Effect of Tra catfish oil and starch sources on nutrient digestibility and methane emission of Charolais crossbred cattle at 17 - 20 months of age

Tran Trung Tuan^1,2 and Nguyen Binh Truong^1,2

¹ An Giang University, An Giang, Vietnam. No 18, Ung Van Khiem street, Long Xuyen ward, An Giang province
tttuan@agu.edu.vn
² Vietnam National University Ho Chi Minh City, Vietnam

Abstract

This study evaluated the effects of dietary Tra catfish oil (TrO) supplementation and different starch sources on nutrient digestibility and methane emissions in Charolais crossbred cattle. A 2 × 2 factorial arrangement in a 4 × 4 Latin square design was used, with four diets comprising two starch sources (maize or cassava chips) and two lipid levels (with or without 3% TrO). Feed intake, nutrient digestibility, methane emission and body weight change were measured in four bulls (average BW=290±41.2 kg). Results indicated that neither starch source nor TrO supplementation significantly affected dry matter intake, organic matter, crude protein, or fiber intake (p>0.05). Apparent nutrient digestibility of DM, OM, CP and NDF showed no significant differences among treatments; however, a significant interaction effect was observed for ADF digestibility (p=0.009), with the highest value in the cassava + TrO group. Methane emissions were significantly reduced by TrO supplementation (P=0.001), regardless of starch type, with reductions of approximately 5–6% in energy loss. There were no significant changes in body weight gain among treatments (p>0.13). These findings suggest that Tra catfish oil can be a promising dietary lipid source to mitigate methane emissions without compromising feed intake or nutrient utilization, especially when combined with rapidly fermentable starch like cassava.

Key words: digestibility, lipid supplementation, methane emission, ruminal fermentation, rumen microbiota, starch fermentability

Introduction

Livestock, particularly ruminants, are essential to global agriculture and food security, converting fibrous plant materials into nutrient rich protein. However, this biologically efficient process is also associated with the release of enteric methane (CH₄), a potent greenhouse gas that contributes to global climate change and represents a significant energy loss between 2% and 12% of the animal's gross energy intake (Johnson and Johnson 1995; Vargas et al 2022). Enteric methane originates from ruminal fermentation, primarily through the activity of methanogenic archaea, whose metabolism is tightly linked to the broader rumen microbial ecosystem (Huws et al 2018). Dietary inputs shape the structure and function of microbial communities, thereby regulating fermentation pathways, volatile fatty acid profiles, nitrogen excretion patterns, and methanogenesis (Matthews et al 2018). Good rumen efficiency begins with supporting fibrolytic microbes through quality forage and proper processing, while providing starch at optimal levels to enhance energy use without hindering fiber digestion or microbial protein synthesis (Firkins 2021).

Mitigating methane emissions while maintaining productivity has become a key priority in livestock nutrition. Among the nutritional interventions, lipid supplementation to suppress methanogens, reduce hydrogen availability for methane formation, and shift volatile fatty acid (VFA) production toward propionate, which is more energetically efficient (Martin et al 2021). Lipid source such as fish and soybean oil, and savory plant additives influence rumen fermentation, nutrient use, and fatty acid profiles, especially by enhancing unsaturated fat metabolism, supporting microbial protein synthesis, and reducing methane emission (Golbotteh et al 2024). The type of fat and its interaction with carbohydrate fermentability are critical to maximizing benefits while avoiding negative effects on feed intake or fiber digestibility.

The Mekong Delta region of Vietnam is the world’s leading producer of Tra catfish, accounting for over 1.5 million tons annually. The region's integrated aquaculture system produces a large volume of Tra catfish oil as a secondary product (Tan et al 2023). It contains about 42-48% saturated fatty acids (SFA), about 33 - 40% monounsaturated fatty acids (MUFA), and about 12-18% polyunsaturated fatty acids (PUFA), including omega-3 fatty acids like EPA and DHA (Binh et al 2009). These properties suggest it could provide methane suppressing effects similar to marine fish oils, but with lower cost and higher availability. Despite this potential, its use in cattle feeding has been little studied.

The fermentability of starch sources in ruminant diets also influences methane emissions and nutrient utilization. Cassava starch, which is rapidly fermented in the rumen, may interact more effectively with dietary lipids than maize starch, which ferments more slowly. According to Andersen et al (2023), diet induced shifts in protozoa and fiber digesting bacteria play a role in reducing methanogenesis, particularly when starch and lipid sources are strategically combined.

Martin et al (2021) demonstrated that supplementing ruminant diets with 5% corn oil and wheat starch reduced methane emissions by over 25% and enhanced propionate production and nutrient digestibility. These findings support the idea that targeted combinations of lipids and fermentable carbohydrates can optimize ruminal fermentation while reducing environmental impact.

The present study aims to evaluate the effects of Tra catfish oil supplementation (3% of dry matter) in combination with two different starch sources (cassava and maize) on nutrient digestibility and methane emission in Charolais crossbred cattle. This research contributes to the development of environmentally sustainable and economically viable feeding strategies in tropical beef production systems, while providing new insights into the interaction between regional lipid sources and dietary starch fermentability.

Materials and methods

Location

The experiment was carried out at the Hanh Cuong cattle farm, Chau Thanh district of An Giang province, Vietnam. Samples were analysed at laboratory E205 (Ruminal animal production techniques – 4) of the Faculty of Animal Sciences, Agriculture University of Can Tho University, the chemical makeup of the experimental diets was examined.

Experimental design

Four Charolais crossbred bulls with an average body weight of 290±41.2 kg were randomly assigned to an experiment, arranged in a 2 × 2 factorial design in a 4 × 4 Latin square design, with four treatments and four replications. Factor one was the carbohydrate source, including maize (Ma) and cassava chips (Ca), while Factor two related to Tra catfish oil (TrO), with or withouth TrO (proposed by Thu and Dong 2021). The nutritional composition of TrO (%DM) analyzed was 98.6% DM, 99.2% OM and 99.8% EE.

Each diet was formulated to provide comparable energy and protein levels, with all ingredients expressed as a percentage of dry matter (DM). The main energy source in the diets was either maize or cassava chip at 15% DM, depending on the treatment. Both starch sources combinated with broken rice (15%) as a complementary carbohydrate source. Soybean meal (5%) was included in all diets to ensure adequate crude protein content. Tra catfish oil was added at a level of 3% DM in the TrO-supplemented diets (MaTrO and CaTrO), while it was excluded in the NoTrO diets. The supplementation of Tra catfish oil aimed to evaluate its effect as a lipid source, particularly on methane emissions and digestibility (Table 1).

Measurements taken

Feed and feeding

Before feeding, all the feed was weighed and supplied to the experimental cattle. All supplements were fed twice at 7:00 am and 1:00 pm. In detail, at first, Tra catfish oil was the drink of beef cattle. Then, the maize and cassava chips were mixed with broken rice, soybean meal, urea, and premix supplements before feeding. The Napier grass was fed at 8:00 am. Rice straw was supplied ad libitum at 2:00 pm. Freshwater was provided ad libitum. Refused feeds were weighed each morning. The daily feed intake and nutrient consumption were determined from feed and refusals.

Chemical analysis: Feeds, refusals, feces and urine were analyzed for chemical composition.

The method of AOAC (1990) was used to analyze of dry matter (DM) and organic matter (OM). The nitrogen (N) in feed, refusals, feces, and urine was specified using the Kjeldahl procedures of AOAC (1990). However, acid detergent fiber (ADF) and neutral detergent fiber (NDF) were analyzed using the method of Van Soest et al (1991).

Energy intakes and methane emissions

The metabolizable energy (ME) in diets was calculated by the suggestion of Bruinenberg et al (2002).

ME (MJ/head/day) = 14.2 x DOM + 5.9 x DCP (with DOM/DCP < 7)

ME (MJ/head/day) = 15.1 x DOM (with DOM/DCP > 7)

Where: DOM was the digestible organic matter, and DCP was the digestible crude protein.

Methane emissions were suggested by Mills et al (2003)

Methane (MJ/day) = 1.06 + 10.27 Dietary forage proportion + 0.87 DMI.

Apparent nutrient digestibility:Each experimental period lasted three weeks: the first week for adaptation, the second for diet stabilization, and the third for feces collection (McDonald et al 2022).

Daily weight gains (DWG):The Charolais crossbred cattle were weighed in the morning (two consecutive days) before feeding, at the beginning and end of each experimental period by using the electrical scale (Tru-Test, Limited Auckland, New Zealand).

Data analysis

The data were analyzed according to a 2x2 factorial arrangement in a 4x4 Latin square design using the ANOVA Linear Model (GLM) of Minitab Reference Manual Release 20 (Minitab, 2021). The statistical model included terms for cattle, period, energy source, Tra fish oil level, and interaction between the energy source and Tra fish oil level. Tukey's pairwise comparisons (p<0.05) were applied to determine differences between treatments.

Results and discussions

The chemical composition data of the ingredients (Table 2) illustrate that the experimental diets were designed to evaluate the effects of starch sources and lipid supplementation on nutrient utilization and methane emissions of the Charolais crossbred cattle. The contrast between maize and cassava chips in terms of CP and fiber content may influence digestibility, rumen fermentation, and feed intake. The low CP content of cassava chips may result in nitrogen deficiency for rumen microbes, potentially affecting fiber digestion. Therefore, the high crude protein content of soybean meal and nitrogen content of urea compensated for the low CP content in cassava chips, while the combination of rice straw and Napier grass ensured adequate fiber for rumen health and function.

Feed and nutrient intake

The results of feed intake (kg/head/day) presented in Table 3 indicate that the experimental diets were formulated with different starch sources (maize or cassava chip) and Tra catfish oil (TrO) supplementation, and had no significant effect on total daily feed intake. The statistical analysis evaluated the main effects of energy source (Es), Tra catfish oil supplementation (TrO), and their interaction (Es×TrO). The intake of other ingredients such as broken rice, soybean meal, Napier grass, rice straw, and premix did not differ significantly among treatments (p>0.05), indicating consistency in the base formulation of the diets.

Among diets, DM intake ranged from 6.32 to 6.34 kg/head/day, with no significant differences from starch source, TrO supplementation, or their interaction (p>0.05). Similarly, the intake of OM, CP, NDF, ADF, and ME showed no significant variation all diets (p>0.05). This indicates that the supplementation of TrO or the replacement of maize with cassava chip did not affect the voluntary feed intake or the overall nutrient consumption. Nutrient intake parameters (DM, OM, CP, NDF, ADF and ME) per 100 kg body weight were also not significantly different between diets (p>0.05). DM intake per 100 kg body weight was approximately 2.02 kg, while ME intake was approximately 19.2 MJ/100 kg body weight, regardless of diet. There were no significant differences in CP and fiber (NDF, ADF) intakes indicating that protein and fiber levels were well balanced across all diets.

The non-significant differences in feed intake between treatments are consistent with the concept that total feed intake tends to be adjusted to meet energy requirements as long as energy concentration and rumen function are not significantly altered. Voluntary feed intake is governed by a complex interaction between dietary energy density, fiber content, palatability, rumen fill, and metabolic signals (McDonald et al 2022). The results did not find any interaction on DM consumption of cattle between starch sources and Tra catfish oil (p>0.05). Previous publications indicated that the effect of fish oil supplementation on the DM intake of cattle was diverse. Thu and Dong (2021) with and without supplemented catfish oil at levels of 1, 2 and 3% DM into diets, containing rice straw and concentrate (coconut cake meal, soybean cake meal, broken rice and rice bran), and reported that DMI had not differed. Ngu et al (2019) investigated the effects of different levels of concentrate (0.5% vs. 1.5% of body weight) and oil supplementation (soybean oil or fish oil 6% DM) on feed intake and nutrient utilization in Brahman crossbred cattle. The study found that oil source had no significant effect on total DMI. Soybean oil was better tolerated than fish oil, with fish oil having some tendency to reduce DMI slightly. Nishimura et al (2025) studied the effect of medium chain fatty acid (MCFA) supplementation (1.5% DM; 80% caprylic acid + 20% capric acid) in dairy cows. The results showed that MCFA supplementation did not significantly affect DMI during either the prepartum or postpartum periods. By contrast, Amorocho et al (2009) conducted two experiments with 1.5% and 3.0% catfish oil supplementation levels in the diets of lactating Holstein dairy cows. The results showed that DMI or DMI per percentage of body weight increased linearly with rising catfish oil levels.

Nutrient digestibility

Digestibility data for DM, OM, CP, NDF, ADF of Charolais crossbred cattle fed diets containing different starch sources (maize or cassava chip), with or without TrO supplementation are shown in Table 4, expressed both as a percentage and per 100 kg body weight. The apparent digestibility coefficients of nutrients ranged from moderate to high all treatments, indicating good rumen fermentation and nutrient utilization. There were no differences among treatments were observed for digestibility of DM, OM, CP, NDF (p>0.5). Only ADF digestibility showed a significant interaction effect between starch source and TrO supplementation (p=0.009). DM digestibility ranged from 64.2% to 69.3%, with the highest digestibility observed in the CaTrO treatment and the lowest in CaNoTro. Although neither the starch source (p=0.982) nor the TrO combination (p=0.359) had a significant effect, there was a tendency for an interaction effect between starch source and oil supplementation (p=0.053), showed a potentially synergistic effect on DM digestibility when cassava was combined with TrO. Similarly, OM digestibility followed the same trend, with values ranging from 66.5% to 71.5%. The highest OM digestibility was also observed in the CaTrO diet. The interaction between starch source and TrO approached significance (p=0.070), while the main effects of starch source and oil were not significant (p>0.30), indicating a combined influence on OM utilization. CP digestibility varied slightly among treatments, ranging from 76.6% to 78.8%. The highest value was found in the CaTrO diet, while the lowest was recorded in the CaNoTro. However, there were no significant difference of starch source (p=0.916), TrO (p=0.703), or their interaction (p=0.548) on CP digestibility. NDF digestibility ranged from 62.3% to 67.2%. Although no significant effects were found for starch source, TrO, or their interaction (p>0.28), numerically higher digestibility was seen in the CaTrO diet. For ADF, a significant interaction was observed between starch source and TrO supplementation (p=0.009). The highest ADF digestibility (61.4%) was seen in the CaTrO diet, whereas the lowest (54.5%) was in the MaTrO diet. This result indicates a notable improvement in ADF digestibility when TrO was combined with cassava, suggesting a positive interaction between fat supplementation and rapidly fermentable starch. When nutrient digestibility was expressed as kg DM per 100 kg body weight (BW), no significant differences were found among treatments for DM, OM, CP, NDF, or ADF digestibility (p>0.19). However, numerically higher digestible DM and OM per unit BW were recorded in the CaTrO treatment, agreeing with the trends observed in percentage digestibility.

The non-significant differences in digestibility of DM, OM, CP and NDF indicated that 3% catfish oil supplementation did not adversely affect the overall digestibility of nutrients. This finding agreed with studies by Ngu et al (2019) and Thu and Dong (2021), reported no significant differences in digestibility when supplementing cattle diets with fish oil at similar levels. However, the numerically lower digestibility values observed in oil-supplemented treatments may be biologically relevant. Unsaturated fatty acids in fish oil have been shown to suppress cellulolytic bacterial such as Ruminococcus spp. and Fibrobacter succinogenes populations in the rumen, PUFA exposure damaged cell membrane integrity and reduced the growth of butyrate and acetate producing microbes, potentially altering fiber fermentation and volatile fatty acid profiles and coat feed particles, which can hinder microbial attachment and fiber degradation (Maia et al 2006). Shingfield et al (2011), reported that fish oil, linseed oil, and their mixture in growing Aberdeen Angus steers fed a maize silage-based diet. Notably, found that lipid supplementation at 3% of DM did not significantly alter the digestibility of DM, OM, CP, starch, or NDF. Darabighane et al (2021), reported the effect of a mixture of sunflower and fish oils on lactating dairy cows and found that oil supplementation at 3% DM had no significant effect on DM, OM, CP, NDF, or ADF digestibility. Kairenius et al (2017), supplementing lactating dairy cow diets with fish oil (FO), or FO combined with sunflower oil or linseed oil, reported total nutrient digestibility remained largely unaffected, with only a slight trend (p=0.09) for increased DM digestibility in lipid supplemented groups and found that fish oil and oil mixtures significantly altered fatty acid biohydrogenation, increasing trans fatty acid intermediates escaping the rumen and slightly modifying the microbial populations. However, overall microbial abundance and protozoal counts remained unaffected. Burdick et al (2022), who evaluated the effects of medium chain fatty acid (MCFA) supplementation on lactating dairy cows. MCFA supplementation did not significantly alter total tract DM, OM, or CP digestibility, but tended to reduce NDF and ADF digestibility. By contrast, Atikah et al (2018) investigated the effects of dietary oil supplementation (palm oil, coconut oil, and fish oil) on ruminal fermentation characteristics and nutrient digestibility in sheep. Fish oil supplementation significantly decreased DM and fiber (NDF and ADF) digestibility compared to control diets. The reduced digestibility was attributed to inhibition of cellulolytic bacteria, including Fibrobacter succinogenes and Ruminococcus albus, by long chain polyunsaturated fatty acids. Vargas et al (2020), investigated plant oil supplementation (sunflower, linseed, and olive oils at 4% DM) in dairy cows, found that DM, OM and CP digestibility were unaffected by oil type. NDF and ADF digestibility decreased significantly with linseed and sunflower oil, attributed to inhibition of fibrolytic bacteria by high levels of unsaturated fatty acids.

Methane emission

Methane (CH₄) emission data, showed in various units, were analyzed to evaluate the influence of starch source (maize vs. cassava chip) and dietary TrO supplementation on rumen methane production in Charolais crossbred cattle. The results in Table 5 as methane energy loss (MJ/head/day), methane yield per unit of body weight and intake, and associated growth performance. CH₄ emissions, measured as MJ/head/day, ranged from 633 to 672 MJ/day. The lowest CH₄ emission was recorded in the MaTrO and CaTrO diets (633 and 634 MJ/day, respectively), while the highest emission occurred in CaNoTrO (672 MJ/day). A statistically significant reduction in CH₄ emission was observed with TrO supplementation (p=0.001), regardless of starch source. However, there was no significant effect of starch source (p=0.672) or their interaction (p=0.726) on CH₄ output. When CH₄ emission was expressed per 100 kg body weight (MJ/100 kg BW/day), no significant differences were observed among treatments (p>0.47), with values ranging from 203 to 217 MJ. Similarly, CH₄ emission per kg of dry matter intake (DMI) and digestible dry matter (DDM) showed no significant differences between starch sources, oil supplementation, or their interaction (p>0.47). However, there was a consistent trend of lower methane emissions in the oil supplemented diets (MaTrO and CaTrO) compared to the non-oil supplemented diets. CH₄ per kg DMI was 100–104 MJ in oil diets compared to 106–110 MJ in non-oil diets. CH₄ per kg DDM was lowest in the CaTrO diet (145 MJ/kg DDM) and highest in the CaNoTrO diet (172 MJ/kg DDM). These results showed that dietary TrO supplementation may have reduced methane yield, likely due to its inhibitory effects on methanogenesis through reduced hydrogen availability and alter in rumen fermentation pathways.

There were no significant differences in initial or final body weight among treatments (p> 0.90). Although weight gain was numerically higher in the CaNoTrO diet (891 g/day) and lower in the CaTrO diet (527 g/day), the variation was not statistically significant (p=0.132). This trend may suggest an improved feed efficiency or nutrient utilization when cassava was combined with dietary TrO.

Supplementation of catfish oil at 3% DM in this study reduced CH₄ emissions without reducing feed intake or energy intake affecting nutrient intake or digestibility (Tables 3 and 4). This suggests a more favorable balance between CH₄ reduction and animal performance, especially when using unsaturated fat sources such as fish oil, which are known to influence rumen CH₄ production through: reduction in fiber digesting microbes and protozoa, competitive hydrogen utilization through biohydrogenation, shifts in rumen volatile fatty acid (VFA) profile towards propionate. Patra and Yu (2013), compared the effects of coconut and fish oils on ruminal fermentation and microbial populations. It found that FO significantly reduced CH₄ production, inhibited methanogens, and altered the abundance of fiber digesting microbes like Ruminococcus flavefaciens and Fibrobacter succinogenes . It also influenced the VFA by increasing propionate levels relative to acetate. Fievez et al (2003), evaluated two FO types FOa (n-3-eicosapentanoic acid (EPA), 18.1%; n-3-docosahexanoic acid (DHA), 11.9%) and FOb (EPA, 5.4%; DHA, 7.5%) and level (0, 12.5, 25, 50, 75, 100 and 125 mg FO) of FO in vitro and in vivo. It reported that FO inhibited CH₄ production, increased propionate, and decreased acetate production. These changes were attributed to the high PUFA content and its role in biohydrogenation, which competes with methanogenesis for hydrogen. Vargas et al (2017), demonstrated using an artificial rumen simulation technique system that CH₄ production was significantly reduced (p<0.05) when marine FO or sunflower oil were added to a ruminant diet. In that study, CH₄ production dropped from 265 mL/day in the control to ~210 mL/day in oil-supplemented treatments, and CH₄ per mL total gas declined significantly as well. The mechanism involves inhibition of methanogenic archaea, suppression of protozoa, and increased propionate production, resulting in lower acetate:propionate ratios and a shift in fermentation pathways.

The findings agreed with Giagnoni et al (2025), reported that increased dietary fat, particularly MCFA from palm kernel, significantly reduced CH₄ yield (g CH₄/kg DMI) in dairy cows. The study showed a linear reduction in CH₄ yield as the concentration of dietary fat increased, with the medium palm kernel fat group achieving greater CH₄ suppression than rapeseed oil groups. However, the reduction in CH₄ was sometimes accompanied by decreased DMI or milk energy yield. Zhang et al (2008) used an in vitro model to test the effect of various octadeca carbon fatty acids (C18:0, C18:1, C18:2, and C18:3) on CH₄ production. Found that unsaturated fatty acids (C18:2 linoleic and C18:3 linolenic acids) had strong inhibitory effects on CH₄ and microbial fermentation. Fatty acid toxicity selectively inhibited methanogens and cellulolytic bacteria, which are essential for acetate production and fiber digestion. Ngu et al (2019) studied Brahman crossbred cattle and showed that fish oil supplementation reduced acetate and propionate concentrations in the rumen and significantly decreased the abundance of Fibrobacter succinogenes, a key acetate producing cellulolytic bacterium, and no significant differences in initial or final body weight or body weight change among treatments. Toral et al (2010) reported that adding FO and sunflower oil to high concentrate sheep diets altered the fatty acid composition of rumen digesta, by inhibiting biohydrogenation of 18-carbon unsaturated fatty acids, increasing the presence of intermediates like trans-18:1 and long chain PUFA (C20:5 n-3, C22:6 n-3). These changes reflect microbial suppression of biohydrogenation pathways, which in turn limit hydrogen availability, a critical substrate for methanogens. Quang et al (2020), studied the effects of vegetable oil (1.5–3% DM) and tannin (0.3–0.5% DM) supplementation on CH₄ emissions in Red Sindhi crossbred cattle. The results showed that CH₄ emissions significantly decreased with higher levels of oil and tannin. Lima et al (2024), lamb study reported that increased dietary soybean oil at 0, 30, 60, 90 and 120 g/ kgDM, reduced ADG linearly. Wistuba et al (2005), supplementing corn-based diets with 1.5% FO at grazing phase (78 days) resulted in significantly reduced ADG and adding 3% FO to a high concentrate finishing diet at growing phase (56 days) led to the decrease in ADG.

Conclusion

The present study demonstrated that supplementation of Tra catfish oil at 3% of dry matter in diets containing either maize or cassava chip starch sources did not significantly alter feed intake or overall nutrient intake in Charolais crossbred cattle. Apparent nutrient digestibility (DM, OM, CP, NDF, ADF) was generally unaffected by the type of starch or the supplementation of Tra catfish oil.

Methane emissions were significantly reduced by Tra catfish oil supplementation, regardless of starch source. This reduction, estimated at 5–6% compared to the unsupplemented diet, suggests that Tra catfish oil may be an effective nutritional strategy to reduce enteric methane production without negatively affecting intake or digestibility.

Acknowledgements

This research is funded by Vietnam National University Ho Chi Minh City (VNU-HCM) under grant number C2024-16-06. The authors thank the Hanh Cuong cattle farm, An Giang University (AGU), Vietnam National University Ho Chi Minh City (VNU-HCM).

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