Effect of probiotic supplementation on growth performance and gut morphology in broiler chickens

Introduction

Commercial broiler production in Mexico grew 3.8% year-on-year between 2018 and 2022, yet the country's 2019 regulation restricting antibiotic growth promoters in poultry feed left producers searching for alternatives that could maintain the feed efficiency gains that subtherapeutic antibiotic once provided [1,2] . Probiotics live microbial feed additives that confer a health benefit on the host when administered in adequate amounts are among the most promising candidates, but their effectiveness varies widely depending on strain combination, dose, bird age, and environmental conditions [3] . The proposed mechanisms by which probiotics improve broiler performance operate at two levels. At the gut-epithelial level, beneficial bacteria stimulate villus elongation, deepen the glycocalyx layer, and tighten intercellular junctions, collectively expanding the absorptive surface area and reducing paracellular leakage of nutrients and pathogens [4] . At the microbialecology level, competitive exclusion of pathogenic genera such as Salmonella, Clostridium, and Escherichia by Lactobacillus and Bacillus species shifts the fermentation profile toward short-chain fatty acid production, which in turn lowers luminal pH and provides energy to colonocytes [5] . Multi-strain formulations combining lactic acid bacteria, spore-forming bacilli, and yeast are theorised to outperform single-strain products because each organism occupies a different niche within the gastrointestinal tract and contributes a different mode of action [6] . Lactobacillus acidophilus colonises the proximal intestine and produces lactic acid; Bacillus subtilis survives pelleting temperatures and germinates in the upper gut to produce antimicrobial lipopeptides; and Saccharomyces cerevisiae ferments undigested carbohydrates in the caecum and provides B-vitamins to the host [7] . However, controlled dose-response trials of such threecomponent blends under Mexican tropical conditions are scarce. A missing link in many probiotic trials is the connection between gut-level histological changes and whole-bird performance outcomes. Villus height and the villus-height-tocrypt-depth (V:C) ratio are standard markers of intestinal absorptive capacity, and their improvement by probiotics has been documented in temperate-climate trials, but whether the same magnitude of change occurs under the higher heat loads and different pathogen pressures of a tropical environment is less certain [8,9] . This research aimed to (a) compare the growth performance of Ross 308 broilers receiving two probiotic dose levels against an unsupplemented control over a 42-day production cycle, (b) quantify jejunal villus height, crypt depth, and V:C ratio at slaughter, (c) characterise caecal bacterial phyla composition across treatments, and (d) test the correlation between villus height and final body weight. The findings are intended to guide Mexican poultry nutritionists on optimal probiotic inclusion rates for antibiotic-free production.

Materials and Methods

Materials

Two hundred-and forty-day-old Ross 308 male broiler chicks were obtained from a commercial hatchery in Zapopan, Jalisco, and transported to the experimental poultry unit at the Guadalajara Research Institute of Animal Sciences (20.68° N, 103.35° W, elevation 1,566 m). Chicks were wing-banded, individually weighed (mean initial weight 42.3 ± 1.8 g), and randomly allocated to three dietary treatments with four replicate pens of 20 birds each. The trial ran from 5 June to 17 July 2023. Ethical clearance was granted by the Institute's Animal Use Committee (Protocol GRIAS-AUC/2023-016, approved 22 May 2023). The commercial multi-strain probiotic (ProBioMax-3, Nutrición Animal del Bajío, León, Mexico) contained Lactobacillus acidophilus (≥ 2 × 10⁹ CFU/g), Bacillus subtilis (≥ 1 × 10⁹ CFU/g), and Saccharomyces cerevisiae (≥ 5 × 10⁸ CFU/g) on a maltodextrin carrier. Treatments were: T1 = basal diet (control), T2 = basal + 0.5 g/kg ProBioMax-3, T3 = basal + 1.0 g/kg ProBioMax-3. The basal diet was a maizesoybean meal formulation meeting Ross 308 nutrient specifications for starter (Day 1-21, 23% CP, 3,050 kcal ME/kg) and finisher (Day 22-42, 20% CP, 3,150 kcal ME/kg) phases [10] .

Field experimental design

The trial used a completely randomised design with three treatments and four replicates per treatment, yielding 12 experimental units. Each pen of 20 birds constituted one experimental unit. Treatments were assigned by random number generation in Excel. Pen positions within the house were randomised to prevent location bias from proximity to exhaust fans or feed storage. A 7-day pre-starter adaptation period was not used, as probiotics were included from Day 1 to maximise early gut colonisation. Mortality was recorded daily and dead birds' weights were used to adjust FCR calculations for the affected replicate.

Methods

Birds were housed in floor pens (2.0 × 1.5 m) with fresh wood-shavings litter, nipple drinkers, and tube feeders. A 23L:1D lighting programme was applied for the first 7 days, transitioning to 18L:6D thereafter. Ambient temperature was maintained at 32 °C during Week 1 and reduced by 2 °C weekly to a final 24 °C. Feed and water were offered ad libitum. Body weight was recorded weekly by pen, and feed intake was measured per replicate to calculate the feed conversion ratio (FCR = feed consumed / body weight gain) [11] . On Day 42, two birds per replicate (8 per treatment) were euthanised by cervical dislocation. A 2-cm segment of midjejunum was excised, fixed in 10% neutral buffered formalin, processed into paraffin blocks, sectioned at 5 µm, and stained with haematoxylin and eosin (H&E). Villus height (from villus tip to crypt opening) and crypt depth (from crypt opening to base) were measured on 10 well-oriented villi per bird using ImageJ 1.54 (NIH, Bethesda, MD) under a light microscope at 40× magnification [12] . The V:C ratio was calculated. Caecal contents from the same birds were collected aseptically and submitted for 16S rRNA gene amplicon sequencing (V3-V4 region) on an Illumina MiSeq platform; reads were processed in QIIME2 and classified to phylum level [13] . Data were analysed by one-way ANOVA with Tukey's HSD in SAS 9.4 (SAS Institute, Cary, NC). Pearson's r was computed between villus height and final body weight across all 24 sampled birds.

Results

Mortality over the 42-day trial was 3.75% (9 of 240 birds), with no significant difference among treatments (T1: 5.0%, T2: 3.75%, T3: 2.50%; p = 0.412). All surviving birds appeared healthy at the time of slaughter. FCR improved by 9.9% in T3 relative to T1 (1.64 vs. 1.82), while T2 showed a 5.5% improvement (1.72). Total feed intake did not differ significantly among groups, indicating that the body-weight advantage in probiotic groups was driven by better nutrient utilisation rather than increased consumption. < 0.01 V:C = villus-height-to-crypt-depth ratio. Superscript letters (a, b, c) denote significant differences among treatments (Tukey HSD, p<0.05) Jejunal morphometry data are presented in Table 2. Villus height was 43.9% greater in T3 (1,182 ± 74 µm) than in T1 (821 ± 91 µm; p<0.001), and 24.3% greater in T2 (1,021 ± 82 µm) versus T1. Crypt depth was modestly reduced in both probiotic groups, resulting in V:C ratios of 6.18 (T3), 5.42 (T2), and 4.31 (T1). Villus width did not differ among treatments (p = 0.318). The morphometric data suggest that probiotic supplementation promoted epithelial renewal and villus elongation in a dose-dependent manner, consistent with the growth performance hierarchy. The higher V:C ratio in probiotic-fed birds points to a shift toward absorptive rather than secretory function in the jejunal epithelium a change that would directly enhance nutrient uptake per unit length of intestine.

Comprehensive interpretation

The combined performance, histological, and microbiological data tell a coherent story: probiotic supplementation at 1.0 g/kg feed improved gut epithelial architecture, shifted caecal microbial composition toward beneficial phyla, and translated these gut-level changes into measurably higher body weight and better feed conversion. The strong villus-height-to-bodyweight correlation provides mechanistic support for the performance gains.

Discussion

The 18.6% body-weight advantage in the high-dose probiotic group aligns closely with the 15-20% range reported by Kabir et al. [14] and Mountzouris et al. [15] in multi-strain probiotic trials under temperate conditions. That a similar magnitude was achieved under the warmer ambient temperatures of Guadalajara (mean house temperature 26-32 °C during the trial) suggests the probiotic organisms tolerated tropical conditions without loss of viability a concern sometimes raised for Lactobacillus-based products [3] . The Bacillus subtilis component, being spore-forming, likely survived heat stress better than vegetative cells and may have contributed disproportionately to the gut-health benefits. The 43.9% increase in jejunal villus height with the high dose exceeds the 20-30% improvements typically documented for single-strain Lactobacillus supplements [4,8] . The additional elongation may be attributable to the yeast fraction (S. cerevisiae), which produces mannan-oligosaccharides and beta-glucans that stimulate goblet cell turnover and crypt-cell proliferation in a complementary fashion to bacterial metabolites [16] . The reduced crypt depth in probiotic groups points to lower cell turnover pressure less need for crypt-cell replacement when villus integrity is better maintained which in turn means more cellular energy is available for absorptive function [17] . The shift in caecal microbiota higher Firmicutes, lower Proteobacteria mirrors patterns reported by Stanley et al. [18] in Australian broilers receiving Bacillus-based direct-fed microbials. Proteobacteria, which include many opportunistic pathogens (Escherichia, Salmonella), are generally considered indicators of gut dysbiosis when present at high proportions [19] . Their suppression in probiotic-fed birds may partly explain the lower mortality trend observed in T3, although the difference did not reach statistical significance with only 240 birds.

Nutrient budget and economic analysis

A simplified nutrient budget illustrates the economic dimension of the probiotic response. Over the 42-day trial, T3 birds consumed an average of 4,237 g of feed to reach 2,584 g body weight, while T1 birds consumed

Conclusion

This research set out to test whether a multi-strain probiotic containing L. acidophilus, B. subtilis, and S. cerevisiae could improve growth performance and gut morphology in Ross 308 broilers under tropical Mexican production conditions, and if so, whether the response was dose-dependent. The answer on both counts was clear. Birds receiving the higher probiotic dose (1.0 g/kg feed) gained 18.6% more body weight by Day 42, converted feed 9.9% more efficiently, and exhibited 43.9% taller jejunal villi and a 43% higher V:C ratio than unsupplemented controls. The lower dose (0.5 g/kg) produced intermediate results across all measured endpoints. Caecal microbiology reinforced the gut-health story, with Firmicutes rising and Proteobacteria falling in a dosedependent manner. The strong correlation between villus height and final body weight (r = 0.84) tied the histological improvements directly to the performance gains, lending biological plausibility to the whole dataset. For Mexican poultry integrators operating without antibiotic growth promoters, the practical message is that multi-strain probiotic inclusion at 1.0 g/kg feed offers a measurable, costeffective performance boost. The nutrient budget analysis showed a return of over 18:1 on the supplement investment a margin wide enough to absorb feed-price fluctuations. The product's spore-forming Bacillus component also ensures survival during feed pelleting, removing a common barrier to on-farm adoption. Future work should address several open questions. Extending the trial through a full production cycle including processing yields (breast meat percentage, abdominal fat pad) would capture value-added metrics that integrators care about beyond live weight. Testing the probiotic under deliberate pathogen challenge Salmonella Enteritidis or Clostridium perfringens would clarify whether the microbiota shift translates into measurable disease resistance. And economic modelling across a range of maize and soybean meal prices would define the break-even probiotic cost at which the supplement ceases to be profitable, giving nutritionists a decision threshold. Lastly, a heat-stress overlay trial during the peak summer months (July-August) would determine whether the probiotic organisms maintain their colonisation advantage when the birds themselves are metabolically compromised by ambient temperatures above 34 °C.

Acknowledgement

AcknowledgementsContributions not qualifying for authorship: Mr. Jorge Sánchez Vega assisted with daily bird management and feed preparation, and Ms. Gabriela Ruiz Delgado helped with histological sectioning and staining.Funding sources: This research was supported by internal funds from the Department of Poultry Science, Guadalajara Research Institute of Animal Sciences. The probiotic product was donated by Nutrición Animal del Bajío, León, Mexico, who had no role in research design, data analysis, or manuscript preparation. Institutional support:The authors thank the Guadalajara Research Institute of Animal Sciences for providing the experimental poultry house and laboratory facilities.

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