Evaluation of Increasing Levels of Acacia mearnsii Tannins on Growth Performance and Intestinal Morphometrics of Broiler Chickens Undergoing a Salmonella Heidelberg Challenge

Abstract

Phytogenic additives such as tannins are characterized as polyphenolic compounds known for their antimicrobial, anti-inflammatory, antioxidant, and immunostimulatory properties that have been used to enhance the performance, intestinal health, and meat quality of broiler chickens. The objective of this experiment was to evaluate the effects of increasing dietary supplementation of tannins from Acacia mearnsii on the intestinal morphometrics, litter moisture, and growth performance of broiler chickens. A total of 1400 Cobb 500 one-day-old male chicks were randomly distributed into five dietary treatments with eight replicates (35 birds/pen) until 42 days of age. The treatments consisted of Salmonella Heidelberg-challenged groups supplemented with 0, 300, 500, 700, or 900 mg/kg tannin from Acacia mearnsii. A four-phase feeding program was used with pre-starter, starter, grower, and finisher feeds. At 3 days of age, birds were orally gavaged with an S. Heidelberg culture. Feed intake, body weight gain (BWG), and feed conversion ratio (FCR) were evaluated until day 42. The morphometry of duodenum, jejunum, and ileum was measured at 7 and 42 days of age. From 1 to 28, 1 to 35, and 1 to 42 days of age, tannin supplementation for broilers under S. Heidelberg challenge led to quadratic increases (p < 0.05) in BWG, with optimal responses at 265, 412, and 456 mg/kg, respectively. No effects of tannin were observed on FCR in all periods. Villus height was similar in all segments on day 7 (p > 0.05); however, on day 42, tannin supplementation that improved villus height of the ileum was 600 mg/kg (p = 0.0100). In conclusion, tannins from Acacia mearnsii were able to improve body weight gain and intestinal morphometry of broiler chickens under an imposed challenge of S. Heidelberg.

1. Introduction

Bacteria from the genus Salmonella spp. (Enterobacteriaceae family) are characterized as short rods, Gram-negative, facultative anaerobes without capsule or spore formation [1]. More than 2600 serotypes of Salmonella are known, and S. enterica subsp. enterica serotype Heidelberg is a paratyphoid serotype that has the ability to infect several hosts, including animals and humans [2]. Although these serotypes seldom induce systemic disease in healthy avian hosts, they can colonize the intestine asymptomatically, contributing to the contamination of animal-derived products and foodborne illnesses in humans.

Salmonella Heidelberg is considered one of the most commonly encountered serotypes in poultry farming worldwide. Among the serovars, S. Heidelberg stands out, particularly in the United States and Canada. In Brazil, it is also an emerging serotype [3,4], described as the third most isolated serotype from swabs collected from commercial poultry houses [5]. In poultry, S. Heidelberg induces moderate pathogenicity, characterized by apathy, drowsiness, diarrhea, and reduced growth performance [6]. Adult broilers and breeders may not exhibit clinical signs of infection, complicating the control of systemic infection as S. Heidelberg can invade muscular tissues, primarily lodging in commercially marketed cuts, or infect eggs through the birds’ reproductive tract [6].

Although the use of antibiotics is still employed prophylactically to control Salmonella spp. in poultry farming, there is a global trend towards reducing or banning this practice, while it can be ineffective in decreasing this pathogen and may also lead to bacterial resistance [7,8,9]. Therefore, there is a constant need to develop and search for more effective methods to control Salmonella, such as disinfectants, biosecurity, vaccines, and the use of feed additives [10,11]. Regarding feed additives, the success of the available commercial products in controlling pathogens depends on their active compounds, mode of action, dose, and frequency of administration. Tannins have been reported as alternatives to reduce bacterial loads [12], to reduce the negative impacts of S. Typhimurium [13], and to improve the intestinal health of broiler chickens [14]. However, no research evaluating the use of tannins in broilers infected with S. Heidelberg has been found in the literature.

Most of the tannins used as feed additives can be obtained from Acacia mearnsii, a plant native to Southeastern Australia and introduced to Europe, Asia, Africa, South America, and North America [15]. In Acacia mearnsii, tannins are predominantly in the condensed form, containing flavan-3,4-diol, and a monomer of the condensed tannins called profisetinidins can be extracted from heartwood. Condensed tannins may contain from two to fifty flavonoid units, with a complex structure that is resistant to hydrolysis but also potentially soluble in aqueous organic solvents, depending on the structure [16]. Thus, studies utilizing tannin products in poultry feeds are needed because the difference between the amount of this additive that may have beneficial effects vs. the amount that will exhibit antinutritional effects for monogastric animals is subtle.

In broiler chickens, tannins have exhibited bacteriostatic and bactericidal effects due to their ability to interact directly with components of the bacterial cell wall and to act on microbial enzymes [12,13]. Additionally, they contribute to improving intestinal health, presenting antimicrobial, anti-inflammatory, and antioxidant functions, as well as their beneficial effects to enhance litter quality by reducing excreta moisture [17]. The hypothesis of this study was that supplemental tannins from Acacia mearnsii might improve the growth performance and intestinal health of broiler chickens challenged with S. Heidelberg. Therefore, the objective of this research was to evaluate the effects of increasing levels of supplemental tannins on growth performance, duodenum, jejunum and ileum morphology, litter moisture, and cecal Salmonella occurrence in broilers undergoing an intestinal challenge with S. Heidelberg.

2. Materials and Methods

The procedures used in the present research were approved (number 12/2021) by the Research and Ethics Committee of Mercolab Laboratory (Cascavel, PR, Brazil).

2.1. Housing, Experimental Feeds, and S. Heidelberg Challenge

A total of 1400 one-day-old male chicks (Cobb 500), vaccinated for Marek’s disease at the hatchery (Globoaves, Garibaldi, RS, Brazil) were distributed into 40-floor pens measuring 2.65 m² in a climate-controlled poultry barn. Tube feeders and nipple drinkers were available in each pen, and the litter was composed of new wood shavings. Broilers had ad libitum access to water and corn-soybean meal feeds provided as mash.

Broilers were distributed in a completely randomized design with 5 experimental diets, 8 replicates, and 35 broilers each. The experimental diets consisted of a challenged control and 4 challenged groups supplemented with increasing levels of tannins from Acacia mearnsii (300, 500, 700, and 900 mg/kg) with a 73.5 g/kg minimum guarantee of condensed tannins (NutreSet PNT, SETA S.A., Estancia Velha, RS, Brazil). The levels of supplemented tannin were set based on the manufacturer’s recommendation (500 mg/kg); however, in order to explore the effects of the product under intestinal challenge conditions, dosages below and above the recommended level were set.

The challenge was carried out with a strain of Salmonella Heidelberg isolated from the field. The bacteria were multiplied in Brain Heart Infusion (BHI) broth for 18 h at 37 °C until it reached a concentration of 1.0 × 10⁹ CFU/mL, and then the dilution procedure was carried out to the desired concentration of 1.0 × 10⁶ CFU/mL. All treatments had birds challenged at 3 days of age with 0.5 mL of S. Heidelberg culture, where 30% of the birds in each pen received an oral gavage and spread to the rest.

The feeding program had four phases: pre-starter (days 1 to 7), starter (days 8 to 21), grower (days 22 to 35), and finisher (days 36 to 42) diets. Feeds were formulated following the broiler genetic management guide [18] and recommendations of the Brazilian broiler industry (

Table 1. Ingredient and nutrient composition of the control diet, as-fed.

Item	Pre-Starter (Day 1 to 7)	Starter (Day 8 to 21)	Grower (Day 22 to 35)	Finisher (Day 36 to 42)
Ingredients, %
Corn	52.00	57.36	66.92	66.08
Soybean meal	40.70	35.29	26.00	26.95
Soybean oil	2.80	3.00	3.50	3.80
Dicalcium phosphate	1.98	1.75	1.16	1.15
Limestone	0.99	0.91	0.77	0.72
Salt	0.47	0.46	0.34	0.34
Sodium bicarbonate	0.01	0.01	0.02	0.02
DL-Methionine, 99%	0.38	0.40	0.58	0.29
Lysine sulphate, 78%	0.27	0.36	0.35	0.31
L-Threonine, 98.5%	0.10	0.16	0.12	0.10
Choline chloride, 60%	0.05	0.05	0.04	0.04
Vitamin and mineral premix 1	0.25	0.25	0.20	0.20
Nutrient and energy composition, % or as shown
ME, kcal/kg	2975	3050	3100	3150
Crude protein	23.10	20.80	19.06	18.17
Ca	0.90	0.84	0.78	0.76
Av. P	0.43	0.40	0.38	0.37
Na	0.21	0.20	0.19	0.18
Choline, mg/kg	1600	1600	1500	1500
Dig. Lys	1.25	1.12	1.02	0.97
Dig. Met + Cys	0.93	0.83	0.75	0.72
Dig. Thr	0.83	0.74	0.67	0.64
Dig. Arg	1.44	1.28	1.15	1.09
Dig. Val	0.96	0.86	0.79	0.75
Dig. Ile	0.90	0.80	0.72	0.68

¹ Composition per kilogram of feed: Vit. A, 9000 IU; Vit. D₃, 2500 IU; Vit. E, 20 IU; Vit. K₃, 2.5 mg; Thiamine, 2 mg; Riboflavin, 6 mg; Pyridoxine, 3.8 mg; Cyanocobalamin, 0.015 mg, Pantothenic acid, 12 mg; Niacin, 35 mg; Folic acid, 1.5 mg; Biotin, 0.1 mg; Fe, 40 mg; Zn, 80 mg; Mn, 80 mg; Cu, 10 mg; I, 0.7 mg; Se, 0.25 mg. Vitamin and mineral premix with salinomycin in the pre-starter and starter diets (55 g/t) and adsorbent (100 g/t).

).

2.2. Growth Performance

Birds’ body weight and feed intake (FI), averaged by pen, were recorded every week. Growth performance evaluated as feed intake, body weight (BW), body weight gain (BWG), and feed conversion ratio (FCR) was presented from 1 to 7, 1 to 14, 1 to 21, 1 to 28, 1 to 35, and 1 to 42 days of age. The FCR was calculated as the average FI of the pen divided by the average BW of the pen.

2.3. Intestinal Measurements

Two samples with approximately 3 cm length of the duodenum, jejunum, and ileum from 1 bird per pen were collected at 7 and 42 days of age for intestinal morphometric analysis. The intestinal lumen was washed with distilled water and kept in 10% buffered formalin solution. One sample from a single bird (1 bird/pen) was selected for staining, and critical regions of the intestinal portions were evaluated by densitometry of sections. The staining method was with myelin, as described by Prophet et al. [19]. At least 4 cross-sections of 5 µm were obtained for each segment. Digital images were captured, and 20 villi and 20 crypts were measured to obtain the villi height and crypt depth in each portion (duodenum, jejunum, and ileum samples at 7 and 42 days of age).

2.4. Litter Moisture and Cecal Content Collection

On days 7, 28, and 42, one representative sample of the litter was collected in each pen and kept at 4 °C until analysis. To determine the litter moisture, each sample was weighed in duplicate (100 g each) and dried in an oven at 105 °C for 16 h [20].

At 28 and 42 days of age, one bird per pen was slaughtered, and the cecum was individually collected and sent for S. Heidelberg identification. To determine the occurrence of S. Heidelberg in each treatment, each bird was classified according to the absence or presence of S. Heidelberg via colony-forming unit counting in the cecal content [21].

2.5. Statistical Analysis

All response variables were subjected to analysis of variance (ANOVA) using the R software version 4.4.0. Shapiro–Wilk and Bartlett tests were used to assess the normality of these data and homogeneity of variance, respectively. Data that did not meet the conditions were transformed, applying the square root and/or the inverse of the square root [22,23]. Results were considered statistically significant at p < 0.05. The effect of broiler diets on the response variables was assessed using linear and quadratic regressions. Models were compared using the Akaike Information Criterion (AIC) and the Bayesian Information Criterion (BIC). All the models were fitted in the “nlme” package [24], and those with the lowest AIC and BIC were used in the ANOVA. Contrasts were used to compare S. Heidelberg’s occurrence between the control diet vs. diets with supplemented tannin products.

3. Results

3.1. Cumulative Growth Performance

Overall averages of BWG, FCR, and FI are presented in the supplementary file Table S1. The effects of increasing levels of tannins on the cumulative BWG of broiler chickens undergoing an S. Heidelberg challenge are presented in Figure 1. Regression equations allowed for the estimation of tannin levels that optimized BWG from 1 to 14, 1 to 28, 1 to 35, and 1 to 42 days of age. The tannin supplementation treatments that maximized BWG from 1 to 28 and 1 to 35 days of age were 265 mg/kg (R² = 0.66; p = 0.0094) and 412 mg/kg (R² = 0.83; p = 0.0224), respectively. In the overall period, 456 mg/kg tannins resulted in the maximum BWG response of broilers (R² = 0.47; p = 0.0042).

Feed intake of broilers under the S. Heidelberg challenge fed with increasing tannin levels is shown in Figure 2. Dietary tannin supplementation did not affect feed intake from 1 to 14, 1 to 21, 1 to 28, and 1 to 35 days of age (p > 0.05). However, in the overall period, the estimation of dietary tannin that increased feed intake was 448 mg/kg (R² = 0.48; p = 0.0127).

Figure 3 presents the results of FCR of S. Heidelberg-challenged broilers fed with increasing dietary supplementation of tannins. In addition to the improvements observed in BWG in most of the cumulative periods, FCR was not affected by the dietary tannin throughout this study.

3.2. Intestinal Measurements

Morphometric measurements of duodenum, jejunum, and ileum of broilers on day 7 are presented in Figure 4. Tannin supplementation led to quadratic increases in the crypt depth of the duodenum at 7 days of age, with the optimized response obtained with 500 mg/kg (R² = 0.66; p = 0.0052). Still, on day 7, no differences were observed in the jejunum and ileum villus heights. Crypt depth was not affected by treatments in all intestinal portions collected from broilers undergoing an S. Heidelberg challenge.

On day 42, no differences were observed in the villus height and crypt depth of the duodenum and jejunum samples (p > 0.05), as shown in Figure 5. However, increasing levels of tannins resulted in improvements in the villus height at 42 days of age. The estimation of dietary tannin that increased villus height on day 42 was 600 mg/kg (R² = 0.95; p = 0.0100).

3.3. Litter Moisture and Cecal Content

Mortality was not affected by either the S. Heidelberg challenge or dietary tannin supplementation throughout this study. No differences in litter moisture were observed in this study (p > 0.05). Overall averages of litter moisture obtained at 7, 28, and 42 days of age were 46.2%, 33.5%, and 29.8%, respectively. At 7 days, litter moisture was 47.5% when broilers were fed the non-supplemented control, whereas 44.9% was observed as an average of all tannin-supplemented groups. At 42 days, 29.9% litter moisture was found in the control diet, while the average litter moisture for the supplemented diets was 29.7%.

The occurrence of S. Heidelberg at 28 and 42 days of age is presented in Figure 6. At 28 days of age, 63% of birds were positive for S. Heidelberg in the non-supplemented control, and the lowest percentage of broilers with S. Heidelberg (38%) was observed with 500 mg/kg tannins. At 42 days, the difference increased between treatments, where the non-supplemented control had 88% of birds with S. Heidelberg, being reduced to 38% occurrence with 300 mg/kg tannins and then reduced to 13% occurrence with 500 mg/kg tannins. The supplementation of 700 and 900 mg/kg tannins resulted in 25% and 50% S. Heidelberg presence, respectively. Contrasts between the non-supplemented control vs. 300, 500, and 700 mg/kg supplemented tannin product indicated significant differences with p-values at 0.0400, 0.0008, and 0.0009, respectively. On the other hand, the non-supplemented control had a similar S. Heidelberg presence to 900 mg/kg tannins (p = 0.1200).

4. Discussion

The prevalence of Salmonella serotypes varies according to different countries, regions, and periods of time. Salmonella Heidelberg is capable of triggering intestinal inflammation, resulting in systemic infection and high fecal excretion, and these effects can cause asymptomatic or symptomatic infections, potentially worsening growth performance [25]. Salmonella Heidelberg primarily affects young birds, where newly hatched chicks infected with S. Heidelberg may exhibit clinical signs of diarrhea, ruffled feathers, and mortality. In some cases, no obvious signs of infection are observed between 5 to 10 days, but the disease may worsen after 7 days of age. A high mortality rate usually occurs during the second or third week, and birds exhibit lethargy and drooping wings [26,27]. However, the epidemiology of Salmonella spp. is complex and involves vertical transmission, triggering the hatching of infected chicks, which may or may not develop the disease.

In the present study, we did not compare challenged vs. unchallenged birds. The expected reduction in the growth performance of broilers under an S. Heidelberg challenge was confirmed by Sausen et al. [28]. The authors evaluated a similar challenge where one-day-old chicks were orally gavaged with S. Heidelberg, also isolated from field samples in Brazil, and observed worse FCR of broilers at 7 and 21 days in the challenged control compared with the non-challenged group. In the present study, all birds were challenged with S. Heidelberg because the negative effects of S. Heidelberg are well-known based on the already mentioned scientific publications and field reports of commercial flocks in Brazil. The effect of S. Heidelberg on the intestinal mucosa depends on the birds’ age, S. Heidelberg infection, and when the infection occurs. This will cause greater or lesser effects on intestinal mucosa, which will also vary according to the portion of the intestinal tract. The intestinal mucosa is the first target site of Salmonella spp. in the host and the first line of defense against pathogens [29].

Salmonella strains isolated in recent years have been reported to present high resistance to antimicrobials [8,9]. The use of disinfectants and biosecurity management are the best means of protection against pathogens [10,11]; however, more recently, feed additives have been applied as alternatives to replace antibiotic growth promoters (AGP) to ameliorate the negative effects of Salmonella spp. infection in broilers and to decrease the occurrence of Salmonella. Tannins and polyphenolic compounds that can precipitate proteins have been considered potential alternatives for AGP mainly because of their antimicrobial effects. Tannins extracted from grape pomace or grape seed are the main condensed tannins evaluated in broiler diets, while chestnut and tannic acid have been studied as main sources of hydrolyzed tannin. Positive results from the use of tannic acid for broilers challenged with S. Typhimurium indicated that the additive promoted antimicrobial and immunostimulatory effects and increased BWG [17]. Godoy et al. [14] demonstrated that tannins from Acacia mearnsii could reduce the negative effects of Clostridium perfringens in broilers, improving growth performance. However, no previous research evaluating the use of tannins in broilers infected with S. Heidelberg has been found in the literature.

Tannins are capable of exerting biologically and pharmacologically relevant activities due to their ability to form complexes with different molecules, such as proteins, carbohydrates, metal ions, bacterial cell membranes, and enzymes [17]. Depending on the tannin dose, type of tannin ingested, and duration of ingestion, studies have highlighted significant antibacterial functions, activity against protozoa and helminths, tissue repair, and enzymatic and protein regulation [17,30]. In an in vitro trial testing quebracho (Schinopsis lorentzii) tannins, the bacteriostatic effect of tannins against S. Enteritidis and S. Gallinarum was reported by Prosdócimo et al. [31]. In addition, Redondo et al. [32], evaluating the same tannins, observed reduced excretion of S. enteritidis in broiler chickens, highlighting its antibacterial effects.

Considering the tested levels, 300 mg/kg of tannins from Acacia mearnsii resulted in a 5% increase in BWG compared with the control diet. Dietary tannin supplementation for broilers under the S. Heidelberg challenge led to quadratic increases in BWG, with optimal supplementation at 265, 412, and 456 mg/kg from 1 to 28, 1 to 35, and 1 to 42 days of age, respectively. Based on a regression model, Godoy et al. [14] observed that low levels of tannins improved growth performance, intestinal permeability, and nutrient digestibility of broilers challenged with C. perfringens. The authors concluded that tannin levels that optimized BWG were 349 and 310 mg/kg from 1 to 21 and 1 to 43 days of age, respectively. On the other hand, the authors observed improvements in the FCR of broilers, with an optimal response at 444 mg/kg tannins.

Tannins are classified as hydrolysable or condensed depending on the composition and plant origin. Condensed tannins or proanthocyanidins are defined as oligomeric or polymeric flavonoids consisting of flavanol units: flavan-3-ols (catechin) or flavan-3,4-diols [16]. Condensed tannins also exhibit a more complex structure compared with hydrolysable tannins [30]. Unlike hydrolysable tannins, condensed tannins are not vulnerable to hydrolysis, which may imply low gastrointestinal tract bioavailability in animals [17]. Tannins demonstrate varying degrees of bioavailability (absorbability) that depend on several factors, including their derivatives, affinity with proteins, molecular structure, and molecular weight [17]. Therefore, high molecular weight condensed tannins are poorly absorbed in the intestine. According to Choi and Kim [17], understanding the bioavailability of tannins is an important characteristic of their functionality and application in diets for monogastric animals. The authors suggested that tannins with low bioavailability potentially have better antimicrobial effects in chickens; however, when it increases, they would be more beneficial as antioxidant and anti-inflammatory agents. This information is more commonly associated with ruminants, with limited information available for poultry.

In the current trial, the digestibility of nutrients was not evaluated. In this context, Godoy et al. [14] found that, in 21-day-old broilers, 466, 316, 294, and 374 mg/kg were the optimal supplementation levels for intestinal integrity and digestibility of dry matter, energy, and protein, respectively. Tannins that optimized the villus height of jejunal samples were 528 and 628 mg/kg at 21 and 43 days of age, respectively, demonstrating that the negative effect of C. perfringens was ameliorated when Acacia mearnsii tannins were supplemented. In the current study, no differences were observed in the villus height measured in the duodenum, jejunum, and ileum on day 7; however, on day 42, tannin supplementation that improved villus height of ileum was 600 mg/kg. As another comparison, Godoy et al. [14] observed linear reductions in litter moisture at 21 and 43 days, collecting five representative samples from each experimental unit at each age. Nonetheless, we did not find differences in litter moisture, but it is already known that the results of litter moisture can be affected by variable factors such as sample collection, environmental conditions, and drinker systems. One possible explanation for this similar result is that only one sample was collected from each pen in our study, increasing variability.

In the present study, the reduced presence of broilers with S. Heidelberg observed through the cecal content evaluation was obtained when 500 mg/kg tannins were supplemented. Since the percentages represent S. Heidelberg’s occurrence, data were not subjected to regression analysis. The reduction in S. Heidelberg counts may help to explain the enhanced BWG and villus height obtained in broiler-fed diets supplemented with increasing levels of tannins. In fact, some other effects of moderate tannin levels have been reported as positive results in intestinal morphology and the modulation of intestinal microbiota [33]. Still, Scalbert [34] andLiu et al. [35] reported more beneficial effects of tannins increasing membrane cell wall permeability in bacteria, reducing substrates that could be required for microbial growth, and presenting antimicrobial activities.

5. Conclusions

In conclusion, tannins from Acacia mearnsii enhanced growth performance and intestinal morphometrics of broiler chickens under an imposed challenge of S. Heidelberg. The optimal tannin supplementation that improved body weight gain from 1 to 42 days of age was at 456 mg/kg, whereas 600 mg/kg was needed to improve ileal villus height, and 500 mg/kg resulted in the lowest presence of S. Heidelberg in cecal samples.