Effect of dietary α-monolauric acid and bacillus subtilis supplementation on growth performance and nutrient digestibility in early-weaned crossbred piglets under heat stress

Introduction

Pig production is an important component of the global livestock industry, with pork accounting for approximately 36% of total meat consumption worldwide (USDA, 2019). Domestic pigs (Sus domesticus) are highly valued for their prolific reproductive performance, rapid growth, efficient feed conversion and high dressing percentage, making them one of the most productive meat animals. In India, however, pigs contribute only 4.8% of total meat production, despite a population of 9.06 million animals reported in the 20th Livestock Census, highlighting significant scope for improving pig productivity and meeting the increasing demand for animal protein.

Weaning is one of the most stressful and critical phases in pig production. Early weaning (30-35 days), although beneficial for increasing sow reproductive efficiency by shortening the farrowing interval, exposes piglets to nutritional, environmental, and social stress. These stressors impair intestinal barrier integrity, reduce villus height, decrease digestive enzyme activity, and increase intestinal permeability, predisposing piglets to post-weaning diarrhea, poor nutrient utilization, reduced growth performance, and higher mortality (Smith et al., 2010; Campbell et al., 2013) [1, 2] . Therefore, nutritional interventions that support gut health and alleviate weaning stress are essential for improving piglet performance. Feed additives such as α-monolauric acid and probiotics have emerged as promising alternatives to antibiotic growth promoters. α-Monolauric acid, a feed additive approved by the U.S. Food and Drug Administration (FDA), possesses broad-spectrum antibacterial and antiviral properties, enhances feed palatability and improves nutrient digestibility by promoting intestinal health (Jiang et al., 2018, Mustafa, 2019, Welch et al., 2020) [3,4,5] . Similarly, the probiotic Bacillus subtilis, a spore-forming bacterium, enhances gut microbial balance by producing digestive enzymes, stimulating beneficial Lactobacillus populations, suppressing enteric pathogens such as Escherichia coli, Salmonella, Campylobacter and Clostridium spp., and improving intestinal health and nutrient utilization (Hosoi et al., 2000, Enthoven and Van der Lee, 2004) [6, 7] . Despite the documented benefits of these additives individually, studies evaluating their combined supplementation, particularly under Indian production conditions and heat stress, remain limited. Therefore, investigating the synergistic effects of α-monolauric acid and Bacillus subtilis on growth performance and nutrient digestibility in early-weaned piglets is of considerable scientific and practical importance.

Materials and Methods

A 92-day feeding trial was conducted from 12 April to 12 July 2025 at the Livestock Farm Complex (LFC), College of Veterinary Science, Rajendranagar, Hyderabad, India, to evaluate the effects of α-monolauric acid (α-MLA) and Bacillus subtilis probiotic supplementation on the performance and nutrient digestibility of early-weaned crossbred piglets under heat stress conditions. Laboratory analyses were carried out in the Department of Animal Nutrition, College of Veterinary Science, Rajendranagar, Hyderabad.

Experimental Diets

Twenty-four healthy crossbred piglets (30-35 days old) with an average initial body weight of 6.3±0.1 kg were randomly allotted to four dietary treatments in a completely randomized design (n = 6). The dietary treatments consisted of: Control (BD)-basal diet without additives, basal diet supplemented with α-monolauric acid at 1000 mg/kg (BA), basal diet supplemented with Bacillus subtilis probiotic (GutCare®, 2 × 10 [8] CFU/g) at 500 g/MT⁰ (BP) and basal diet supplemented with both α-monolauric acid (1000 mg/kg) and probiotic (500 g/MT) (BAP). The basal diet was formulated according to the nutrient requirements of growing piglets as recommended by ICAR (2024).

Table 1: The four experimental diets include

Diets

Description of diets BD Basal diet (without antibiotic or any feed additive) BA Basal diet + α-Monolauric acid @ 1000 mg/kg of diet BP Basal diet + Probiotic (Bacillus Subtilis) @500 gm/MT of feed

BAP

Basal diet + α -Monolauric acid @ 1000 mg/kg of diet + Probiotic (Bacillus Subtilis) @ 500 gm/MT of feed

Growth Performance Parameters

In this study, growth performance of the piglets was analysed by calculating fortnightly body weight gain, average daily gain and finally total weight gain of all piglets fed experimental diets.

Body Weight Gain

The body weights of the piglets were measured at fortnightly intervals.

Total body weight gain = Final body weight -Initial body weight

Average Daily Gain 𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴 = 𝑇𝑇𝑇𝑇𝑇𝑇𝐴𝐴𝐴𝐴 𝑏𝑏𝑇𝑇𝐴𝐴𝐴𝐴 𝑤𝑤𝐴𝐴𝐴𝐴𝐴𝐴ℎ𝑇𝑇 𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴(𝑘𝑘𝐴𝐴)

𝑇𝑇𝑇𝑇𝑇𝑇𝐴𝐴𝐴𝐴 𝐴𝐴𝑇𝑇 𝑇𝑇𝑜𝑜 𝐴𝐴𝐴𝐴𝐴𝐴𝑑𝑑 (90)

Digestion Trail

A digestibility trial was conducted during the final 12 days of the experiment (days 81-92), comprising a 5-day adaptation period followed by a 7-day collection period. Four piglets from each treatment were selected for digestibility assessment using the double-indicator method. Chromic oxide (Cr₂O₃ ; 3g / day) , administered in hydroxypropyl methylcellulose capsules before feeding, served as the external marker. Fresh feacal samples were collected by rectal massage three times daily, pooled for each animal, processed and stored for chemical analysis. Feed and feacal samples were analyzed for dry matter (DM), crude protein (CP), ether extract (EE), crude fiber (CF), total ash (TA), and nitrogen-free extract (NFE) according to AOAC (2012) [9] procedures. Chromic oxide concentration was determined using the wet digestion method and apparent nutrient digestibility coefficients were calculated using marker ratios. These analyses were used to estimate nutrient digestibility and retention in piglets receiving different dietary treatments.

Digestibility of DM, CP, CF, EE were calculated using internal indicator acid insoluble ash (AIA) using the following formula

Statistical Analysis

Data analysis was performed using the General Linear Model (GLM) procedure in the Statistical Package for Social Sciences (SPSS) version 20. Means were compared using Duncan's multiple range test (Duncan, 1955) [10] , with statistical significance set at P<0.05. The experimental data were analyzed under a completely randomized design employing one-way analysis of variance (ANOVA) as described by Snedecor and Cochran (1989).

Iii. Results and Discussion

The study evaluated the effects of dietary supplementation with α-monolauric acid (α-MLA), Bacillus subtilis probiotic, and their combination on growth performance and nutrient digestibility. The findings obtained are presented and discussed in the following sections.

Nutrient Compostion of the Diet

The chemical composition of the experimental diets is presented in Table 3. All four dietary treatments were formulated to be isonitrogenous and isocaloric, providing comparable levels of metabolizable energy (3291.6 kcal/kg), crude protein (22.06%), calcium (0.70%), phosphorus (0.60%), lysine (0.95%), and methionine (0.66%), thereby meeting the nutrient requirements of growing piglets. [14,15,16] . The greatest response observed in the combined supplementation group suggests a synergistic effect, where reduced pathogen load and improved probiotic colonization together enhance digestive efficiency and growth performance, consistent with previous findings of Jackman et al., 2020 [11] ; Tang et al., 2024 [16] .

Fortnight Body Weight Gain Significantly (P < 0.05) higher fortnightly body weight gains were observed in piglets supplemented with α-monolauric acid, Bacillus subtilis, and their combination ( [20,21,22] . These discrepancies may be attributed to differences in additive type and dosage, supplementation period, diet composition, animal age and experimental conditions. [20,21,22,26,27] . These variations are likely due to differences in the type and inclusion level of feed additives, supplementation strategies, experimental duration, pig genetics, health status and management conditions. [28,18,19,24,29,30,31,32,20,33] . In contrast, Li et al., 2022 [21] and Cui et al., 2019 [20] observed no significant effect of glycerol monolaurate or Bacillus subtilis supplementation on ADG. Such inconsistencies may be attributed to differences in feed additive type, dosage, duration of supplementation, pig genotype, age, health status, and management or environmental conditions.

Feed Intake Parameters Feed Intake and Dry Matter Intake

No significant (P > 0.05) differences were observed in feed intake or dry matter intake among the treatment groups, although piglets receiving the combined supplementation of αmonolauric acid and Bacillus subtilis (Table 7) showed numerically higher values. These findings are consistent with several studies reporting that supplementation with mediumchain fatty acids, probiotics, organic acids, or their combinations had no significant effect on feed intake ( [29,17,18,20,21,24,31,33,34,35] . However, Liu et al., 2025 [19] , Ficagna et al., 2025 [36] and da Silva et al., 2021 [20] reported significant improvements in feed intake following supplementation with Bacillus subtilis, organic acid-yeast cell wall blends, or benzoic acid-probiotic combinations. These variations among studies are likely due to differences in the type and dosage of feed additives, supplementation strategies, experimental duration, animal age and physiological status, and environmental and management conditions.

Feed Conversion Ratio

Piglets supplemented with α-monolauric acid, Bacillus subtilis, and their combination exhibited an improved feed conversion ratio (FCR) compared with the control group, with the greatest improvement observed in the combined supplementation group (Table 7). Despite no significant (P>0.005) differences in feed intake, the lower FCR indicates enhanced nutrient utilization and growth efficiency, likely resulting from improved intestinal health, nutrient digestibility, and gut microbial balance. These findings agree with previous studies reporting improved FCR following supplementation with probiotics, organic acids, medium-chain fatty acids, or their combinations ( [17,19,34,36] . However, Cui et al., 2019 [20] , da Silva et al., 2021 [20] and Li et al., 2022 [21] reported no significant effects of similar feed additives on FCR. Such variations may be attributed to differences in dietary composition, additive type and inclusion level, experimental duration, animal age and health status, and environmental and management conditions. [37,38,39] . Bacillus subtilis further enhances digestion by producing extracellular enzymes, improving gut microbial balance and stimulating intestinal epithelial development through shortchain fatty acid production (Hosoi et al., 2000, Wang and Gu, 2010) [6,40] . These findings are consistent with previous studies demonstrating improved nutrient digestibility following supplementation with medium-chain fatty acids, probiotics, or their combinations ( [29,18,19,21,22,24,32,41] . However, some studies reported selective or non-significant effects on certain nutrients, particularly with medium-chain fatty acids alone (Gebhardt et al., 2020; Fan et al., 2025) [42,27] , likely due to differences in additive type, dosage, diet composition and experimental conditions.

Iv. Conclusion

Dietary supplementation with α-monolauric acid, Bacillus subtilis, and their combination improved the growth performance and nutrient digestibility of early-weaned crossbred piglets under heat stress conditions. The combined supplementation produced the best results, with significantly higher body weight gain, average daily gain, improved feed conversion ratio, and greater digestibility of dry matter, crude protein, ether extract, and organic matter. These findings suggest that the combination of α-monolauric acid (1000 mg/kg feed) and Bacillus subtilis probiotic (500 g/metric ton of feed) is an effective nutritional strategy to enhance piglet performance and nutrient utilization, and may serve as a promising alternative to antibiotic growth promoters.

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