Dietary α-galactosidase and xylanase to improve the nutritional value of corn-soybean-rapeseed meal diets in broiler chickens

ABSTRACT

Two experiments were performed to assess the effects of an enzyme preparation (containing α-galactosidase and xylanase) on chicken performance, carcass traits, footpad dermatitis score, and nutrient digestibility. During first experiment, chickens were fed four diets: (1) control diet (PC): corn and soybean-rapeseed meal; (2) PC supplemented with 350 ppm of the enzyme; (3) Negative control (NC): PC with a 4% reduction of apparent metabolizable energy (AME) and crude protein content; (4) NC supplemented with 500 ppm of the enzyme. In the second experiment, chickens were fed PC or PC + enzyme 350 ppm and a digestibility balance was performed. The enzyme supplementation at 350 and 500 ppm significantly improved the overall feed conversion ratio in PC and NC diet, respectively (P < 0.01). Regarding footpad dermatitis score, animals fed NC + enzyme 500 ppm tended to present a lower value than animals fed NC (P < 0.1). Concerning the digestibility balance, PC + enzyme 350 ppm diet presented a higher feed AME and AMEn value than PC diet (P < 0.05). The enzyme also improved the apparent ileal digestibility (AID) of proline (P < 0.05) and tended to increase AID of cysteine (P < 0.1). In conclusion, the enzyme preparation improves birds feed efficiency by increasing energy utilization.

KEYWORDS:

• alpha-galactosidase
• xylanase
• soybean meal
• raffinose
• stachyose
• rapeseed meal

Introduction

Feed production represents the most important cost in the broiler chicken industry, comprising more than 60–70% of the total production costs. Furthermore, the current global context is highly influenced by the non-stability of raw material prices and stocks due to phenomena caused by global warming or armed conflicts (FAO 2023). In fact, soybeans used for soybean meal (SBM) production, one of the most common ingredients included in broiler chicken feed as a source of protein and energy, has experienced an important price increase in recent years (Comparison among Corn, Soybeans and Wheat prices (CLAL.it)). In Europe, the foreign reliance reduction in soybean is essential for keeping the poultry sector competitive, with national initiatives for increasing internal availability by maximizing the nutrient content and profitability of feed formulas, as well as by including local and alternative raw materials such as rapeseed meal (RSM).

Regarding the optimization of feed formulas, it is essential to consider the antinutritional factors (ANF) present in each raw material that are responsible for reducing nutrients digestion and absorption (Cowieson et al. 2010; Slominski 2011). Soybean meal and RSM present important contents of non starch polysaccharides (NSP), as does the raffinose family of oligosaccharides (RFOs), a group of soluble carbohydrates that includes raffinose and stachyose, and they are indigestible due to the lack of endogenous enzymes that degrade them in poultry species (Aftab 2009; Pettersson and Pontoppidan 2013). It has been proven that RFOs are indigestible in the upper intestinal tracts of broiler chickens, causing flatulence and intestinal discomfort due to enteric fermentations, while lowering energy utilization and nutrient digestibility (Coon et al. 1990; Choct et al. 2010). The use of specific carbohydrases, such an α-galactosidase, allows for RFO hydrolysis on their α-(1→6)-glycosidic linkages into galactose and sucrose, increasing the available energetic content of SBM and RSM for the animals and reducing their negative effects on the gastrointestinal tract (Brenes et al. 1993; Zhang et al. 2010). On the other hand, cereal grains, such as corn and wheat, contain arabinoxylans, which are responsible for increasing digesta viscosity and altering gastrointestinal microbiota, thereby impairing nutrient digestibility (González-Ortiz et al. 2016; Kim et al. 2022). In this case, the routine inclusion of exogenous xylanases, which hydrolyze the 1,4-b-D-xylosidic linkage between xylose residues (Mendis et al. 2016), has been demonstrated to be useful for improving nutrient digestibility in broiler chickens (Morgan et al. 2021).

In recent years, the available literature referring to the negative effects of NSPs, which are present in wheat and other cereals, and the benefits of adding exogenous xylanase in broiler chicken feed to improve performance has grown. Although several works focused on the effects of α-galactosidases in broiler feeding have been published, particularly its effects in SBM based diets, conclusions may be inconsistent (Waldroup et al. 2006; Amer et al. 2020) and there is a lack of published reports assessing the effect of this enzyme in SBM-RSM based diets. Furthermore, due to NSP variability, the literature suggests that it is desirable to combine two or more NSPases to achieve a wider range of effects on different ANFs instead of including a single enzyme to hydrolyze NSPs (Munyaka et al. 2016; Woyengo et al. 2019). Therefore, the aim of the present experiments was to assess the effects of combining α-galactosidase and xylanase on broiler chicken feed based on corn-SBM-RSM and its impacts on performance, carcass traits, and energy and nutrient utilization (crude protein and amino acids).

Materials and methods

The experiments were performed in accordance with the animal welfare standards stated in the Ethical Principles and Guidelines for the Use of Animals for Scientific Purposes from the National Research Council of Thailand (National Research Council of Thailand 1999). Both experiments were reviewed and approved by the Bangkok Animal Research Center (protocol number AB18447A, November 2018).

Experimental diets, animal husbandry, and sampling

Regarding temperature, light, and humidity, the birds were maintained following the specifications in the Ross 308 lineage management handbook (Aviagen 2014a). All birds were vaccinated for Newcastle and infectious bronchitis diseases at 7 days (d) of age and for Gumboro disease at 14 d of age.

Experiment 1. Performance parameters, carcass traits, and footpad scores

A total of 320 newly hatched Ross 308 male broiler chickens were used in a 35-d study. The animals were obtained from a commercial hatchery, weighed, and randomly distributed to 4 experimental treatments, with 8 replicates (pens) and 10 birds per replicate. The replicates were allocated for a homogeneous distribution of treatments and started with a similar body weight (BW) mean. Each pen consisted of 1 m² (using rice hull as the bedding material) and equipped with a tubular feeder and three nipple water drinkers. Feed and water were provided ad libitum throughout the trial.

The feeding programme was divided in three phases: starter (from 0 to 14 d), grower (from 15 to 28 d), and finisher (from 29 to 35 d). A practical diet, used as the positive control (PC) diet, was formulated to meet the Aviagen nutrient requirements for broiler chickens (Aviagen 2014b), either with or without enzyme supplementation at 350 ppm (CAPSOZYME® SB PLUS; Industrial Técnica Pecuaria, S.A., Barcelona, Spain). The enzyme preparation used in the present trials has been approved as a zootechnical additive in the European Union in the category of digestibility enhancers (Regulation EU 2022/1470). The enzyme had a 1,6-α-galactosidase activity of 40,000 GALU/kg (1 GALU is the amount of enzyme capable of degrading 1 micromole/min of para-nitrophenyl-alpha-D-galactopyranoside at a pH of 5.5 and 37°C) and an endo-1,4-b-xylanase activity of 50,000 AXC/kg (1 AXC is the amount of enzyme which liberates 0.058 micromoles/min of xylose equivalents from a wheat arabinoxylan substrate at a pH of 4.7 and 30 °C). A negative control (NC) diet was formulated with a 4% restriction in both metabolizable energy (∼120 kcal/kg) and crude protein (CP) in comparison to the PC diet. In the case of the NC diet, the enzyme preparation was added at 500 ppm. All experimental diets were based on corn-SBM-RSM, and the composition and calculated nutrient contents of the diets for each feeding phase are presented in Table 1. All diets were produced with a conditioning temperature of 80°C and a pellet size of 3 mm in diameter. During the first 14 d, feed was provided to birds in crumble form, and thereafter, it was provided in pellet form until the end of the trial.

Table 1. Ingredients and calculated compositions of the positive control (PC) and negative control (NC) basal diets used in experiments 1 and 2 (only grower phases) on an as-fed basis.

	Starter phase (0–14 d)		Grower phase (15–28 d)		Finisher phase (29–35 d)
Ingredient (%)	PC	NC	PC	NC	PC	NC
Corn (CP 7.5%)	50.25	55.56	51.57	56.98	54.28	59.40
SBM dehulled (CP 48.5%)	34.32	31.60	30.12	27.18	24.83	22.17
Rapeseed meal (CP 38%)	4.00	4.00	5.00	5.00	6.00	6.00
Rice bran, S.E (CP 18.3%)	3.00	3.00	4.00	4.00	5.00	5.00
Soybean oil (ME 8,800)	3.55	0.97	4.99	2.32	6.20	3.57
A MDCP (16.6% Ca/22.0% P)	1.98	1.99	1.72	1.74	1.48	1.49
Limestone (38.9% Ca)	1.18	1.20	1.07	1.09	0.97	0.98
Salt	0.24	0.21	0.26	0.23	0.30	0.27
Sepiolite	0.30	0.30	0.30	0.30	0.30	0.30
Vitamin/mineral premix^a	0.20	0.20	0.20	0.20	0.20	0.20
Dl-Methionine	0.33	0.36	0.28	0.30	0.19	0.21
L-Lysine HCl	0.23	0.31	0.16	0.25	0.06	0.14
L-Threonine	0.15	0.18	0.11	0.14	0.03	0.07
Sodium bicarbonate	0.19	0.23	0.15	0.20	0.10	0.13
Salinomycin 12%	0.05	0.05	0.05	0.05	0.05	0.05
Choline chloride 60%	0.03	0.04	0.02	0.02	0.01	0.02
Calculated composition:
ME for poultry (kcal/kg)	2,955	2,835	3,055	2,930	3,150	3,025
Crude protein (%)	23.0	22.1	21.5	20.6	19.5	18.7
Dig. lysine for poultry (%)	1.28	1.28	1.15	1.15	0.96	0.96
Dig. methionine for poultry (%)	0.64	0.65	0.57	0.58	0.47	0.48
Dig. M + C for poultry (%)	0.95	0.95	0.87	0.87	0.75	0.75
Calcium (%)	0.96	0.96	0.87	0.87	0.78	0.78
Phosphorus (available) (%)	0.48	0.48	0.44	0.44	0.39	0.39

^a Supplied per kg of diet: vitamin A/D3, 12,000,000 IU; vitamin E, 20 mg; vitamin K, 2.45 mg; vitamin B1, 1.9 mg; vitamin B2, 4.99 mg; vitamin B6, 1.94 mg; vitamin B12, 0.02 mg; niacin, 49 mg; cal-D-pan, 14.78 mg; biotin 0.05 mg; folic acid, 0.98 mg; copper, 9 mg; ferrous, 38.75 mg; manga-nese, 60 mg; zinc, 45 mg; iodine, 0.75 mg; selenium, 1 mg; and antioxidant, 2.5 mg.

The individual weights of the animals and the feed intake per pen (10 birds) were recorded at 14, 28, and 35 days of age. The data were used to calculate the average daily gain (ADG), the average daily feed intake (ADFI), and the feed conversion ratio (FCR) within each period (0–14 d, 15–28 d, and 29–35 d) and in the overall study (0–35 d). Dead and culled birds were recorded daily to adjust the ADFI and ADG. At the end of the experiment, 2 birds from each pen with BWs close to the pen mean were slaughtered for carcass yield measurements (dressing yield, breast meat, thigh, and abdominal fat). Internal organs and gut tracts were collected for weighing (with and without contents), and small intestinal samples from each intestinal section (duodenum, jejunum, and ileum) were obtained. Each intestinal section was weighed, and their lengths were measured. Footpad dermatitis was measured macroscopically by using a scoring range from 0 to 2 [0 = smooth, no lesions, discoloured papillae, and no ulcers; 1 = minor to larger discolouration and superficial lesions; and 2 = severe lesions with scabs or ulcerations (Global Animal Partnership)].

Experiment 2. Digestibility balance

In experiment 2, a total of 96 newly hatched Ross 308 male broiler chickens, provided by a commercial hatchery, were fed with the same starter and grower diets formulated in experiment 1 (Table 1). In the case of the grower diets (15–28 d), chromic oxide was added at 0.3% as an indigestible marker. Then, at day 24, the animals were weighed and randomly distributed to 2 experimental treatments, with 6 replicates (metabolic cages) and 8 birds in each cage. The replicates were allocated for a homogeneous distribution of treatments within the barn and started with a similar BW mean. Each metabolic cage had a surface area of 0.45 m × 0.60 m and was equipped with a feeder and a nipple drinker, and the housing had an air conditioner to maintain the room temperature and humidity, following the Ross 308 lineage management handbook (Aviagen 2014a). The experimental treatments consisted of the PC grower phase diet formulated in experiment 1, either with or without the enzyme supplementation at 350 ppm. The assessment of the enzyme addition on energy and nutrient utilization was performed over a 4 d collection period in a digestibility balance between days 24 and 28 of the trial. During the collection period, all feeders were removed, the amount of test diet in each feeder was recorded, and all the feeders were put back on. This was considered as the starting point of the excreta collection period. During the 96-hour collection period, the BWs, ADGs, and ADFIs of the animals in each cage were recorded. Furthermore, all wet excreta from each cage were collected daily, cleaned of contaminants, homogenized, freeze-dried, ground, and kept at 4 °C for further analysis.

On the other hand, to assess the effects of the enzyme preparation on CP and amino acid (AA) utilization, at the end of the experiment (28 d), all birds were sacrificed for ileal digesta collection. The ileal digesta contents (from Meckel’s diverticulum to a point 1 cm proximal to the ileocecal junction) obtained from the birds in each cage were pooled, freeze dried, ground, and kept at 4°C for further analysis.

Laboratory analyses

Samples of the experimental diets from both trials were taken at the beginning and end of each experimental period, and they were ground and kept at 4°C for further analysis. The experimental feed samples (experiments 1 and 2), dried excreta, and ileal contents (experiment 2) were analyzed according to the methods of AOAC International (AOAC 2005): dry matter (method 934.01) and CP (method 968.06), besides gross energy (GE) contents were determined by adiabatic bomb calorimeter (Leco model AC-350, isoperibol method). The inert marker (chromic oxide) was analyzed in feed and excreta/ileal samples following the methodology described in Czarnocki et al. (1961).

The amino acid contents in the feed and the ileal contents were determined with a Hitachi Amino Acid Analyzer (Tokyo, Japan) using ninhydrin for the post-column derivatization and norleucine as the internal standard. The samples were hydrolyzed with 6 N HCl for 24 h at 110 °C before analysis (AOAC 982.30). The methionine and cysteine were quantified after oxidizing the samples with performic acid hydrolysis (AOAC 994.12), whereas an alkaline hydrolysis was carried out for tryptophan, followed by reverse phase HPLC (AOAC 988.15).

Calculations and statistical analysis

The apparent ileal digestibility (AID) of the nutrients evaluated was calculated using the following equation: $\mathrm{AID}\left(\text{\%}\right)=100\ast \left\{\left[{\left(\mathrm{Cr}/\mathrm{N}\right)}_{\mathrm{d}}\text{–}{\left(\mathrm{Cr}/\mathrm{N}\right)}_{\mathrm{i}}\right]/{\left(\mathrm{Cr}/\mathrm{N}\right)}_{\mathrm{d}}\right\},$

where (Cr/N)_d is the concentration of the inert marker (chromic oxide) in the corresponding nutrient in the diet and (Cr/N)_i is the concentration of the inert marker in the corresponding nutrient in the pooled ileal content of each replicate. The apparent metabolizable energy (AME) of the experimental diets and its nitrogen corrected equivalent (AMEn) were calculated as follows: $\mathrm{AME}\left(\mathrm{kcal}/\mathrm{kg}\right)=\left(\mathrm{GE}\mathrm{intake}\text{–}\mathrm{GE}\mathrm{outtake}\right)/\mathrm{feed}\mathrm{intake}\mathrm{and}$ $\mathrm{AMEn}\left(\mathrm{kcal}/\mathrm{kg}\right)=\left\{\right(\mathrm{GE}\mathrm{intake}\text{–}\mathrm{GE}\mathrm{outtake}\left)\text{–}\right(\mathrm{NR}\ast \mathrm{K})/\mathrm{feed}\mathrm{intake},$where the NR is the nitrogen retention, which was assumed to be 20% of the body weight gain/6.25, and K is the constant which is equal to 8.21 kcal/g of nitrogen retention.

The cage or pen means were used as the experimental units in the performance parameters, carcass yields, footpad scores, and the digestibility balances for both trials. A Shapiro – Wilk test indicated a normal distribution of the data. The data were analyzed by one-way ANOVA using R Statistics (version 4.1.0; R Core Team, Vienna, Austria), with treatment as the main factor and a residual standard error to estimate the average deviation of any point from the statistical model. Duncan’s multiple range test was performed to determine whether the means were significantly different (P ≤ 0.05) or tended to be different (0.05 < P < 0.1).

Results

The effects of the dietary enzyme supplementation on the performance parameters of the chickens are shown in Table 2, and the effects of the enzyme addition on the carcass yields, organ weights, intestinal segment lengths, and footpad dermatitis scores are presented in Table 3. Regarding the performance parameters, the energy and protein reductions in the NC diet caused a significant increase in FCR in the starter (0–14 d) and grower (14–28 d) phases and during the overall trial (0–35 d) in comparison to the animals fed the PC diet (P < 0.001). On the other hand, the enzyme supplementation at 350 ppm with the PC diet significantly improved the FCR at the end of the 35 d study (P < 0.001). Likewise, the enzyme supplementation at 500 ppm with the NC diet significantly improved the FCR in the starter phase and for the global period regarding the NC treatment (P < 0.001). Nevertheless, no significant effects were associated with the nutrient reductions (PC vs. NC) or the enzyme supplementation on the animals’ BW and ADFI (P > 0.05). With respect to the carcass yield parameters and intestinal segment lengths, no effects were associated with the nutrient restriction content of the diets (P > 0.05; PC vs. NC) and the enzyme supplementation (P > 0.05), except for the relative weights of the jejunums and ileums, where the animals fed the PC + enzyme 500 ppm tended to have increased measurements (P < 0.1). Furthermore, the enzyme addition in the PC and NC diets tended to reduce the relative weights of the abdominal fat pads in relation to the carcass weights, and it tended to reduce the footpad scores of the animals fed the NC diet (P < 0.1). Finally, significant reductions in the relative weights of the kidneys in relation to the carcass weights were observed in the animals fed the NC + enzyme 500 ppm diet with respect to the NC diet (P < 0.05), although this was not different from the PC diet.

Table 2. Growth performance of the broiler chickens fed with the experimental diets in experiment 1.

Item	Treatments				RSE	P-value
	PC	PC + Enzyme 350 ppm	NC	NC + Enzyme 500 ppm
From 0 to 14 days (starter phase)
BW at 0 days (g)	40.7	40.7	40.7	40.7	0.32	0.998
BW at 14 days (g)	459	465	453	461	17.84	0.588
ADG (g/bird/day)	29.9	30.3	29.4	30.1	1.27	0.580
ADFI (g/bird/day)	33.9	34.3	34.5	34.8	1.52	0.746
FCR	1.137^A	1.134^A	1.170^C	1.154^B	0.0127	<0.001
From 14 to 28 days (grower phase)
BW at 28 days (g)	1,722	1,732	1,694	1,727	52.54	0.491
ADG (g/bird/day)	90.2	90.5	88.7	90.4	3.08	0.612
ADFI (g/bird/day)	121.4	120.8	124.1	124.9	3.90	0.118
FCR	1.346^A	1.335^A	1.400^B	1.383^B	0.0207	<0.001
From 28 to 35 days (finisher phase)
BW at 35 days (g)	2,560	2,562	2,503	2,550	56.04	0.141
ADG (g/bird/day)	119.7	118.6	115.5	117.6	3.91	0.195
ADFI (g/bird/day)	193.3	189.0	189.1	190.9	5.97	0.436
FCR	1.615	1.593	1.635	1.624	0.0381	0.172
From 0 to 35 days (global results)
ADG (g/bird/day)	72.0	72.0	70.3	71.7	1.60	0.140
ADFI (g/bird/day)	100.7	99.8	101.2	102.0	2.09	0.251
FCR	1.400^B	1.385^A	1.438^D	1.423^C	0.0145	<0.001
Mortality (%)	2.50	1.25	1.25	1.25	4.677	0.933

PC, positive control; NC, negative control; RSE, residual standard error; BW, body weight; ADG, average daily gain; ADFI, average daily feed intake; FCR, feed conversion ratio. ^{A – D} indicate that the values within the same row with no common superscripts were significantly different, P ≤ 0.05 (Duncan’s test).

Table 3. Carcass yield measurements, organ weights, intestinal segment lengths, and footpad dermatitis scores of the broiler chickens fed the experimental diets in experiment 1.

Item	Treatments				RSE	P-value
	PC	PC + Enzyme 350 ppm	NC	NC + Enzyme 500 ppm
Dressing^1 (%)	75.04	75.09	75.33	75.64	0.767	0.401
Breast^2 (%)	30.64	31.40	31.57	31.52	1.291	0.454
Thigh^2,3 (%)	18.37	18.13	17.92	18.15	0.505	0.399
Fat pad^2 (%)	1.76^ab	1.57^b	1.87^a	1.59^ab	0.264	0.089
Kidney^2 (%)	0.66^AB	0.69^A	0.69^A	0.63^B	0.045	0.045
Liver^2 (%)	2.21	2.24	2.32	2.11	0.183	0.166
Gizzard^2 (%)	1.13	1.14	1.17	1.15	0.141	0.960
Proventriculus^2 (%)	0.31	0.32	0.31	0.31	0.043	0.970
Duodenum^2 (%)	0.58	0.43	0.40	0.40	0.221	0.296
Jejunum^2 (%)	0.79^b	0.90^a	0.84^ab	0.80^b	0.085	0.061
Ileum^2 (%)	0.59^ab	0.67^a	0.58^b	0.57^b	0.086	0.098
Duodenum length (cm)	31.7	32.4	29.9	30.9	2.35	0.212
Jejunum length (cm)	80.4	82.9	80.3	80.6	5.68	0.763
Ileum length (cm)	86.1	90.5	86.1	86.1	6.00	0.373
Footpad score^4	23.4^a	25.0^a	50.0^b	37.5^ab	22.02	0.077

PC, positive control; NC, negative control; RSE, residual standard error. ^{A – B} indicate that the values within the same row with no common superscripts were significantly different, P ≤ 0.05 (Duncan’s test). ^{a – b} indicate that the values within the same row with no common superscripts tended to be different, 0.05 < P < 0.1 (Duncan’s test).¹ indicates dressing as a percentage of live weight (without head, neck, and feet). ² indicates relative weight as a percentage of carcass weight. ³ indicates thigh with bone. ⁴ indicates that foot pad dermatitis was calculated as: [(0 × the total number of birds with score 0) + (0.5 × the total number of birds with score 1) + (2 × the total number of birds with score 2)/total number of scored birds].

The effects of the inclusion of the enzyme preparation in the PC diet on the energy utilization and the CP and AA ileal utilization are shown in Table 4. The results extracted from the digestibility balance indicated that the enzyme supplementation at 350 ppm significantly improved the AME and AMEn values of the feed (P < 0.05) at 28 and 24 kcal/kg, respectively; therefore, a significant improvement was observed even if the factual AME was 43 kcal/kg higher than the calculated composition (Table 1). Numerical improvements were observed for the digestibility of all AAs analyzed, with an overall improvement of 3.26%, although no significant differences were detected other than for proline (P < 0.05) and a tendency for cysteine (P < 0.1).

Table 4. Energy, crude protein, and amino acid utilization of the broiler chickens fed with the experimental diets during the digestibility balance phase, as well as the performance parameters in experiment 2 (24–28 d).

	Treatments
Item	PC	PC + Enzyme 350 ppm	RSE	P-value
AME ‘as fed’ (kcal/kg)	3,098	3,126	19.2	0.034
AMEn ‘as fed’ (kcal/kg)	2,918	2,942	18.0	0.042
Crude protein (%)	70.33	72.08	2.335	0.223
Total AA (%):	61.55	64.81	0.157	0.202
Lys (%)	74.76	77.03	2.554	0.154
Met (%)	65.00	68.03	4.139	0.234
Cys (%)	63.49	66.79	2.842	0.072
Thr (%)	59.06	62.38	4.532	0.233
Arg (%)	58.05	61.55	6.541	0.376
Ile (%)	59.78	63.03	5.450	0.326
Leu (%)	59.89	63.58	4.808	0.213
Phe (%)	60.58	63.39	4.959	0.350
His (%)	58.55	62.83	4.560	0.135
Ala (%)	58.90	62.83	5.503	0.244
Tyr (%)	59.40	62.08	4.749	0.352
Pro (%)	59.46	63.56	3.047	0.042
Trp (%)	69.02	71.05	4.421	0.446
Performance parameters (24–28 d):
BW at 24 days (g)	1,288	1,302	14.7	0.126
BW at 28 days (g)	1,663	1,694	22.2	0.036
ADG (g/bird/day)	93.8	98.0	4.12	0.111
ADFI (g/bird/day)	136.6	140.3	3.04	0.066
FCR	1.460	1.433	0.0581	0.443
Mortality (%)	0.00	0.00	–	–

PC, positive control; RSE, residual standard error; BW, body weight; ADG, average daily gain; ADFI, average daily feed intake.

Discussion

Regarding the experimental diets, due to the matrix application that considered the enzymes supplementation, a reduction in soybean oil inclusion was performed on the NC diet to optimize the formula’s cost. However, under commercial conditions, the energetic reduction should consider the minimum required inclusion of added fats to ensure the proper physical quality of the pellets and the correct pigmentation of the animals by facilitating carotenoid pigment absorption, which is a fat-soluble molecule, in the small intestine. On the other hand, RSM inclusion responds to the necessity of using alternative protein ingredients for broiler chicken feed formulations. However, it is well known that RSM contains glucosinolates (Tripathi and Mishra 2007) and other ANFs such as RFO and NSP, with negative effects on performance (Mikulski et al. 2012). The rapeseed meal levels in the starter and grower diets were used according to FEDNA recommendations (FEDNA), with a maximum inclusion level of 5.0% for young chickens (0–18 d of life). In the case of the finisher phase of experiment 1, the RSM was increased only one point (i.e. up to 6.0%) to avoid possible negative effects on carcass quality, as described in (Leeson and Summers 1976; Mikulski et al. 2012; FEDNA). Despite the abovementioned restrictions, recent published studies have demonstrated that RSM may represent an efficient alternative to SBM for broiler chicken feed if its inclusion is complemented by a suitable formulation strategy (Wu et al. 2022; Wiśniewska et al. 2023).

The results observed in experiment 1 demonstrated that the inclusion of an α-galactosidase and xylanase preparation in corn-SBM-RSM-based diets (PC and NC) improved the overall FCR regarding their respective non-supplemented treatments (−10.7% and – 10.5% for the PC and NC diets, respectively). On the other hand, despite that no significant differences were observed in the BWs between the treatments, the animals fed the NC + enzyme 500 ppm diet presented higher numerical values in comparison to those fed the control in all the feeding phases (14, 28, and 35 d). It is also important to highlight that the animals fed the NC diet presented higher overall FCRs than those fed the PC diet (+27.1%), confirming the impact of the dietary challenge in terms of energy and protein deficiency.

Though the available literature about the effects of combining α-galactosidase and xylanase in broiler chicken feed is scarce, several authors have confirmed the positive effects of its dietary addition on performance parameters and, particularly, on feed conversion. Kidd et al. (2001) indicated that supplementation with an enzyme preparation mainly based on α-galactosidase (but also presenting α-amylase, β-glucanase, protease, xylanase, and cellulase activity) for broiler chickens on an SBM-corn based diet improved the adjusted feed-to-gain ratios when compared to a non-supplemented group. A study conducted by Zou et al. (2013) assessed the effect of supplementing different enzymes, including α-galactosidase (100 ppm; activity of 750,000 U/kg), on SBM-corn-based diets for broiler chickens, and in the case of their restricted diets (−100 kcal/kg compared to the control treatment), they concluded that its inclusion significantly improved the feed-to-gain ratio regarding when compared to the animals fed the control restricted diet. However, Zou et al. (2013) did not find significant differences between the FCRs of the supplemented and non-supplemented animals fed diets with normal metabolizable energy levels, unlike the results obtained in the present work. Amer et al. (2020) reported that the inclusion of α-galactosidase at a dose of 50 ppm on an SBM-corn-based diet for broiler chickens significantly improved the animals’ final BWs (40 d) and the overall FCR in the animals fed an energy reduced diet (−120 kcal/kg compared to the control diet). Nonetheless, the animals fed the normal diet (without an energy reduction) showed no significant differences in any of the performance parameters linked to the enzyme addition, despite the supplemented animals showing numerical increases in final BW and overall ADG, as well as numerical reductions in overall ADFI and FCR Amer et al. (2020). On the other hand, it is important to remark that previous works also have shown contrasting results. Jasek et al. (2018) reported that the inclusion of an enzyme preparation containing α-galactosidase and xylanase at 200 ppm (α-galactosidase and xylanase minimum activity of 8 and 300 U/g, respectively) in a normal diet and in energy/AA-reduced SBM-corn-based diets did not have any significant effect on the overall FCR of the broiler chickens used in the trial (0–21 d). Similarly, other studies have indicated the absence of positive effects associated with the inclusion of α-galactosidase in corn-SBM diets on a broiler chicken’s feed efficiency (Waldroup et al. 2005, 2006). Nevertheless, most of the reviewed studies indicated the product dose but not the activity of each enzyme, nor did they define the units expressed; thus, the value of comparing the different results to assess the beneficial effects of including NSPases on performance, feed conversion, and the consequences on cost formulas due to nutrient restrictions is limited due to a lack of essential information.

The performance improvements observed in our study may be explained by the ability of α-galactosidase to degrade RFOs from SBM and RSM into digestible monosaccharides (galactose, glucose, and fructose), which increases their nutritional value. In the case of SBM, its content of RFOs may be variable depending on several factors; however, Choct et al. (2010) reviewed the chemical composition of different SBMs used for monogastric feeding and reported that the RFO contents reached 1.5% and 6.0% for raffinose and stachyose, respectively. Further, previous studies have demonstrated that the use of SBM free from RFOs or the inclusion of α-galactosidase in monogastric diets based on corn-SBM increases the energy and nutrient contents of the feed (Irish et al. 1995; Amer et al. 2020). In addition, some recent experiments have demonstrated the deleterious effects of RFOs on broiler chickens’ performances, indicating a relationship between RFO dietary concentrations and a reduction in ADG and FCR, particularly in young animals (Zhu et al. 2021; Teague et al. 2023). Also, it is important to remark that in the case of including ingredients in a diet other than corn, such as wheat, with higher arabinoxylan contents (Ward 2021), a greater effect on performance due to the enzyme preparation could be expected because of the xylanase activity. The reduction in overall FCR observed in the animals on the PC and NC diets due to the enzyme preparation demonstrated the improvement in the feed efficiency of both the standard and the nutrient restricted diets, confirming the room for improvement in both cases on animal’s performance, and this might have a significant impact on cost formulation.

Regarding the carcass yield measurements and organ weights, no effects were associated with the enzyme preparation on most of the parameters assessed. In the case of the kidney to carcass relative weights, differences were found between treatments NC + enzyme 500 ppm against the NC diet, whereas no differences were observed between the PC vs. NC and PC vs. PC + enzyme 350 ppm. Thus, the differences observed in the relative kidney weights appear to have been artifacts and were not related to the use of the enzymes. On the other hand, in the case of the fat pad relative weights, the enzyme supplementation tended to reduce the fat depositions in the PC – and NC-fed animals (−10.8% and – 15.0% for the PC + enzyme and NC + enzyme treatments, respectively; P < 0.1), showing a hypolipidemic effect. It is known that abdominal fat pads can be direct indicators of body fat content (Butterwith 1989), and thus, the enzyme preparation may have reduced the total body fat contents of the animals, which is a way to improve carcass yields. Previous authors have indicated that increases in abdominal fat depositions may be caused by dietary imbalances, particularly in terms of energy-to-protein relations (Nahas and Lefrancois 2001; Wu and Ravindran 2004). In this sense, a study conducted by van Emous et al. (2013) reported that diets with high values of energy-to-protein ratios resulted in increased fat depositions in broiler breeder females due to the rising conversion of dietary carbohydrates to lipid depositions. Thus, α-galactosidase plus xylanase supplementation might reduce fat percentages in broiler chicken carcasses by improving nutrient digestion and absorption. However, this hypothesis should be confirmed in further studies, along with complementary assessments of lipidic serum markers (total cholesterol, HDL, and LDL, among others) and the regulation of related genes on the liver, such as acetyl-CoA carboxylase and fatty acid synthase (Rosebrough et al. 2002). Unfortunately, the available literature on the use of a similar enzyme preparation on broiler chicken carcass parameters is scarce and, in some cases, inconsistent. A study conducted by Wang et al. (2005) evaluated the effect of including α-galactosidase (at 250 and 500 mg/kg; enzyme activity of 90.2 U/g) in a corn-SBM-based diet for broiler chickens using several carcass parameters, and they described no effects of the enzyme on fat depositions, which was in contrast to the tendency observed in the present experiment. However, the results reported by Jasek et al. (2018) also indicated that α-galactosidase did not have any remarkable effect on the rest of the carcass parameters (breast and leg muscle percentages), which was consistent with the results stated by Attia et al. (2021) and with the results observed in the present experiment. On the other hand, a study conducted by Llamas-Moya et al. (2020) assessed the effect of an enzyme preparation that combined α-galactosidase, xylanase, and cellulase on the carcass parameters of broilers fed corn-SBM-based diets, and they reported that the enzyme supplementation increased breast and meat percentages in relation to carcass weights, contrasting with our results. The authors discussed that the improvements were associated not only with the enzyme supplementation but also with the energy restrictions that they applied to the experimental treatments. However, in our case, no effect of nutritional restriction on dressing parameters was detected.

Regarding the effects of the enzyme preparation on footpad scores, the enzyme preparation tended to reduce the scores in animals fed the NC diet, lowering the incidence and severity of pododermatitis. Several studies have demonstrated that the inclusion of NSPases and, particularly, xylanase, reduces feed intestinal viscosity, which has an impact on improvements in litter quality (reducing moisture) and the incidence and severity of pododermatitis (Nagaraj et al. 2007; González-Ortiz et al. 2016). In the case of α-galactosidase, the degradation of RFO soluble sugars (Liu et al. 2021) with an osmotic effect in the intestinal lumen appeared to have an impact on the reduction in excreta moisture, and consequently, this led to better litter quality and a lower incidence of animal pododermatitis. Indeed, the direct relationship between dietary RFOs and higher excreta moisture levels has been indicated, and thus, it may have increased the incidence of pododermatitis (Teague et al. 2023). More specifically, Teague et al. (2023) demonstrated a linear increase in young broiler (from 0 to 21 d) excreta moisture levels with rising levels of dietary RFOs compared to a control treatment that used enzymatically treated SBM that was free from ANFs, and this had several implications, such worsening litter quality. Furthermore, the foot pad scores may also have been affected by the nutrient restriction in the NC diet, it has been demonstrated that nutrient deficiencies, such as some AA, lead to increased chyme viscosity and food pad dermatitis (Shepherd and Fairchild 2010).

In relation to experiment 2, the results indicated that the enzyme preparation addition increased the AME and AMEn value of the feed (28 and 24 kcal/kg, respectively), and numerically increased the apparent ileal utilization of CP and all the AAs assessed (from 2.03 up to 4.28 percentage points), including the total AAs (3.26 percentage points). As mentioned before, the analytical results of the PC diet showed higher feed AME values (3,098 kcal/kg) than the intended values of the grower phase diet (Table 1; 3,055 kcal/kg). This fact may have affected the improvement linked to the enzyme ‘on top’ addition; probably, in diets more adjusted to their intended values for energy content the improvement observed expected might have been higher than that reported in the experiment.

The improvements in energy utilization were attributed to reductions in ANFs, such as the RFOs, which increased the availability of nutrients (Pettersson and Pontoppidan 2013). Furthermore, the xylanase present in the enzyme preparation not only reduces feed viscosity by degrading the hydrolysable arabinoxylans (González-Ortiz et al. 2016), but also is capable to degrade the cell wall in vegetal raw materials, rich in NSP, reducing its nutrient-encapsulation effect (Meng and Slominski 2005). Similar to results shown in the present experiment, Coon et al. (1990) demonstrated that diets based on SBMs with lower levels of oligosaccharides (by an ethanol extraction) increased the AMEn values of the feed in roosters when compared to diets with non-treated SBMs. Additionally, they also indicated that the animals fed the treated SBM presented higher digestibility of CP, AAs, and dry matter. Other authors also have described similar effects on energy utilization with similar enzyme preparations. A study conducted by Jasek et al. (2018) reported that the ileal digestible energy content of experimental feeds offered to broiler chickens, independent of the nutrient value of the diets based on corn-SBM (normal and energy/AA-restricted diets), was improved by the supplementation of 200 ppm of α-galactosidase combined with xylanase. Similarly, two studies conducted by Wang et al. (2005) indicated that different supplementations with α-galactosidase (500 and 1000 mg/kg in the first experiment and 250 and 500 mg/kg in the second) increased the AME, AMEn, and TMEn contents of broiler chicken diets based on corn-SBM when compared to non-supplemented animals. An experiment conducted by Attia et al. (2021) tested the efficacy of a multi-carbohydrase (xylanase, α-galactosidase, β-glucanase, and β-mannanase) at 0.05% on corn-SBM-based diets for broiler chickens, and it indicated that there was a significant improvement in gross energy utilization.

Regarding the CP and AA utilization, the numerical improvements could be attributable to reductions in the ANF concentrations, including RFOs and arabinoxylans, and increasing the concentrations of available proteins and AAs in the diet mainly from the SBM, RSM, and corn. In our study, the AA digestibility did not reach a significant improvement between the treatments (except for proline); however, the sensitivity of the statistical test could have been affected by the dispersion of the residuals in the statistical model (RSE values) despite having obtained numerical differences from 2.03% to 4.28%. Other authors have confirmed the beneficial effect of adding similar enzyme combinations on CP and AA utilization (Wang et al. 2005; Gallardo et al. 2018; Jasek et al. 2018; Liu et al. 2021). The results obtained in the present experiments agreed with those of Jasek et al. (2018), who indicated that adding 200 g/ton of an enzyme preparation based on α-galactosidase and xylanase increased the total AA utilization in normal and energy/AA-reduced diets based on corn-SBM for broiler chickens (3.9% and 1.8% for normal and restricted diets, respectively). Similarly, the results reported by Liu et al. (2021) indicated that supplementing a corn-SBM diet for broiler chickens with the same enzyme preparation in the present study at 300 and 500 g/ton improved the total AA utilization and a great portion of AAs assessed when compared to a non-supplemented group.

Conclusions

In conclusion, the addition of an enzymatic preparation composed of α-galactosidase and xylanase in broiler chicken diets based on corn-SBM-RSM improved the feed conversion in diets with standard and restricted levels of energy and crude protein, whereas did not impact the final bodyweights and carcass dressings of the animals. In addition, the enzyme preparation tended to reduce the abdominal fat pads of the carcasses and the incidence and severity of footpad dermatitis. Regarding the digestibility balance, the enzyme supplementation increased the feed apparent metabolizable energy content (AME and AMEn), and numerically increased the apparent ileal CP and AA utilization. Therefore, the data suggest that the enzyme preparation increased the energetic content of broiler chicken diets based on SBM-RSM and corn.

Data availability statement

The data presented in this study are available on request from the corresponding author.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

This work was supported and funded by Industrial Técnica Pecuaria S.A. (ITPSA).