Dietary Saccharomyces cerevisiae Ameliorates the Adverse Effects of Aflatoxin B1 on Growth Performance, Haematological and Biochemical Parameters in Broiler Chickens

Abstract

Aflatoxin contamination of animal feeds may impact broiler chicken health and production. The adverse impact of aflatoxin can be ameliorated and detoxified by adding capable binding agents, such as Saccharomyces cerevisiae. A total of 648 mixed gender 1-day-old Ross-308 were assigned to a 3 × 2 factorial experiment to investigate the effect of aflatoxin B1 (AF) and Saccharomyces cerevisiae (SAC) on growth performance, blood parameters and carcass characteristics. Chickens were randomly allocated to dietary treatments consisting of three levels of AF at 0, 20 and 60 µg/kg, and with or without SAC (10¹⁰ cells/kg) supplementation of 1 g/kg of dried yeast. Results showed that both AF and SAC increased average daily feed intake (both, p < 0.001) and reduced feed efficiency (p < 0.001 and p = 0.035, respectively), while only AF reduced average daily gain (p = 0.009). Supplementation with SAC improved the average daily feed intake in chickens subjected to AF60 (interaction, p < 0.001), suggesting that SAC could improve the appetite of broilers. Chickens fed AF had a lower carcass weight (p = 0.028) and heart weight (p = 0.031), but higher carcass-normalized weight of gizzard (p = 0.038) and liver (p = 0.010). Aflatoxin administration reduced white blood cells (p = 0.030), lymphocytes (p = 0.029) and basophils (p < 0.001), while increasing neutrophils (p = 0.009). SAC reduced neutrophils (p = 0.004) and mean corpuscular haemoglobin (p < 0.001) while increasing lymphocytes (p = 0.003) and basophils (p = 0.015). The haematological results suggest that AF caused a disturbance in the immune system, compromising the health of the chicken, whereas SAC potentially mitigates these alterations. Dietary AF increased the activity of glutamate oxaloacetate transaminase (p = 0.009). These findings suggest a potential use of Saccharomyces cerevisiae as a natural binder to reduce aflatoxicosis in poultry production systems.

1. Introduction

Vietnam’s hot and humid climate favours the proliferation of mycotoxin-producing fungi in agricultural products [1,2]. Mycotoxins are toxic secondary metabolites produced by certain moulds that commonly infest crops. Among all the mycotoxins, aflatoxins B1 (AF) are the most known mycotoxins due to their high toxicity and widespread occurrence in Southeast Asia [3]. Aflatoxins are mainly produced by Aspergillus flavus, Aspergillus parasiticcus, Aspergillus niminus and Aspergillus pseudotamarii [3,4]. Aflatoxin might contaminate a variety of foods, such as corn, peanuts and other agricultural products. Aflatoxin B1 contamination in poultry diets impairs growth performance and physiological health [3,5]. Dietary AF had negative effects on growth performance, antioxidant capacity, blood biochemistry and liver metabolism in broiler chickens [6]. Aflatoxin also affects the metabolism of carbohydrates, proteins, lipids and vitamins and induces the generation of excess reactive oxygen species [7,8].

Currently, some substances can absorb toxins when added to food and act in the animal’s digestive tract. Substances capable of absorbing toxins include activated carbon [9] and hydrated calcium sodium aluminosilicate [10]. Biological methods using microorganisms against fungi have been applied in practice. Previous studies have reported the use of the cell wall and (1-3)-β-D-Glucan of Baker’s yeast (Saccharomyces cerevisiae, SAC) to bind aflatoxin B1 [11]. The Mannan component in Mannan–oligosaccharides in the cell wall of the yeast SAC can combine with aflatoxin and reduce the effects of the toxin when added to poultry feed. Previous studies demonstrate that dietary supplementation with SAC improved growth performance and gut health of broiler chicken, as evidenced by upregulation of tight junction protein genes and downregulation of immunomodulatory gene [12]. Furthermore, dietary SAC improves bacterial diversity in the cecum of broilers by increasing the abundance of beneficial bacteria while reducing the harmful bacteria [12]. Therefore, in this study, we investigated the ability of yeast (SAC) to bind AF in feed and the effect of AFB1 on the growth performance, carcass traits and blood biochemistry of broiler chicken.

2. Materials and Methods

2.1. Animals and Experimental Design

All the care and uses of animals in this study were approved by the Animal Ethics Committee of the Faculty of Animal Science, Vietnam National University of Agriculture (Protocol number: T2022-02-03TĐ).

A total of 648 1-day-old male and female broiler chickens (Ross 308) were obtained from a commercial hatchery and transported to the experimental site (Trau Quy, Gia Lam, Hanoi, 12406, Vietnam). The chicks were individually weighed, leg-tagged and allocated to 18 pens with 36 chicks/pen (18 males + 18 females). The chicks were assigned to a 3 × 2 factorial experiment, three levels of AF (0, 20 and 60 µg/kg as AF0, AF20 and AF60, respectively) and with or without supplementation of SAC (10¹⁰ cells/kg) (represented as CON and SAC, respectively). The combination of AF and SAC generated six dietary treatments. The chicks were randomly allocated to one of the six treatments, with three replicates of 36 chicks, as mentioned earlier. The base diets were formulated according to a three-phase feeding program as follows: starters (days 1 to 14), growers (days 15 to 28) and finishers (days 29 to 42). The nutritional compositions of base diets are presented in

Table 1. Nutritional compositions of the base diets (as fresh as-fed basis).

Ingredient, g/kg	Starter	Grower	Finisher
Corn	495	484	526
Cassava	50	100	100
DDGS corn	60	60	60
Palm oil	21	24.7	24.3
Limestone	10.65	9.00	9.05
Soybean meal	226	221	180
Meat and bone meal 45%	20	30	30
Extruded soybean	90	50	50
Monocalcium phosphate	5.3	1	0
Salt	2.3	2.2	2.2
Methionine 98%	2.9	2.9	3.2
Threonine 98%	0.9	1	1.6
Lysine HCL 98.5%	3.1	3,3	4.6
Tryptophan	0.1	0.15	0.4
Choline chloride 60%	1	1	1
Premix *	11.75	9.75	7.65
Total Batch	1000	1000	1000
Calculated nutrient levels
Crude protein (min, %)	21.0	19.0	18.0
Moisture (max, %)	13.0	13.0	13.0
Lysine (min, %)	1.1	1.0	0.9
Met + Cys (min, %)	0.8	0.7	0.6
ME (Kcal/kg)	3000	3000	3100
Phosphorous (min-max, %)	0.6–1.0	0.6–1.0	0.6–1.0
Calcium (min-max, %)	0.5–2.5	0.5–2.5	0.5–2.5
Crude fibre (max, %)	4.0	4.0	4.5

* Supplied per kg of diet: Vitamin A: 13,700 IU; Vitamin D₃: 3500 IU; Vitamin E: 55 mg; Vitamin K₃: 2.5 mg; Vitamin B₁: 2.5 mg; Vitamin B₂: 5.0 mg; Vitamin B₃ (Niacin): 40 mg; Vitamin B₅ (Pantothenic acid): 12.5 mg; Vitamin B₆: 4.0 mg; Vitamin B₁₂: 0.02 mg; Biotin: 0.2 mg; Folic acid: 1.0 mg; Zinc: 70 mg; Iron: 60 mg; Copper: 5 mg; Manganese: 95 mg; Iodine: 0.3 mg; Selenium: 0.3 mg; Cobalt: 0.15 mg; ME: Metabolizable Energy; Met: Methionine; Cys: Cysteine.

.

2.1.1. AF Production and Diet Preparation

Aflatoxins were produced via fermentation of corn by Aspergillus parasiticus VTCC 31,543 (VNU Institute of Microbiology and Biotechnology, Hanoi, Vietnam). The fungus was cultured on potato dextrose agar and incubated at 28–30 °C for 7 days. It was then inoculated onto sterilized moist corn (60–70% moisture) and incubated under the same conditions. Humidity and temperature were monitored daily. After 7 days, the mould was harvested, dried at 50 °C and ground into a fine powder. The aflatoxins levels in the corn powder were measured by HPLC. The milled substrate was added to the basal diet to provide three levels of aflatoxins: 0, 20 and 60 µg/kg of feed. The concentrations of aflatoxins in each diet were confirmed by HPLC.

2.1.2. Saccharomyces cerevisiae Culture

Saccharomyces cerevisiae var. boulardii VTCC 22,083 was obtained from the VNU Institute of Microbiology and Biotechnology, Hanoi, Vietnam. The strain was stored in YPD medium (HiMedia, Wagle Industrial Estate, Maharashtra, India) supplemented with 25% glycerol at –40 °C. For activation, it was cultured on YPD agar plates at 30 °C for 24–48 h, then transferred to liquid YPD medium and incubated at 30 °C for 48 h on a rotary shaker at 150× g. Yeast biomass was harvested by centrifugation at 3000× g for 30 min and washed twice with sterile water. Cell concentration was determined using the plate count method with an automatic colony counter (SphereFlash^®, IUL, Barcelona, Spain). The final biomass was adjusted to a concentration of 10¹⁰ cells/g. After drying at <40 °C to a moisture content below 12%, the active dry yeast was incorporated into the experimental diet at a rate of 1 g/kg of feed.

2.2. Data Collection and Measurements

The chickens had free access to feed and water throughout the experimental period. Body weight, average daily feed intake (ADFI) and feed consumption ratio (FCR) were recorded weekly for 6 weeks. Average daily gain (ADG) of each bird was calculated as the difference in body weight over each period. ADFI was calculated on pen basis, by monitoring the feed supplied and number of refusals in each period. Similarly, FCR was determined on a pen basis as a ratio of total feed intake to total weight gain.

At 42 days of age, two chickens (1 male + 1 female) from each replicate were randomly selected to measure body weight. Blood samples were collected by vein puncture. Blood from each bird (4 mL) was divided between EDTA-coated glass tubes and heparinized tubes. Blood samples were collected in EDTA-coated glass tubes to determine the haematocrit (Hct) haemoglobin (Hb), red blood cell (RBC) and white blood cell (WBC) count analysis. The WBCs and RBCs were manually counted in chambers of a haemocytometer using a microscope. Another set of blood samples was gathered in heparinized tubes. The plasma was obtained by blood centrifugation in glass tubes (3000× g for 10 min at room temperature) and stored at −20 °C for further analysis. After blood collection, chickens were weighed and slaughtered using the approved method. The weight of carcass, heart, liver, thigh and breast, liver and abdominal fat weights were measured and relative weight to the total body weight of broiler chickens was determined. Breast and abdominal relatives were determined as a percentage of carcass weight.

Plasma samples were analysed individually for total protein, albumin, creatinine, glucose, urea and activities of Glutamate Pyruvate Transaminase (GPT) and Glutamate Oxaloacetate Transaminase (GOT) using a plasma biochemical analyser as per the instruction in the manufacturer’s kit (BS 120 Chemistry analyser).

2.3. Statistical Analysis

This study consisted of a 3 × 2 factorial experiment, so data were analysed for the main effects of AF (AF0, AF20 and AF60), SAC (CON vs. SAC) and the interaction between AF and SAC using SAS Version.9. Differences among treatment means were analysed using Duncan’s Multiple Range Test. In the text, the results were expressed as presented means and standard error of the difference (SED) for the pooled main effects of AF (AF0, AF20 and AF60) and SAC (CON vs. SAC), while in tables, data were presented as adjusted means and SED for the full interactions (AF × SAC). Differences were considered significant if the p-value ≤ 0.05, while a trend was considered if the p-value ≤ 0.10. Fisher’s least significant difference test was used post hoc to compare multiple means, with alphabetical superscripts used to identify different groups. For the main effects of AF, differences among AF0, AF20 and AF60 are denoted by uppercase superscript letters (A–C), whereas interactions between AF and SAC are denoted by lowercase superscript letters (a–c).

3. Results

3.1. Growth Performance

During 0–21 days of age, ADFI was not affected by AF, whereas it was increased by SAC (53.0 vs. 53.8 g/day, for CON vs. SAC, respectively, p = 0.009,

Table 2. Effects of dietary aflatoxin (AF) and Saccharomyces cerevisiae (SAC) on growth performance of broiler chickens.

Parameter	CON 1			SAC 1			SED	p-Value			AF 2			SAC 3
	AF0	AF20	AF60	AF0	AF20	AF60		AF	SAC	AF × SAC	AF0	AF20	AF60	CON	SAC
0–21 days
ADFI, g/day	53.6 bc	53.1 ab	52.4 a	53.5 bc	54.1 c	53.9 bc	0.46	0.33	0.009	0.085	53.6	53.6	53.1	53.0	53.8
ADG, g/day	34.1 d	26.4 abc	25.6 a	27.4 c	26.7 bc	25.7 ab	0.72	<0.001	<0.001	<0.001	30.7 A	26.6 B	25.5 C	28.6	26.6
FCR	1.57 a	2.01 b	2.08 b	1.96 b	2.03 b	2.10 b	0.088	<0.001	0.016	0.020	1.77 A	2.02 B	2.09 B	1.89	2.03
21–42 days
ADFI, g/day	122 b	125 e	123 c	120 a	124 d	132 f	0.12	<0.001	<0.001	<0.001	121 A	125 B	127 C	123	125
ADG, g/day	70.4	73.6	69.1	69.4	73.7	71.1	2.17	0.053	0.76	0.63	70.0 A	73.7 B	70.1 A	71.0	71.4
FCR	1.74	1.70	1.78	1.73	1.69	1.85	0.054	0.027	0.54	0.47	1.74 AB	1.70 A	1.82 B	1.74	1.76
1–42 days
ADFI, g/day	94.1 b	96.0 c	94.2 b	92.9 a	95.5 c	100 d	0.25	<0.001	<0.001	<0.001	93.5 A	95.8 B	97.1 C	94.8	96.2
ADG, g/day	53.5 c	51.3 bc	48.4 a	49.6 ab	51.4 bc	49.6 ab	1.07	0.009	0.21	0.014	51.5 A	51.3 A	49.0 B	51.0	50.2
FCR	1.76	1.87	1.95	1.87	1.86	2.02	0.041	<0.001	0.035	0.13	1.82 A	1.87 A	1.99 B	1.86	1.92

ADFI: average daily feed intake; ADG: average daily gain; FCR: feed conversion ratio; ¹ the interactions between AF and SAC; ² the main effect of AF; ³ the main effect of SAC; SED: standard error of the difference for the interaction between AF × SAC. ^{a–f, A–C} Differing superscripts denote groups p < 0.05.

). However, there was an indication of interaction between AF and SAC (p = 0.085) such that SAC increased ADFI in birds subjected to AF20 and AF60, but the differences were not observed in the AF0 group. Average daily weight gain was decreased by inclusion with AF and the decrease was more significant when the dose of AF increased (30.7 ^A, 26.6 ^B and 25.5 ^C g/day for AF0, AF20 and AF60, respectively, p < 0.001). The growth rate was also reduced for SAC (28.6 vs. 26.6 g/day, for CON vs. SAC, respectively, p < 0.001, Table 2) and the reduction was greater when SAC combined with AF (interaction, p < 0.001). FCR was increased for AF (1.77 ^A, 2.02 ^B and 2.09 ^B kg feed/kg weight gain AF0, AF20 and AF60, respectively, p < 0.001, Table 2) and for SAC (1.89 vs. 2.03 kg feed/kg weight gain, p = 0.016, Table 2). However, there was an interaction (p = 0.020) such that addition with AF increased FCR in the chicken-fed CON diet, but the differences were not observed in the SAC group (Table 2).

In the period of 21–42 days of age, ADFI was increased linearly for AF (121 ^A, 125 ^B and 127 ^C g/day, for AF0, AF20 and AF60, respectively, p < 0.001, Table 2) and SAC (123 vs. 125 g/day, for CON vs. SAC, respectively, p < 0.001, Table 2). There was an interaction (p < 0.001), whereas SAC increased ADFI in chicken-fed diet with AF60, while it reduced ADFI in those subjected to both AF0 and AF20 (Table 2), but the differences were not observed in the SAC group (Table 2). Average daily weight gain tended to be increased in response to AF (70.0 ^A, 73.7 ^B and 70.1 ^A g/day for AF0, AF20 and AF60, respectively, p = 0.053, Table 2), whereas it was not impacted by SAC or interaction. The feed conversion ratio was increased when the higher level of AF was added to the diet (1.74 ^AB, 1.70 ^A and 1.82 ^B kg feed/kg weight gain for AF0, AF20 and AF60, respectively, p = 0.027, Table 2) while it was not influenced by the main effect of SAC or interaction.

For the duration of the experiment, ADFI was increased linearly for AF (93.5 ^A, 95.8 ^B and 97.1 ^C g/day for AF0, AF20 and AF60, respectively, p < 0.001, Table 2) and SAC (94.8 vs. 96.2 g/day, p < 0.001, Table 2). There was an interaction between AF and SAC (p < 0.001) such that the improvement in ADFI was observed in chickens fed AF60 and SAC but not in other groups. Dietary AF reduced ADG (51.5 ^A, 51.3 ^A and 49.0 ^B g/day, for AF0, AF20 and AF60, respectively, p = 0.009, Table 2), whereas SAC did not influence this parameter. However, there was an interaction (p = 0.014) in which dietary SAC reduced ADG in chickens given AF0, but no differences were observed in other groups. FCR was increased for AF (1.82 ^A, 1.87 ^A and 1.99 ^B kg feed/kg weight gain for AF0, AF20 and AF60, respectively, p < 0.001, Table 2) and SAC (1.86 vs. 1.92, p = 0.035, Table 2) but no interactive effects were observed.

3.2. Haematological Properties

Although there were no main effects of AF and SAC, there was an indication of an interaction (p = 0.069) such that birds fed dietary supplementation with SAC exhibited lower blood haematocrit in combination with AF0 administration but not in AF20 and AF60 (

Table 3. Effects of aflatoxin (AF) and Saccharomyces cerevisiae (SAC) on haematological properties of broiler chickens.

Parameter	CON 1			SAC 1			SED	p-Value			AF 2			SAC 3
	AF0	AF20	AF60	AF0	AF20	AF60		AF	SAC	AF × SAC	AF0	AF20	AF60	CON	SAC
Hwt (%)	27.5 b	25.5 ab	26.4 b	21.7 a	25.7 ab	27.8 b	2.21	0.30	0.30	0.069	24.6	25.6	27.1	26.4	25.1
WBC (109/L)	23.6 b	16.5 a	13.3 a	13.3 a	24.2 b	15.7 a	2.90	0.030	0.98	0.001	18.5 A	20.4 B	14.5 AB	17.8	17.8
MCV (fL)	98.8 d	91.1 ab	93.1 bc	86.9 a	92.3 bc	96.7 cd	2.33	0.17	0.095	<0.001	92.8	91.7	94.9	94.3	92.0
MCH (pg)	44.0 c	39.3 a	40.2 ab	38.8 a	41.5 b	40.4 ab	0.85	0.17	0.058	<0.001	41.4	40.4	40.3	41.2 A	40.2 B
LYM (109/L)	14.70 c	4.00 a	9.38 b	9.01 b	10.93 bc	12.10 bc	2.19	0.029	0.32	0.003	11.80 A	7.43 B	10,73 A	9.34 A	10.62 B
BASO (109/L)	1.062 c	0.135 a	0.063 a	0.925 bc	0.658 b	0.548 b	0.187	<0.001	0.015	0.039	0.994 A	0.396 B	0.305 B	0.420 A	0.710 B

Hwt: haematocrit; WBC: white blood cell; MCV: mean corpuscular volume; MCH: mean corpuscular haemoglobin; LYM: lymphocyte; BASO: basophil; ¹ the interactions between AF and SAC; ² the main effect of AF; ³ the main effect of SAC; SED: standard error of the difference for the interaction between AF × SAC. ^{a–d, A,B} Differing superscripts denote groups p < 0.05.

). White blood cells were increased for AF (18.5 ^A, 20.4 ^B and 14.5 ^AB 10⁹/L for AF0, Af20 and AF60, respectively, p = 0.030, Table 3), but they were unchanged with SAC. Mean corpuscular volume (MCV) was not affected by dietary AF, but it tended to be decreased with SAC (94.3 vs. 92.0, p = 0.095 fL, Table 3, Figure 1a). However, there was an interaction (p < 0.001) such that while MCV decreased in birds fed dietary AF exclusion of SAC, it was only improved in those given SAC and AF60 (Table 3, Figure 1a). Dietary AF did not impact MCH, while SAC tended to decrease this parameter (41.2 vs. 40.2 pg, p = 0.058, Table 3). However, there was an interaction between AF and SAC (p < 0.001) such that supplementation with SAC improved MCH in chicken fed AF20 but not in AF0 or AF60 (Table 3, Figure 1b). Lymphocyte (LYM) was decreased by AF inclusion (11.8 ^A, 7.43 ^B and 10.7 ^A 10⁹/L for AF0, AF20 and AF60, respectively, p = 0.029, Table 3,) but it was not affected by SAC. However, there was an interaction between AF and SAC (p = 0.003) such that supplementation with SAC improved LYM in birds fed AF20 and AF60 but not AF0 (Table 3, Figure 1c). Basophil (BASO) was decreased by AF (0.994 ^A, 0.396 ^B and 0.305 ^B 10⁹/L for AF0, AF20 and AF60, respectively, p < 0.001), whereas it was increased in response to SAC (0.420 vs. 0.710 10⁹/L, p = 0.015, Table 3). There was an interaction between AF and SAC (p = 0.039) such that supplementation with SAC improved BASO in birds administered with AF20 and AF60, but the improvement was not observed in the AF0 group (Table 3, Figure 1d). Red blood cells tended to be decreased by dietary AF (2.65 ^A, 2.87 ^AB and 3.08 ^B 10¹²/L for AF0, AF20 and AF60, respectively, p = 0.062,

Table 4. Effects of aflatoxin (AF) and Saccharomyces cerevisiae (SAC) on haematological properties of broiler chickens.

Parameter	CON 1			SAC 1			SED	p-Value			AF 2			SAC 3
	AF0	AF20	AF60	AF0	AF20	AF60		AF	SAC	AF × SAC	AF0	AF20	AF60	CON	SAC
Hb (g/L)	118	101	110	103	105	107	10.6	0.59	0.47	0.46	111	103	109	110	105
RBC (1012/L)	2.79	2.92	3.09	2.51	2.82	3.08	0.24	0.062	0.37	0.72	2.65 A	2.87 AB	3.08 B	2.93	2.80
RDW (%)	12.1	12.0	12.3	12.1	12.2	12.3	0.47	0.78	0.86	0.97	12.1	12.1	12.3	12.1	12.2
PLT (109/L)	5.04	4.78	5.59	5.14	5.70	7.03	0.84	0.106	0.107	0.53	5.09	5.24	6.31	5.14	5.96
NEU (109/L)	7.80	16.2	15.1	4.90	11.1	6.16	2.96	0.009	0.004	0.37	6.4 A	13.6 B	10.6 AB	13.0	7.1
MONO (109/L)	0.400	0.461	0.445	0.338	0.506	0.395	0.1184	0.41	0.75	0.78	0.369	0.484	0.420	0.435	0.413
MCHC (g/L)	442	435	438	463	420	401	18.60	0.054	0.35	0.105	453 A	428 AB	420 B	439	428
EOS (109/L)	0.048	0.053	0.034	0.034	0.038	0.036	0.014	0.57	0.28	0.67	0.041	0.046	0.035	0.045	0.036

Hb: haemoglobin; RBC: red blood cell; RDW: red cell distribution width; PLT: platelet count; NEU: neutrophil; MONO: monocyte; MCHC: mean corpuscular haemoglobin concentration; EOS: eosinophil; ¹ the interactions between AF and SAC; ² the main effect of AF; ³ the main effect of SAC; SED: standard error of the difference for the interaction between AF × SAC. ^A,B Differing superscripts denote groups p < 0.05.

), while they were not affected by SAC or interactions. Blood neutrophils (NEU) were decreased in response to dietary AF (6.35 ^A, 13.6 ^B and 10.6 ^AB 10⁹/L for AF0, AF20 and AF60, respectively, p = 0.009) and SAC (13.0 vs. 7.39 10⁹/L for CON vs. SAC, respectively, p = 0.004) but no interaction was observed (Table 4). Mean corpuscular haemoglobin concentration (MCHC) was decreased in response to AF (453 ^A, 428 ^AB and 420 ^B for AF0, AF20 and AF60 g/L, respectively, p = 0.054, Table 3), but it was not influenced by SAC or interaction. There were no main or interactive effects on haemoglobin, red cell distribution width (RDW), platelet count (PLT), monocyte (MONO), or eosinophil (EOS) (p > 0.05 for all, Table 4).

3.3. Blood Biochemistry

Dietary AF significantly increased the blood level of GOT (3875 ^A, 9899 ^B and 9706 ^B, for AF0, AF20 and AF60, respectively, p = 0.009,

Table 5. Effects of dietary aflatoxin (AF) and Saccharomyces cerevisiae (SAC) on blood biochemistry of broiler chickens.

Parameter	CON 1			SAC 1			SED	p-Value			AF 2			SAC 3
	AF0	AF20	AF60	AF0	AF20	AF60		AF	SAC	AF × SAC	AF0	AF20	AF60	CON	SAC
Protein (g/L)	34.8	29.5	27.8	28.1	31.3	26.5	5.15	0.48	0.49	0.51	31.5	30.4	27.1	30.7	28.6
Albumin (g/L)	8.43	7.67	6.84	7.09	8.17	6.94	1.344	0.52	0.75	0.61	7.76	7.92	6.89	7.65	7.40
Creatine (µmol/L)	1946	11,875	12,314	6803	7500	8243	5128	0.23	0.69	0.37	4375	9687	10,278	8711	7515
Urea (mmol/L)	0.675	0.678	0.620	0.568	0.370	0.553	0.1443	0.63	0.069	0.47	0.621	0.524	0.586	0.658	0.497
Glucose (mmol/L)	13.5	10.7	11.7	12.3	11.6	10.8	2.01	0.40	0.71	0.72	12.9	11.2	11.3	12.0	11.6
GOT (U/L)	2016	10,143	11,918	5733	9654	7494	2732	0.009	0.80	0.14	3875 A	9899 B	9706 B	8026	7627
GPT (U/L)	4.45	2.98	3.51	3.97	3.26	3.54	0.72	0.13	0.89	0.75	4.21	3.12	3.52	3.65	3.59

GOT: glutamate oxaloacetate transaminase; GPT: glutamate pyruvate transaminase; ¹ the interactions between AF and SAC; ² the main effect of AF; ³ the main effect of SAC; SED: standard error of the difference for the interaction between AF × SAC. ^A,B Differing superscripts denote groups p < 0.05.

, Figure 2), but no main effects of SAC or interaction were observed. Blood urea tended to decrease with SAC (0.658 vs. 0.497, p = 0.069, Table 5), while it was not impacted by AF or interaction. There were no main effects of either AF or SAC or interaction blood levels of protein, albumin, creatine, glucose and GPT (p > 0.05 for all, Table 5).

3.4. Carcass Characteristics

Dietary AF reduced the live weight of broiler chickens (2604 ^A, 2416 ^AB and 2305 ^B g/bird for AF0, AF20 and AF60, respectively, p = 0.033,

Table 6. Effects of dietary aflatoxin (AF) and Saccharomyces cerevisiae (SAC) on carcass characteristic of broiler chickens.

Parameter	CON 1			SAC 1			SED	p-Value			AF 2			SAC 3
	AF0	AF20	AF60	AF0	AF20	AF60		AF	SAC	AF × SAC	AF0	AF20	AF60	CON	SAC
Live weight (g)	2633	2378	2290	2575	2455	2320	148	0.033	0.85	0.81	2604 A	2416 AB	2305 B	2433	2540
Carcass (g)	2029	1767	1600	1859	1777	1722	123	0.028	0.65	0.39	1944 A	1772 AB	1691 B	1819	1786
Carcass (%)	77.1 b	74.5 ab	72.4 a	72.1 a	72.3 a	74.2 ab	1.65	0.47	0.083	0.028	74.6	73.4	73.3	74.6	72.9
Thigh (g)	228	204	183	201	175	181	19.5	0.078	0.11	0.58	214 A	190 AB	182 B	205	186
Thigh (%)	11.2	11.5	11.0	10.8	9.92	10.5	0.777	0.85	0.080	0.47	11.0	10.7	10.8	11.3	10.4
Breast (g)	266	247	204	253	217	216	28.8	0.074	0.55	0.58	260 A	232 AB	210 B	238	229
Breast (%)	13.1	14.0	12.3	13.5	12.2	12.6	1.30	0.63	0.62	0.40	13.3	13.1	12.5	13.1	12.8
Fat (g)	15.1	14.6	16.6	16.6	18.3	20.7	4.65	0.68	0.26	0.92	15.8	16.5	18.6	15.4	18.5
Fat (%)	0.734	0.841	1.002	0.868	1.021	1.180	0.2203	0.20	0.21	0.99	0.801	0.931	1.091	0.859	1.023
Gizzard (g)	37.8	42.1	38.1	36.2	41.6	38.4	4.54	0.31	0.83	0.96	37.0	41.8	38.2	39.3	38.7
Gizzard (%)	1.86	2.36	2.31	1.97	2.37	2.23	0.24	0.038	0.92	0.86	1.91 A	2.36 B	2.27 AB	2.17	2.19
Heart (g)	10.60	11.91	8.85	12.03	9.37	9.05	1.18	0.031	0.65	0.083	11.31 A	10.60 AB	8.95 B	10.5	10.1
Heart (%)	0.522 a	0.670 c	0.536 ab	0.654 bc	0.528 ab	0.526 ab	0.0618	0.28	0.84	0.020	0.588	0.599	0.531	0.576	0.569
Liver (g)	59.6	63.7	62.2	59.6	72.1	55.9	6.83	0.15	0.87	0.33	59.6	67.9	59.0	61.9	62.5
Liver (%)	2.94	3.59	3.80	3.21	4.06	3.20	0.307	0.010	0.79	0.055	3.08 A	3.83 B	3.50 AB	3.44	3.49
Spleen (g)	3.15	2.90	30.5	2.88	2.18	2.83	0.559	0.45	0.22	0.79	3.01	2.54	2.94	3.03	2.63
Spleen (%)	0.155	0.165	0.185	0.154	0.125	0.165	0.0312	0.39	0.278	0.68	0.155	0.145	0.175	0.168	0.148

¹ The interactions between AF and SAC; ² the main effect of AF; ³ the main effect of SAC; SED: standard error of the difference for the interaction between AF × SAC. ^{a–c, A,B} Differing superscripts denote groups p < 0.05.

) while no main effects of SAC or interaction were observed. Similarly, carcass weight was reduced by AF (1944 ^A, 1772 ^AB, 1691 ^B g/bird for AF0, AF20 and AF60, respectively, p = 0.028, Table 6), whereas it was not affected by SAC or interaction. Carcass percentage was not impacted by AF, but it tended to be reduced with SAC supplementation (74.6 vs. 72.9 %, p = 0.083, Table 6). However, there was an interaction between AF and SAC (p = 0.028) such that dietary SAC reduced carcass percentage in chickens fed AF0, but the differences were not observed in other groups. Thigh weight tended to be lowered by AF (214 ^A, 190 ^AB and 182 ^B g/bird for AF0, AF20 and AF60, respectively, p = 0.078, Table 6), whereas it was not affected by SAC or interactions. Thigh percentage was not impacted by AF, but it tended to be reduced by SAC (11.3 vs. 10.4%, p = 0.080, Table 6). Feeding AF tended to reduce breast meat (260 ^A, 232 ^AB and 210 ^B g/bird for AF0, AF20 and AF60, respectively, p = 0.074, Table 6) while there were no effects of SAC or interaction. Dietary inclusion of AF increased the percentage of gizzard of broiler chicken (1.91 ^A, 2.36 ^B and 2.27 ^AB % for AF0, AF20 and AF60, respectively, p = 0.038, Table 6), but no effects of SAC or interaction were observed. Broilers’ heart weight was reduced in response to dietary AF (11.31 ^A, 10.60 ^AB and 8.95 ^B g for AF0, AF20 and AF60, respectively, p = 0.031, Table 6), but it was unaffected by SAC. However, there was an indication of an interaction between AF and SAC (p = 0.083), such that supplementation with SAC decreased heart weights of chickens subjected to AF20 but not in other groups. The normalization of heart weight based on carcass was not influenced by either AF or SAC (p > 0.05 for both), but there was an interaction (p = 0.020) such that SAC supplementation resulted in the differences in heart percentage in broiler chicken given AF0 and AF20 only. Dietary AF increased the percentage of the liver (3.08 ^A, 3.83 ^B and 3.50 ^AB % for AF0, AF20 and AF60, respectively, p = 0.010, Table 6) while SAC did not impact this parameter. There was an indication of an interaction (p = 0.055) such that SAC tended to increase the percentage of the heart of chicken in AF20, but the differences were not observed in other groups.

4. Discussion

The key findings of this study were that feeding a diet including AF increased GOT activity while SAC tended to decrease blood urea. Additionally, AF increased carcass-relative liver weight, suggesting that AF compromised liver functions. Aflatoxin is known as a potent liver toxin [13]; it can be absorbed by enterocytes and travel through the bloodstream. The negative effects of AF on growth performance and meat quality of broiler chicken are well reported [6,14,15]. It has been reported that AF can negatively impact performance by reducing feed intake and compromising intestinal barrier integrity, including digestive and absorptive functions, which leads to poor growth rates and reduced feed efficiency [6,16,17]. Furthermore, AF may induce oxidative stress, impair immunity and trigger inflammatory responses, which affects carcass characteristics and quality of poultry meat [18,19]. Consistent with previous studies, the growth performance of broiler chicken in this study was reduced by AF, evidenced by lower ADFI and increased FCR throughout the experiment, although overall, ADFI was increased with AF. While the mechanism of improvement in feed intake remains unknown, the compromised growth performance may reflect health issues, demonstrated by increased liver enzyme activity and alteration in haematological profile. Additionally, the impacts of AF become worse in the early life of a chick (in the first 21 days), indicating that young birds are more susceptible to AF than older ones [20]. However, the effects of SAC were unexpected when it was not able to ameliorate the negative influence of AF on productive traits; regardless of that, it improved the ADFI of broiler chickens. This could be due to the fact that SAC mostly has positive effects on the intestine by improving the immune response and regulating the normal gut microbiota [21].

The changes in haematological properties have been associated with health issues or abnormalities in haematologic organs [22]. In this study, the increase in WBC coupled with a reduction in NEUs, LYMs and BASOs in chickens subjected to AF diets suggests a disturbance in the immune system. Aflatoxin is known as an immunotoxic agent that can cause various immune system disorders [23]. In pigs, aflatoxin contamination can disrupt intestinal barrier function by reducing the expression of tight junction proteins, such as ZO-1, claudin-1 and occluding, and promoting cell apoptosis [24]. Aflatoxin exposure induces intestinal oxidative stress and triggers an inflammatory response by modulating the NF-kβ pathway [25,26]. In the current study, the increase in WBC could be an indicator of inflammation and the reduction in immune cells like NEUs, LYMs and BASOs indicates an abnormal immunity caused by AF, affecting the resistance of chickens to infection and mortality. Interestingly, dietary SAC improved MCV, MCH, LYMs and BASOs, suggesting that SAC potentially mitigates haematological alterations induced by AF. These findings are supported by [27,28,29], who reported that diet supplementation with SAC improves haematological parameters, immunity, antioxidant capacity and health status of animals.

The contamination of AF in animal feed increases the risk of diseases associated with liver function, such as cirrhosis and cancer [30], possibly by disrupting the structure and function of hepatic mitochondria [31]. As a metabolic factory of the body, the liver plays a vital role in breaking down and metabolizing toxic metabolites or substances, including aflatoxin [32]. GOT is an enzyme mainly produced by liver cells and plays a crucial role in regulating the metabolism of amino acids and the urea cycle. When liver cells are damaged or injured, they release liver enzymes into the bloodstream, resulting in an elevation of liver enzyme levels. The increased GOT by AF in this study indicates that AF caused liver damage, leading to the elevation of GOT activity. The use of SAC is considered a method to reduce adverse effects of AF due to its binding ability [15,33]. In this experiment, SAC did not appear to ameliorate the elevated activity of GOT, suggesting that the SAC was unable to prevent liver damage induced by aflatoxin. However, the reduced blood urea suggests that SAC improves nitrogen metabolism and utilization. Although previous studies showed that dietary SAC had no effects on nitrogen intake, output and retention in pigs [34,35] and cattle [36], the lower blood urea of chickens fed SAC is supported by [37,38] who reported that yeast culture (SAC) supplementation improved feed efficiency and reduced blood urea concentration. The lower blood urea may also suggest that SAC supports the function of the liver and kidney; the organs play a vital role in metabolizing and excretion of urea to maintain the concentration of this metabolite within the normal range [39,40].

5. Conclusions

Dietary inclusion AF negatively affected growth performance, haematological profiles and carcass traits in broiler chicken. Supplementation with SAC appeared to ameliorate adverse impacts of AF by improving appetite and ameliorating alteration of haematological parameters induced by AF. Saccharomyces cerevisiae could be a beneficial feed additive for improving the health and productivity of broilers, especially under conditions of high risk of AF contamination.