] observed a decline at 0.5% BW supplementation.

The doubling of EED in the 0.5WCS group, compared to controls, is consistent with Chuntrakort et al. [33]. Nogueira et al. [36] also reported a 17% increase in EED with WCS supplementation in dairy cows fed sugarcane bagasse.

3.3. Animal Performance

WCS supplementation significantly improved ADG (p < 0.01) with supplemented animals gaining 340 g/day more than controls (Table 3). This resulted in a final body weight up to 44 kg higher in the supplemented group. Control animals lost 100 g/day, while 0.5WCS animals gained 240 g/day.

The weight loss in controls contrasts with findings from other studies [8, 32, 35], where non-supplemented groups showed weight gain, albeit lower than supplemented groups. This discrepancy likely reflects differences in baseline diet quality; in this study, the control diet was likely nutritionally inferior, highlighting the importance of diet composition in performance outcomes.

The observed increase in ADG underscores the dual benefits of WCS as a source of energy and metabolizable protein. These results emphasize the need for optimized supplementation strategies, particularly in low-quality forage-based systems, to improve animal performance.

3.4. Enteric Methane Emission

As shown in Table 4, enteric CH₄ emissions (g/d) decreased by approximately 30% in both periods with WCS supplementation. Methane yield (g CH₄/kg DMI) showed a tendency to decrease (p < 0.01). Methane intensity (g CH₄/kg BW) significantly declined by 33% and 36% in Periods 1 and 2, respectively, while Ym (%) decreased by 22% and 30% (p < 0.05). The period effect observed in enteric CH₄ emissions (g/d) and CH₄ intensity (g CH₄/kg BW and g/kg BW⁰·⁷⁵) (p < 0.05) could be attributed to variations in animal weight across periods. The treatment–period interaction did not show statistical significance (p > 0.10).

These reductions are consistent with findings by Beck et al. [8] and Chuntrakort et al. [33], who reported similar long-term effects. Beck et al. [14] observed the lowest CH₄ emissions with 0.6% BW WCS supplementation. Other studies [6, 34, 39] reported reductions in CH₄ emissions with WCS inclusion, particularly when it replaced a substantial portion of the diet.

Mechanisms for reduced CH₄ emissions include disruption of methanogens, enhanced biohydrogenation of unsaturated fatty acids, and a shift in rumen fermentation towards propionate production [40, 41, 42]. Additionally, high-fat diets reduce substrate availability for methanogenesis, as noted by Muñoz et al. [23] and Knapp et al. [43].

Studies conducted in Argentina on pasture-fed beef cattle have reported variable Ym values, ranging from 4.0% to 8.2% [44] and 4.3% to 6.2% [45]. The CH₄ yield observed in this study, particularly in the control treatment, exceeded the average values reported in these previous works as well as the IPCC default value of 6.5%. This underscores the urgent need for targeted mitigation strategies in cattle systems relying on low-quality forages, such as Guinea grass.

These findings demonstrate the potential of WCS as an effective CH₄ mitigation strategy in ruminant diets. Future research should also investigate the effects of WCS supplementation on animal performance, rumen fermentation, microbiology, and the scalability of this approach in commercial livestock systems.

4. Conclusions

This study highlights the substantial potential of whole cottonseed (WCS) supplementation as an effective strategy for mitigating enteric CH₄ emissions in cattle consuming low-quality tropical forages. WCS supplementation not only reduced CH₄ emissions but also enhanced animal performance and forage utilization efficiency.

Supplementing WCS at 0.5% of body weight proved to be a sustainable and effective intervention, reducing enteric CH₄ emissions while simultaneously enhancing animal growth and feed efficiency. These findings contribute to the development of more sustainable livestock production systems by addressing both environmental and productivity challenges. Future research should explore the scalability of WCS supplementation across diverse production environments and evaluate its long-term effects on animal health, rumen microbiota, and overall system sustainability.