Effects of palm stearin enriched diets on rumen fermentation in growing buffaloes

Introduction

In ruminant nutrition, dietary energy is a critical component influencing growth performance, feed efficiency and overall animal productivity. Traditionally, cereal grains and other carbohydrate rich feedstuffs have served as primary energy sources; however, rising costs and competition with the human food supply have prompted the exploration of alternative, cost-effective energy supplements. One such alternative is the use of lipid based byproducts (Behan et al., 2019) [5] , which can enhance the energy density of ruminant diets without adversely affecting rumen function when used appropriately (Jenkins, 1993) [13] . Palm stearin (PS), a byproduct of the Palm oil refining industry, is a solid fraction obtained during the fractionation of palm oil and represents a promising lipid supplement due to its high energy content (Tomkins & Drackley, 2010) [21] . The primary constituents of palm stearin are long-chain saturated fatty acids, with palmitic acid being the most abundant (Norliza et al., 2016) [15] . As a byproduct of the palm oil refining industry, PS offers a sustainable approach to utilising agro-industrial waste while potentially improving dietary energy efficiency in livestock production systems. However, the inclusion of dietary fats in ruminant diets must be approached with caution, as excessive unsaturated fats or inappropriate dosages of fat supplementation can disrupt rumen microbial activity (Jenkins, 1993) [13] and thereby alter the fermentation patterns and impair fibre digestion in ruminants (Allen, 2000) [2] . Rumen fermentation is a complex microbial-driven process that produces volatile fatty acids (VFAs), ammonia nitrogen (NH₃-N) and other metabolites essential for the host animal's energy supply and protein synthesis. Monitoring parameters such as rumen pH, NH₃-N concentration, total VFA levels, and the molar proportions of acetate, propionate, and butyrate provides valuable insights into the effects of dietary interventions on rumen health and functionality. Despite the potential advantages of Palm Stearin as a dietary fat source, limited information is available regarding its impact on rumen fermentation characteristics, particularly in buffaloes, a species with growing importance in global meat and milk production. Therefore, the present study was undertaken to evaluate the effects of dietary palm stearin supplementation on rumen fermentation profile in growing buffaloes. This investigation aims to determine whether palm stearin can be safely incorporated into ruminant diets without compromising rumen microbial activity and fermentation efficiency.

Materials and Methods

The current study was conducted to evaluate the impact of dietary supplementation with Palm Stearin, which is a byproduct derived from the palm oil refining process, on the rumen fermentation parameters of growing buffaloes. The experimental trial was performed at the Experimental Animal Shed of the Animal Nutrition Division, ICAR-Indian Veterinary Research Institute (IVRI), located in Izatnagar, Bareilly, Uttar Pradesh. The institute is positioned at a geographical coordinate of 28° 23′ 18.98″ N latitude and 79° 25′ 18.47″ E longitude, with an altitude of 240 meters above mean sea level.

Experimental animal, designs and housing

A total of 18 growing buffalo calves were enrolled in the experiment and randomly assigned to one of three dietary treatment groups: T₀ (control), T₁, and T₂, with six calves in each group. To ensure unbiased allocation and balanced distribution among groups, the animals were assigned using randomised block design (RBD). The study was carried out at the Experimental Animal Shed, Animal Nutrition Division, ICAR-Indian Veterinary Research Institute (ICAR-IVRI), located in Izatnagar. The feeding trial lasted for 165 days. Before the trial began, all calves underwent a routine deworming regimen to eliminate internal parasitic infections. Throughout the study, the animals were housed in well-ventilated, clean, and hygienic conditions to support their wellbeing and minimize external stress factors that could influence the study's results. These preliminary steps were taken to ensure the animals remained in good health throughout the experimental period, thereby enabling a reliable evaluation of the effects of the dietary treatments.

Feeding management

The buffalo calves in the control group (T₀) were provided with a standard diet comprising a 50:50 ratio of concentrate and wheat straw, formulated to meet the nutritional requirements recommended by ICAR (2013) feeding standards. In the treatment groups, T₁ and T₂, palm stearin was included in the concentrate mixtures at levels of 3.0% and 6.0%, respectively. This was achieved by partially substituting maize and wheat bran, while maintaining the diets as iso-nitrogenous to ensure a consistent protein content across all groups. The composition of the concentrate mixtures, detailed in Table 1, consisted of maize, deoiled soybean meal (DSBM), wheat bran, mineral mixture, salt, and the respective levels of palm stearin. To support vitamin A intake, green fodder was supplied twice a week. Clean and fresh drinking water was made available ad libitum, with routine distribution occurring twice daily at 10:00 AM and 3:00 PM throughout the duration of the study.

Rumen liquor collection and analysis

Rumen liquor samples were collected from the experimental buffaloes on the last day of the feeding trial using the stomach tube technique, and the samples were preserved at-20°C for subsequent analysis. Rumen pH was measured immediately after sample collection using a precalibrated digital pH meter. A portion of each rumen liquor sample was preserved with two drops of 20% sulfuric acid (H₂SO₄) for the determination of ammonia nitrogen (NH₃-N),

following the method outlined by Weatherburn (1967) [23] . The concentration of volatile fatty acids (VFAs) in the rumen liquor was analysed using a Nucon-5765 gas chromatograph fitted with a dual flame ionization detector and a glass column packed with Chromosorb 101, as per the protocol described by Cottyn and Boucque (1968) [8] .

Statistical analyses

Statistical analyses were performed in accordance with the standard procedures described by Snedecor and Cochran (2004), utilising SPSS software (version 26.0). All tests were evaluated at a significance threshold of p<0.05.

Results pH and ammonia nitrogen

The data on pH and ammonia nitrogen (NH₃-N) levels in relation to the effects of PS on rumen fermentation are presented in Table 2. The pH values were similar across the dietary groups, with recorded levels of 6.42 in T 0 , 6.40 in T 1 , and 6.38 in T 2 . Statistical analysis indicated no significant differences (p>0.05) in pH values among the treatment groups compared to the control. Similarly, NH₃-N concentrations (mg/dL) in T 0 , T 1 , and T 2 were 12.38, 12.31, and 12.24, respectively. The statistical evaluation confirmed that NH₃-N levels were comparable across all dietary groups, with no significant differences (p>0.05) observed in comparison to the control group.

Total volatile fatty acid and its fractions

Table 3 presents data on total volatile fatty acids (TVFA) and their fractions. The mean TVFA concentrations (mM/dL) were comparable across the treatment groups, with values of 9.58 mM/dL in T 0 , 9.49 mM/dL in T 1 , and 9.45 mM/dL in T 2 . Statistical analysis revealed no significant differences (p>0.05) among the dietary treatments. Similarly, acetate percentages were recorded as 72.40% in T 0 , 71.88% in T 1 , and 71.21% in T 2 , with no statistically significant differences (p>0.05) observed between groups. The propionate proportions were 17.46% in T 0 , 17.69% in T 1 , and 18.10% in T 2 , with statistical analysis confirming no significant variation (p>0.05) across treatments. Additionally, butyrate levels were measured at 6.37% in T 0 , 6.31% in T 1 , and 6.30% in T 2 , showing no significant differences (p>0.05) among the groups. Furthermore, the acetate-to-propionate (A:P) ratio was assessed and remained statistically similar across treatments. No significant differences (p>0.05) were detected in the A:P ratio, indicating that dietary supplementation with PS did not significantly (p>0.05) influence this parameter.

Control (T0), Treatment (T1) & Treatment (T2):

Dietary groups having concentrate containing (0, 3 & 6% PS) along with wheat straw in the diet. TVFA-Total volatile fatty acid, mM/dL-millimoles per decilitre, A:P ratio-Acetate to Propionate ratio.

Discussions pH and Ammonia nitrogen (NH3-N)

In the present study, dietary supplementation with PS did not significantly affect rumen pH levels, as the values remained comparable across all treatment groups. Similarly, the ammonia nitrogen (NH₃-N) concentrations were consistent across treatments, with no significant variations (p>0.05) observed. These findings align with previous studies that have investigated the effects of various fat supplements on rumen fermentation parameters. Teh et al. (1994) [20] reported no significant (p>0.05) alterations in ruminal pH or NH₃-N levels when bypass fat was incorporated into the diet, suggesting that such supplementation does not interfere with microbial fermentation processes. Likewise, Vandoni et al. (2010) [22] found that supplementing beef cattle diets with three distinct bypass fat sources-calcium salts (CaS), hydrogenated fatty acids (HF), and triglycerides did not lead to any significant differences in rumen fermentation characteristics or pH levels. Similar findings were reported by Demirel et al.

(2004) [9] , who observed no discernible changes in rumen pH when animals were fed three different types of rumenprotected fat (RPF). Behan et al. (2019) [5] also demonstrated that supplementation with prill fat had no significant (p>0. [19] reported comparable results in rams supplemented with 6% dietary tallow and Bergafat (a fat source rich in palmitic acid (C16:0) and stearic acid (C18:0)), where ammonia-N levels remained unaffected. Similarly, Behan et al. (2019) [5] observed no significant changes in ammonia nitrogen concentrations when animals were fed 5% dietary prilled fat containing 72.98% palmitic acid. Conversely, Baee et al. (2023) [3] investigated the effects of palmitic and stearic acid supplementation in lamb diets and found that while these fatty acids did not alter rumen pH, ammonia nitrogen concentrations significantly increased (p<0.05) in lambs fed 3% stearic acid or a mixture of palmitic and stearic acids. However, other studies, such as that by Wu et al. (2016) [24] , reported no significant changes in rumen pH or ammonia nitrogen levels following bypass fat supplementation.

In addition to these studies, several other researchers have reported similar findings, highlighting the lack of significant changes in rumen fermentation parameters following protected fat supplementation. Research by Ngidi et al.

(1990) [14] , Bhatt et al. (2013) [6] , Schauff et al. (1992) [18] , Hightshoe et al. (1991) [11] , Grummer et al. (1988) [10] , and Bhatt et al. (2011) [7] further corroborates that ruminal pH remains stable across different dietary treatments, reinforcing the consensus that fat supplementation in recommended levels does not adversely affect rumen fermentation dynamics. These collective findings suggest that fat supplementation at recommended levels, including PS, does not significantly influence ruminal pH or ammonia nitrogen concentrations, thus maintaining normal fermentation processes in ruminants.

Volatile fatty acid and its fractions (VFA)

The findings of the present study indicate that dietary supplementation with PS did not lead to any statistically significant (p>0.05) alterations in total volatile fatty acid (TVFA) concentrations or their individual fractions. The mean TVFA levels remained consistent across all treatment groups, demonstrating that the dietary intervention had no substantial impact on ruminal fermentation. Furthermore, the proportions of acetate, propionate and butyrate were stable among the treatment groups, with no significant differences (p>0.05) detected. Similarly, the acetate-topropionate (A:P) ratio was comparable to that of the control group, with statistical analysis confirming the absence of significant (p>0.05) variations. These results align with previous research that has examined the impact of fat supplementation on ruminal fermentation. Behan et al. (2019) [5] reported that TVFA concentrations were uniform across all treatment groups, consistent with the earlier findings of Hightshoe et al. (1991) [11] . Hightshoe et al. (1991) [11] also demonstrated that neither the source nor the level of rumen-protected fat (RPF) significantly influenced TVFA concentrations or the A:P ratio in steers fed calcium soaps of fatty acids and hydrogenated animal fats. Similarly, Demirel et al. (2004) [9] observed no significant differences in ruminal pH and VFA concentrations when animals were supplemented with three different types of rumen-protected fats (RPF). Patra and Yu (2012) [16] further corroborated these findings by demonstrating that increasing dietary fat levels did not significantly (p>0.05) affect TVFA concentrations or acetate proportions. In a subsequent study, Patra (2013) [17] confirmed that neither TVFA levels nor acetate percentages in ruminal fluid were significantly influenced (p>0.10) by dietary fat supplementation. However, a linear increase in propionate levels (P=0.03) was observed with higher fat inclusion. Additionally, dietary fats exhibited a tendency to reduce butyrate proportions (P=0.10) and the A:P ratio (P=0.07), though these effects were relatively weak. [19] investigated the inclusion of bypass fat in dairy sheep diets and reported no significant effects on individual VFA levels at a 4% dietary inclusion rate (containing 47-53% palmitic acid and 39-44% stearic acid). However, at a 6% inclusion rate, a significant increase in the molar proportion of acetate was observed compared to the control group, suggesting that higher levels of supplementation might influence specific VFA ratios. Furthermore, in vitro studies conducted by Hristov et al.

(2004) [12] demonstrated that palmitic stearic acids did not exert significant effects on total or individual VFA concentrations.

Collectively, these studies support the findings of the present research, indicating that ruminal fermentation parameters, including NH₃-N, TVFA and the molar proportions of propionate and butyrate, remain largely unaffected by dietary fat supplementation. The lack of significant changes suggests that rumen protected fat (RPF) remains inert within the rumen environment and does not disrupt microbial activity at the tested inclusion levels. These results further reinforce the hypothesis that fat based dietary interventions, including PS supplementation, do not adversely impact ruminal fermentation dynamics, as supported by Behan et el. (2019) [5] .

Conclusion

The findings of the present study indicate that supplementation with palm stearin does not significantly influence rumen fermentation characteristics in ruminants. Specifically, measurements of rumen pH, ammonia nitrogen (NH₃-N) concentration, total volatile fatty acid (VFA) concentrations, and the molar proportions of individual VFAs namely acetate, propionate, and butyrate remained statistically unaltered among the different dietary treatment groups (p>0.05). Additionally, the ratio of acetate to propionate, a commonly used indicator of fermentative balance and energy metabolism in the rumen, was not significantly affected by palm stearin supplementation. These results suggest that the supplementation of ruminant diets with palm stearin does not interfere with the normal microbial activity or fermentation dynamics within the rumen, suggesting its potential as a viable dietary fat source in ruminant nutrition.

Acknowledgement

AcknowledgmentThe authors would like to thank the Director, ICAR-Indian Veterinary Research Institute, Izatnagar, Bareilly (India) for providing all the facilities required to conduct the experiment.

References

1. Hristov AN, Ivan M, McAllister TA. in vitro effects of individual fatty acids on protozoal numbers and on fermentation products in ruminal fluid from cattle fed a high-concentrate, barley-based diet. Journal of Animal Science. 2004 Sep;82(9):2693-2704.
2. Jenkins TC. Lipid metabolism in the rumen. Journal of Dairy Science. 1993 Dec;76(12):3851-3863.
3. Ngidi ME, Loerch SC, Fluharty FL, Palmquist DL. Effects of calcium soaps of long-chain fatty acids on feedLot performance, carcass characteristics and ruminal metabolism of steers. Journal of Animal Science. 1990 Aug;68(8):2555-2565.
4. Norliza S, Zahariah I, Ahmadilfitri MN, Mohd Suria AY, Mardhati M, Wong HK. Effect of feeding fractionated RBD palm stearin on milk yield and quality of dairy cattle. Malaysian Journal of Animal Science. 2016 Jun;19(1):9-13.
5. Patra AK, Yu Z. Effects of essential oils on methane production and fermentation by, and abundance and diversity of, rumen microbial populations. Applied and Environmental Microbiology. 2012 Jun;78(12):42714280.
6. Patra AK. The effect of dietary fats on methane emissions, and its other effects on digestibility, rumen fermentation and lactation performance in cattle: A meta-analysis. Livestock Science. 2013 Aug;155(23):244-254.
7. Schauff DJ, Clark JH, Drackley JK. Effects of feeding lactating dairy cows diets containing extruded soybeans and calcium salts of long-chain fatty acids. Journal of Dairy Science. 1992 Nov;75(11):3003-3019.
8. Szumacher-Strabel M, Cieslak A, Potkanski A, Kowalczyk J, Czauderna M. The effects of different amounts and types of fat on rumen microbial protein synthesis in sheep. Journal of Animal and Feed Sciences. 2001 Jun;10:97-102.
9. Teh TH, Trung LT, Jia ZH, Gipson TA, Ogden KB, Sweeney TF. Varying amounts of rumen-inert fat for high producing goats in early lactation. Journal of Dairy Science. 1994 Jan;77(1):253-258.
10. Tomkins T, Drackley JK. Applications of palm oil in animal nutrition. Journal of Oil Palm Research. 2010 Dec;22(3):835-845.
11. Vandoni SL, Dell'Orto V, Sgoifo Rossi CA. Effects of administration of three different by-pass lipids on growth performance, rumen activity and feeding behaviour of beef cattle. Italian Journal of Animal Science. 2010 Feb;9(2):e44.
12. Weatherburn M. Phenol-hypochlorite reaction for determination of ammonia. Analytical Chemistry. 1967 Aug;39(8):971-974.
13. Wu D, Xu L, Tang S, Guan L, He Z, Guan Y, et al. Influence of oleic acid on rumen fermentation and fatty acid formation in vitro. PLoS One. 2016 Jun;11(6):e0156835.