An assessment of Bacillus thuringiensis var. israelensis effects on hematology, intestinal morphology, and testicular growth in chickens

Uma avaliação dos efeitos de Bacillus thuringiensis var. israelensis na hematologia, morfologia intestinal e crescimento testicular em frangos

ABSTRACT

This study aimed to evaluate the effects of dietary supplementation with Bacillus thuringiensis var. israelensis (Bti) on hematological parameters, intestinal morphometry, and testicular growth in chickens. A total of 288 Cobb chicks, comprising both males and females, were included in the study and fed diets supplemented with Bti at a concentration of 1x10⁸ CFU per gram of feed for 42 days. The experimental design consisted of four treatments, each with 12 replicates of six birds, as follows: T1: males without Bti; T2: females without Bti; T3: males with Bti; and T4: females with Bti. Bti supplementation significantly reduced crypt depth and increased the villus-to-crypt ratio (P<0.05). However, other variables, including hematological parameters and testicular growth, were not significantly affected. In conclusion, while Bti supplementation improved certain aspects of intestinal morphology, its effects on hematological parameters and testicular growth were minimal in this study.

Keywords
blood cell count; broilers; growth; health; probiotics

RESUMO

Este estudo teve como objetivo avaliar os efeitos da suplementação da dieta com Bacillus thuringiensis var. israelensis (Bti) na hematologia, morfometria intestinal e crescimento testicular em frangos. Um total de 288 pintos Cobb, incluindo machos e fêmeas, foram incluídos no estudo e alimentados com dietas suplementadas com Bti na concentração de 1x10⁸ UFC por grama de ração durante 42 dias. O delineamento experimental consistiu de quatro tratamentos com 12 repetições de seis aves cada: T1: machos sem Bti; T2: fêmeas sem Bti; T3: machos com Bti; e T4: fêmeas com Bti. A suplementação com Bti reduziu significativamente a profundidade das criptas e aumentou a relação vilos:cripta (P<0,05). Entretanto, outras variáveis, incluindo parâmetros hematológicos e crescimento testicular, não foram significativamente afetados. Em conclusão, embora a suplementação com Bti tenha melhorado certos aspectos da morfologia intestinal, seus efeitos nos parâmetros hematológicos e no crescimento testicular foi limitado neste estudo.

Palavras-chave
contagem de células sanguíneas; frangos; crescimento; saúde; probióticos

1. Introduction

Growing consumer awareness and preference for poultry products free from chemical residues have driven global efforts to identify healthier alternatives to enhance animal health and performance (Wolfenden et al., 2010). Probiotics, defined as live microorganisms that provide health benefits to the host when consumed in adequate amounts, are among these alternatives (Manafi et al., 2018). When added to diets, probiotics can positively modify the microbiota through implantation or colonization (Schrezenmeir & de Vrese, 2001), and in this way, they can prevent diseases and stimulate growth (Coppola & Gil-Turnes, 2004).

Recent research has demonstrated promising effects of probiotic supplementation in broiler diets, including the use of Lactobacillus. The genus Bacillus, a spore-forming, thermotolerant bacterium capable of surviving the gastric barrier, has applications in animal nutrition, dietary supplements, and pharmaceuticals (Cutting, 2011). Its aerobic, spore-forming nature allows it to withstand heat stress and tolerate bile salts, making it an ideal candidate for animal feed (Wu et al., 2011).

Among Bacillus species, Bti is notable for its robust endospore formation, making it one of the hardiest bacteria on Earth (Mandic-Mulec et al., 2015). Primarily used as an entomopathogenic agent in biological pest control, Bti is distinguished by its ability to produce protein crystals during sporulation (Angelo et al., 2010). It accounts for approximately 90% of the global bioinsecticide market (Vilas-Bôas et al., 2007). Despite its extensive use in pest biocontrol, the potential probiotic effects of Bti in broiler chickens remain largely unexplored.

In this context, the present study aimed to evaluate the effects of dietary supplementation with Bti on hematological parameters, intestinal morphometry, and testicular growth in chickens.

2. Material and methods

2.1. Animals and treatments

The experiment was conducted at the poultry facility of the Laboratory of Teaching and Zootechnical Experimentation Prof. Dr. Renato Rodrigues Peixoto – UFPEL, following the principles outlined in the Declaration of Helsinki and approved by the Animal Experimentation Ethics Committee (CEEA) at UFPEL under protocol number 9053. A total of 288 one-day-old Cobb broiler chicks (males and females) were used. The chicks were randomly assigned to four treatments, each with 12 replicates of six birds. Each replicate group was housed in a 1 m² experimental pen with 10 cm high rice husk bedding, with ad libitum access to feed and water for 42 days. Diets were formulated based on corn and soybean meal to meet the nutritional requirements of broiler chickens with regular performance (Rostagno et al., 2011) for the initial (1−20 days), growth (21−35 days), and final (36−42 days) phases, as presented in Table 1. The treatments were as follows: T1: males without Bti; T2: females without Bti; T3: males with Bti (1x10⁸ CFU per gram of feed); and T4: females with Bti (1x10⁸ CFU per gram of feed).

2.2. Production of Bti

The Bti used in this study was obtained from the microorganism collection of the Microbiology Laboratory at the Biotechnology Centre of the Federal University of Pelotas (UFPel). For production, Bti was cultured on Brain Heart Infusion (BHI) agar (Difco, Baltimore, USA) and incubated at 28°C for 24 hours. After growth, isolated colonies were transferred to 200 mL of BHI broth and incubated on an orbital shaker at 200 rpm for 24 hours. The resulting cultures were then inoculated into 4 L of Nutrient Yeast Extract Salt Medium (NYSM medium, Sigma-Aldrich, Saint Louis, USA) and incubated at 28°C with continuous agitation (250 rpm) and aeration (1vvm) for 72 hours. The cells were harvested by centrifugation at 6000 × g for 20 minutes and concentrated into a final volume of 1 L in phosphate-buffered saline (PBS, Sigma-Aldrich, Saint Louis, USA). Bti was then added to the feed at a concentration of 1 x 10⁸ Colony-Forming Units (CFU) per gram of feed, provided daily for 42 days.

2.3. Intestinal histomorphometry

To assess intestinal histomorphometry, 2 cm samples from the distal portion of the duodenal loop were collected from chickens euthanized at 42 days of age and fixed in a 10% formalin solution for 24 hours. The samples were then dehydrated in alcohol solutions with progressively increasing concentrations, culminating in absolute concentration. Following dehydration, the specimens were cleared through the diaphanization process and embedded in paraffin. The paraffin-embedded samples were sectioned into 5-µm-thick slices, with 12 semi-serial cross-sections prepared for each sample. The villus height and crypt depth were measured by capturing optical images using a Nikon Eclipse E200 microscope equipped with a Motican 5 5.0 MP camera and a 4x/0.25 objective lens (Figure 1A). Ten images were captured per replicate, and measurements were performed using Image Pro-Plus 4.5 (IPP4.5) software (Media Cybernetics, Silver Spring, USA, 2002).

Table 1
Nutritional composition and calculated values of the experimental diets.

Ingredient (kg)	Starter Diet* (1−20 days)	Grower Diet** (21−35 days)	Finisher Diet** (36−42 days)
Corn	57.07	61.05	64.41
Soybean meal	38.03	33.51	30.6
Oil	2.5	3.2	3.0
Calcium carbonate	1.40	1.33	1.20
Salt	0.43	0.42	0.39
L-Lisine	0.164	0.169	0.148
DL-Methionine	0.322	0.256	0.192
L-Treonine	0.085	0.065	0.060
Total	100.0	100.0	100.0
Calculated composition
Metabolizable energy (Kcal/Kg)	2980	3070	3100
Crude protein (%)	20.8	19.0	18.0
Calcium (%)	0.95	0.96	0.90
Sodium (%)	0.20	0.19	0.18
Available phosphorus (%)	0.46	0.41	0.41
Digestible Met + Cys (%)	0.95	0.90	0.86
Digestible lysine (%)	1.20	1.05	0.98
Digestible Threonine (%)	0.85	1324	1261

• ^*
  Starter: Choline 7,430 mg;
• Folic acid 127.76 mg; Vitamin E 311.72 IU; Vitamin K3 26.28 mg; Vitamin B2 85.41 mg; Vitamin B6 43.80 mg; Iron 1,800 mg; Zinc 1,667 mg; Vitamin D3 38,180 IU; Vitamin A 172,170 IU; Vitamin B1 26.28 mg; Copper 300 mg; Iodine 20 mg; Nicotinic acid 569.40 mg; Pantothenic acid 255.65 mg; Vitamin B12 222.20 mcg; Manganese 2,100 mg; Selenium 10 mg; Calcium 247 g; Phosphorus 110.57 g.
• ^**
  Grower and finisher: Choline (Min) 4,780 mg;
• Folic acid (Min) 58.56 mg; Vitamin E (Min) 365.85 IU; Vitamin K3 (Min) 25.18 mg; Vitamin B2 (Min) 80 mg; Vitamin B6 (Min) 44.36 mg; Iron (Min) 1,800 mg; Zinc (Min) 1,667 mg; Vitamin D3 (Min) 25,182 IU; Vitamin A (Min) 110,040 IU; Vitamin B1 (Min) 24.27 mg; Copper (Min) 300 mg; Iodine (Min) 20 mg; Nicotinic acid (Min) 482.34 mg; Pantothenic acid (Min) 226.35 mg; Vitamin B12 (Min) 203.85 mcg; Manganese (Min) 2,100 mg; Selenium (Min) 10 mg; Calcium (Min) 221.7 g; Phosphorus (Min) 98.70 g.

2.4. Seminal tubule histomorphometry

At 42 days, the testicles of euthanized chickens were collected, weighed, and pre-fixed in a 4% paraformaldehyde solution for 2 hours. The samples were then sliced into 2-mm-thick sections and further fixed for an additional 12 hours. Following fixation, the tissue was placed in dehydrated cassettes, cleared, and impregnated using an automated tissue processor (ASP 200, Leica Germany) before being embedded in Paraplast Xtra (P3808 − Sigma Aldrich). The embedded samples were sectioned into 5 µm slices using an automated rotary microtome (RM2245 Leica Germany). The sections were mounted on slides and stained with Hematoxylin and Eosin following the protocol described by Carson and Cappellano (2015). Images of the seminiferous tubules, including their diameter and epithelial thickness (Figure 1B), were captured using a biological microscope (BX 51 Olympus) equipped with a cooled digital camera (DP72 Olympus). To assess the seminiferous tubules' mean diameter and epithelial thickness, two slides were prepared for each animal, with ten seminiferous tubules photographed per slide. The smallest diameter and epithelial thickness of each tubule were measured. To standardise the measurement scale, a micrometer ruler was photographed, and a pixel-to-micron conversion was performed using ImageJ software.

Figure 1
Histological micrographs illustrating the measurement of villus height and crypt depth in the intestine (A). CD: crypt depth; VH: villus height. Histological micrographs illustrating the measurement of seminiferous tubules (B). D: diameter; T: thickness.

2.5. Hematological parameters

At 42 days of age, 12 birds per treatment, selected based on body weights representative of their respective groups, were chosen for blood collection. One milliliter of blood was obtained via puncture of the ulnar vein. A blood smear was immediately prepared, stained using rapid panoptic staining, and examined under an optical microscope by a trained technician. The differential white blood cell count was performed by identifying and classifying 200 fixed white blood cells in the smear as heterophils, eosinophils, basophils, lymphocytes, or monocytes.

2.6. Statistical Analysis

The statistical analysis was performed using the following mathematical model: Yijkl = µ + Ai + βj + Aβij + Eijkl, where: µ = overall mean; Ai = probiotic (Bti) effect (i =1,2); βj = sex effect (j = 1,2); Aβij= interaction (probiotic x sex); and Eijkl = random error. The data were analyzed using ANOVA, with Bti and sex as fixed factors, employing the R package emmeans: Estimated Marginal Means (EMMs) (R Core Team, 2020). When a significant F-value was observed, pairwise comparisons of mean values were conducted using the Tukey test, with a significance level set at P<0.05. Before conducting ANOVA, the assumptions of homogeneity of variance and normality of residuals were evaluated. The homogeneity of variance was assessed using Levene's test, while the normality of residuals was verified using the Shapiro-Wilk test.

3. Results and Discussion

Table 2 summarizes the histological analysis findings for the duodenum. No significant differences (P>0.05) were observed in villus length with respect to Bti supplementation or sex. However, Bti supplementation significantly reduced crypt depth (684 µm vs 762 µm; P<0.05). Similarly, Dong et al. (2020) reported that supplementing Ross chickens with Bacillus subtilis BYS2 increased villus height in the duodenum and jejunum and enhanced the villus-to-crypt ratio at week 5 in the treated group. They attributed these effects to reduced crypt depth, which facilitated longer villi, increased villus surface area, and a higher number of absorptive epithelial cells. Crypts serve as the sites of enterocyte proliferation and differentiation, contributing to villus growth through upward cell migration (Uni, 2006).

Table 2
Histological analysis of the duodenum in male and female chickens supplemented with Bacillus thuringiensis var. israelensis (Bti).

Variables	Effect of Bti		Effect of sex		SEM1	P-Value
	Without Bti	With Bti	Male	Female		Bti	Sex	Bti:Sex
Villus Length (µm)	3423	3339	3335	3427	103	0.58	0.53	0.80
Crypt Depth (µm)	762a	684b	729	717	25.2	0.04	0.71	0.14
Villus:Crypt	4.49a	4.88b	4.57	4.77	0.11	0.04	0.28	0.37

• ¹
  SEM: standard error of the mean.
• Means followed by different letters in the same row are statistically different by the Tukey test at 0.05 probability level.

Deeper crypts are often indicative of intestinal challenges such as inflammation, infection, or reduced nutrient absorption capacity. Therefore, increasing villus height and reducing crypt depth is associated with improved nutrient absorption and reduced cell turnover, making the villus-to-crypt ratio a valuable indicator of intestinal health (Arruda et al., 2008). Manafi et al. (2018) reported that probiotic supplementation increased villus height and the villus-to-crypt ratio, which contributed to improved weight gain and efficiency by enhancing food digestion and absorption.

Cell turnover in the intestinal lining involves continuous renewal through mitosis of totipotent cells in the crypts and along the villi (Uni et al., 2000) and cell extrusion at the villus tip. This balance maintains villus size and, consequently, intestinal digestion and absorption capacity. Any imbalance in this process can affect villus height and circumference (Pluske et al., 1997). Probiotics support intestinal health by competing with pathogens for adhesion sites in the mucosa, promoting mucosal restoration, modulating immune responses, and improving villus height, nutrient absorption, and overall performance (Abd El-Hack et al., 2020).

In our study, the villus-to-crypt ratio was higher in the Bti-supplemented group (4.88) compared to the non-supplemented group (4.49), indicating that Bti supplementation helped maintain intestinal integrity by reducing the demand on crypt proliferation. This suggests a probiotic effect of Bti. It is worth noting that the use of probiotics in chicken diets has gained traction in recent years, yielding both positive and variable results. Differences in outcomes are often attributed to variations in probiotic strains, administration methods, dosages, preparation techniques, bird age, diet composition, and hygiene status (Lee et al., 2010; Zhang et al., 2012).

Hematological parameters are summarized in Table 3. Although it was hypothesized that Bti supplementation might enhance key blood cell types and improve immune response, no significant effects (P>0.05) were observed for the experimental factors on blood components or the heterophil-to-lymphocytes ratio.

Table 3
Hematological values of male and female chickens supplemented with Bacillus thuringiensis var. israelensis (Bti).

Variables	Effect of Bti		Effect of sex		SEM1	P-Value
	Without Bti	With Bti	Male	Female		Bti	Sex	Bti:Sex
Heterophils	92.7	85.1	89.4	88.4	2.66	0.05	0.76	0.11
Eosinophils	29.6	31.2	29.1	31.7	2.6	0.67	0.45	0.05
Lymphocytes	76.4	82.4	80.1	78.7	3.4	0.23	0.76	0.71
Monocytes	1.92	1.81	2.1	1.62	0.28	0.90	0.22	0.93
H:L2	1.29	1.10	1.19	1.20	0.08	0.10	0.93	0.63

• ¹
  SEM: standard error of the mean,
• ²
  Heterophils/Lymphocytes ratio.

Of the total blood leukocytes in chickens, 60−65% are lymphocytes, 25−30% are heterophils, 2% are eosinophils, 1.7% are basophils, and 10% are monocytes. These values can vary based on species, environment, sex, age, and hormonal activity, as well as the methods used for blood collection and analysis (Campbell & Joshua Dein, 1984; Meluzzi et al., 1992). Probiotics are often associated with reducing stress in chickens by increasing lymphocyte counts, thereby enhancing resistance to infections (Paryad & Mahmoudi, 2008). However, in this study, lymphocyte values were not significantly affected by Bti supplementation.

Physiological changes in chickens can be assessed through the count of defensive blood cells, with the heterophil-to-lymphocyte (H:L) ratio commonly used as an indicator of prolonged stress (Gross & Siegel, 1983). This ratio is considered a reliable measure for assessing bird welfare in relation to plasma corticosterone concentrations (Macari et al., 2002; Nicol et al., 2009; Prieto & Campo, 2010) and for estimating immune status (Al-Murrani et al., 1997; Patterson & Siegel, 1998). In this study, the H:L ratio fell outside the expected value of 0.5, as suggested by Macari and Luquetti (2002). However, it aligned with findings from Laganá and Ribeiro (2007), who reported H:L ratios greater than 0.62 in chickens exposed to transportation for more than 3 hours and high temperatures.

Significant changes in avian leukocyte numbers often occur in response to infectious processes, making the interpretation of such changes valuable for assessing immune responses (Schmidt et al., 2007). Hematological analysis is essential for early disease detection and underscores the importance of studies that characterize and establish reference values for avian hematological parameters. These reference values can provide critical guidance by accounting for physiological factors such as sex, nutrition, and age, which helps distinguish pathological alterations and enable early, accurate diagnoses.

The results of the seminiferous tubule morphometry are presented in Table 4. No significant effects (P>0.05) of Bti supplementation were observed on testicular weight, seminiferous tubule diameter, or seminiferous epithelium thickness. The onset of spermatogenic activity occurs as early as the 10th hour of incubation, initiated by the differentiation of germ cells. Testicular development and spermatogenesis take place in two distinct phases: the pre-pubertal and pubertal periods. During the pre-pubertal phase, there is intense proliferation of Sertoli cells, with their number per testicle increasing from 1−5 million on the first day of age to over 100 million by 10 weeks. This number remains relatively constant in adulthood, indicating that sperm production capacity is largely determined at an early age (Sauveur & Reviers, 1988).

Table 4
Morphometry (diameter and epithelial thickness) of the seminiferous tubules of chickens supplemented with Bacillus thuringiensis var. israelensis (Bti).

Variables	Treatments
	Without Bti	With Bti	SEM1	P-value
Weight (g)	0.75	0.59	0.11	0.33
Diameter (µm)	184	162	15.6	0.30
Thickness (µm)	53.8	54.4	3.6	0.91

• ¹
  SEM: standard error of the mean.

At birth, the average testicular weight of a one-day-old chick ranges from 3 to 5 mg, with seminiferous tubules comprising approximately 60% of the total testicular volume. The development of seminiferous tubules is driven by the proliferation of Sertoli cells, with testicular weight increasing from 3 to 100 mg by 7 weeks. During this period, the seminiferous tubule diameter doubles from 30 to 60 µm, and subsequently quadruples or quintuples, while the proportion of seminiferous tubules within the testicular volume increases from 60 to 90% (Sauveur & Reviers, 1988).

In adult boars, Colenbrander and Kemp (1990) established a correlation between testicular weight and sperm production. Similarly, studies by Oyeyemi and Okediran (2007) on West African Dwarf bucks and Oyeyemi et al. (2002) on chinchilla rabbits demonstrated that larger testicles tend to produce more sperm than smaller ones. In birds, Moller (1988) reported that testicle weight positively influences ejaculate volume, sperm number, and sperm concentration.

Our results revealed no significant effects of Bti supplementation on testicular weight or seminiferous tubule diameter, suggesting that the supplementation did not influence fertility-related parameters. Berndtson et al. (1987) noted that in young beef bulls, testicles with a higher number of Sertoli cells are larger and tend to produce more sperm compared to those with fewer Sertoli cells.

Tubular measurements are traditionally used as key indicators of spermatogenesis in studies investigating testicular function (Silva Júnior et al., 2006), underscoring the importance of research evaluating these characteristics.

4. Conclusions

This study demonstrated that Bacillus thuringiensis var. israelensis supplementation has some beneficial effects on intestinal morphology, particularly in reducing crypt depth and increasing the villus-to-crypt ratio. The impact of Bacillus thuringiensis var. israelensis supplementation on hematological parameters and testicular morphometry in this study was limited.

These findings suggest that Bti supplementation could enhance nutrient absorption and gut health in poultry, potentially improving feed efficiency and overall performance. Future research should explore the long-term effects of Bti supplementation on production parameters, immune responses, and reproductive performance under diverse farming conditions and across different poultry species.

Acknowledgments

The author, Paola de Freitas Feltrin, acknowledges the support of a Ph.D. scholarship from the CAPES Foundation (Coordination for the Improvement of Higher Education Personnel). The author, Aline Piccini Roll, acknowledges support from CAPES through a Postdoctoral scholarship (PNPD-CAPES).

The authors Fabio Pereira Leivas Leite, Carine Dahl Corcini, Antonio Sergio Varela, Eduardo Gonçalves Xavier, and Victor Fernando Büttow Roll acknowledge the support of grants from the National Council for Scientific and Technological Development - CNPq (CNPq/Produtividade em Pesquisa).

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Edited by

• Editor:
  Sergio Santos de Azevedo

Publication Dates

• Publication in this collection
  21 Mar 2025
• Date of issue
  2025

History

• Received
  03 Sept 2024
• Accepted
  03 Feb 2025