Nutrient and Energy Balance, and Amino Acid Digestibility in Broilers Fed Canola Meal and an Exogenous Enzyme Combination

ABSTRACT

The apparent total tract digestibility (ATTD; trial 1) and the apparent (AID) and standardized (SID) ileal digestibility of the amino acids (AA) (trial 2) in canola meal (CM) were evaluated with the addition of exogenous multi-carbohydrase (MC) and phytase (Phy). A total of 80 28-day-old broilers were allotted in a completely randomized design to receive treatments up to 35 days of age. A 2 × 2 factorial design was used to determine the enzyme effects on the ATTD of dry matter, nitrogen, calcium, phosphorus and fibre; as well as energy use and the AID and SID of AA, in five replicate cages. Supplementation with exogenous enzymes showed a positive interaction (p<0.05) between MC and Phy for nitrogen and energy. The isolated inclusion of Phy or MC showed a favorable effect (p<0.05) for dry matter, calcium, phosphorus and fiber. The enzyme inclusion on the AID and SID of AA in CM, established by comparing the means, suggested a better response to the addition of MC or Phy. Supplementation with Phy or MC was shown to be a viable alternative to increase the ATTD of nutrients and energy. The isolated inclusion of Phy or carbohydrate resulted in higher apparent and standardized digestibility of AA from CM.

Keywords: Amino acid digestibility; broiler chicks; canola meal; multi-carbohydrases; phytase

INTRODUCTION

Canola meal (CM) is the secondary product of the extraction of seed oil by methods that commonly use the combination of mechanical pressure and chemical solvent extraction. This is the preferred and most effective method of extracting oil from oilseeds for most large-scale plants. CM is an important ingredient in the formulation of poultry feed; with soybean meal being considered the better and most used protein alternative in poultry feed, followed by CM. This protein source is rich in fiber and anti-nutritional factors (Canola Council of Canada, 2023), the digestibility of which is limited in non-ruminants. Therefore, it can only be included at moderate levels in poultry feed formulations. Compared with soybean meal, canola meal contains more anti-nutritional factors, such as glucosinolates and fibers (Mejicanos et al., 2016). Glucosinolates and fiber in canola meal may reduce digestibility of energy and amino acids, and feed palatability (Maison et al., 2015; Mosenthin et al., 2016; Landero et al., 2018). CM is rich in sulfur amino acids; however, it contains less metabolizable energy than soybean meal and crude fiber (Rostagno et al., 2017). CM contains anti-nutritional factors such as glucosinolates (Mejicanos et al., 2016). Although their content has been decreased in seed of modern canola cultivars (Landero et al., 2018), glucosinolates in CM may reduce feed palatability, and the digestibility of energy and amino acids (AA) (Mosenthin et al., 2016).

The possibility of using MC was considered as a means to reduce the negative effects of fiber on digestion, by breaking down the non-starch polysaccharides (NSP) of the cell wall; paired with Phy to attenuate the negative effects of phytate, increasing the availability of complexed phosphorus (Woyengo & Nyachoti, 2011). Intestinal transit time and nutrient use can be negatively affected when birds increase their NSP intake due to increased intestinal viscosity (Kiarie et al., 2016; Gallardo et al., 2020; Araujo et al., 2021, 2022). On the other hand, studies have shown that exogenous enzymes can increase the digestibility of vegetable ingredients used as alternatives in the diets of poultry and swine. Therefore, dietary supplementation with enzymes can increase the efficiency of nutrient and energy utilization by non-ruminants, based on apparent or standardized digestibility assessments (Dadalt et al., 2017; Gallardo et al., 2020; Araujo et al., 2021, 2022).

As reported by Araujo et al. (2021), (2022) and Gallardo et al. (2020), Phy reduces the negative effects of phytic acid, increases the release of P from phytate and multi-carbohydrases, and reduces the negative effects of fiber on digestion. Therefore, it was hypothesized that the use of nutrients from CM may be favored by the inclusion of the tested ingredients in the diet of broilers at 35 days of age. Previous studies have shown an improvement in nutrient use, energy, and digestibility of AA with the use of carbohydrates and Phy in the diets of swine and broilers (Dadalt et al., 2017; Gallardo et al., 2020; Trindade et al., 2020; Araujo et al., 2021, 2022).

Thus, we evaluated the nutritional and energy balance, and the digestibility of the AA in CM, with and without MC and Phy in broilers at 35 days of age.

MATERIAL AND METHODS

All research methods and procedures were approved by the ethics committee for the use of animals of the São Paulo State University “Júlio de Mesquita Filho”, UNESP - School of Veterinary Medicine and Animal Science - FMVZ, Botucatu / SP. (Registration No. 0094/2018), Brazil, and were followed to ensure animal welfare. The methodology has been described in detail by Gallardo et al. (2020).

Canola meal and enzymes

The CM used for this study was obtained from Hi-Tech Feeds, Pelotas, Rio Grande do Sul, Brazil, and its chemical composition is shown in Table 1. The nutritional composition of the CM was similar to that described by Rostagno et al. (2017).

The enzymes used were a blend of MC (700 U α-galactosidase, 2,200 U galactomannanase, 3,000 U xylanase and 22,000 U β-glucanase kg / diet) from Endopower Beta, and Phy (500 FTU kg / diet) was sourced from Genophos, as reported in Gallardo et al. (2017). Exogenous enzyme activities were suggested by GNC Bioferm Inc. (Saskatoon, Saskatchewan, Canada), and technical information was provided by Uniquimica, São Paulo, Brazil.

Table 1
Nutrient composition (as-fed basis) of canola meal used in the study.

Nutrient component (%)	Canola Meal
Dry matter	89.61
Crude protein	38.56
Gross energy (MJ/kg)	17.82
Calcium	0.92
Phosphorus	0.97
Neutral detergent fiber	26.55
Essential amino acids (%)
Arginine	2.37
Histidine	1.05
Isoleucine	1.74
Leucine	2.43
Lysine	1.95
Methionine	0.62
Phenylalanine	1.26
Threonine	1.49
Tryptophan	1.64
Valine	1.78
Non-essential amino acids (%)
Alanine	1.48
Aspartic acid	2.29
Cystine	0.70
Glutamine	5.74
Glycine	1.56
Proline	2.07
Serine	1.37
Tyrosine	1.03

Diets and experimental design

The apparent total tract digestibility (ATTD; Trial 1) and the apparent (AID) and standardized (SID) ileal digestibility of the AA (Trial 2) of CM were evaluated with the addition of exogenous MC and Phy. A total of 80 male broilers, at 28 days of age, were allotted in a completely randomized design to receive treatments up to 35 days of age. All experiments were conducted in a completely randomized design in a 2 x 2 factorial arrangement of treatments (5 replicate cages with 16 birds per treatment). The factors were MC (0 and 200 mg/kg) and Phy (0 and 50 mg/kg). Basal diets were used for an additional group of 24 birds kept in 6 cages, which was used to determine the enzyme effects, alone or in combination. A basal corn diet (BD₁) was used for ATTD determination, and a corn-starch basal diet (BD₂) containing 5% casein was used to estimate endogenous losses and SID of AA, as described by Gallardo et al. (2020). The test diets were made by mixing BD and CM in an 8:2 (wt/wt) ratio.

From 21 days of age, the birds were fed with experimental diets until the beginning of the experiment. In trial 1 (days 28 to 33), excreta were collected, and from day 34 to day 35 (trial 2), the birds received a new diet until slaughter at the end the experimental period, when the ileal content was collected. Chromium oxide III (Cr₂O₃) was added at 0.3% to all diets as an indigestible marker. In trials 1 and 2, all diets (Table 2) were supplemented with vitamins and minerals, meeting the nutritional requirements of broilers in the growth phase as recommended by Rostagno et al. (2017).

Table 2
Composition of diets, as fed basis (g/kg diet).

Ingredients	Total collection	Ileal collection diet
	Basal diet (BD1)	Basal diet (BD2)
Yellow corn	850.1	-
Amido	-	502.7
Soybean oil	20.0	12.0
Choline chloride 60	0.2	0.2
Salt	2.5	3.2
Sodium bicarbonate	3.8	3.5
Limestone	8.1	6.9
Bicalcium phosphate	24.0	23.7
Cellulose	50.0	50.0
Dextrose	-	300.0
Casein	-	50.0
Chromic oxide	-	3.0
Vitamin-mineral premix3	5.0	5.0
Kaolin4	36.3	39.8
Total	1000	1000
Calculated composition
Dry matter	888.0	929.3
Crude protein	67.0	42.1
Calcium	9.2	8.6
Available phosphorus	4.0	3.4
Metabolizable energy (MJ/kg)	12.77	12.98
Sodium	2.2	2.2
Choline (mg/kg)	2000	1900
Linoleic acid	26.8	6.3

• ¹
  Corn based diet; ²Cornstarch and casein diet; ³Vitamin-mineral premix per kg of feed: vitamin A 9 000 UI; vitamin D3 1 600 UI; vitamin E 14 UI; vitamin K3 1 5 mg; vitamin B₁ 1 mg; vitamin B₂ 4 mg; pantothenic acid 8 28 mg; vitamin B₆ 18 mg; vitamin B₁₂ 12 mμg; niacin 0 03 mg; folic acid 0 3 mg; biotin 0 05 mg; Se 0 25 mg; Cu 9 mg; Fe 30 mg; I 1 mg; Zn 60 mg; Mn 60mg. ⁴Kaolin: mineral kaolinite, used as inert ingredient to adjust the formulation of diet.

In trial 1, to assess the balance of nutrients and apparent metabolizable energy (EMA) in the CM, samples of excreta were collected twice a day (8 am and 5 pm) for 5 consecutive days. To determine the digestibility coefficients of the AA in trial 2, all birds were weighed and slaughtered for ileal collection in the last 10 cm, before the 2 cm proximal to the ileocecal junction. Prior to analysis, excreta were stored in a freezer at -20 °C, and at the end of the experiment, they were homogenized and freeze-dried.

At 35 days of age, five birds per treatment were collected, and liver and pancreas weights were taken to determine the relative weights of these organs in relation to the post-fasting weight, expressed as a percentage. The following equation was used:

$\mathrm{Re}lativeorganweight=(organweight/post-fastingweight)\times 100$

Sample analyses and data processing

The excreted and ileal samples were freeze-dried for 72 h at -40ºC (LH 0401, Terroni, São Carlos, BR) as described by Gallardo et al. (2017). The diets, CM, excreta and ileal digesta samples were finely milled and analyzed, according to Association of Official Analytical Chemists (AOAC, 2005) for determinations of dry matter (DM), gross energy (GE), nitrogen (N), calcium (Ca), phosphorus (P) and neutral detergent fiber (NDF). Test diets and ileal digesta samples were processed and analyzed to determine the digestibility coefficients of AA as described by Gallardo et al. (2017). Tryptophan was determined by the colorimetric method of Spies (1967), using a standard curve of pure tryptophan (Merck, Germany), and detected at 590 nm with a spectrophotometer (DU-640 UV/Vis; Beckman Coulter, Basking Ridge, New Jersey, (USA). Cystine was expressed as cysteine. All analyses were performed in duplicate.

Calculations

As reported by Dadalt et al. (2017), all formulas related to apparent nutrient digestibility and amino acid digestibility (AID and SID) were as follows:

$Digestibilityofnutrients\left(\text{\%}\right)=100\times [\left(\text{NI}-{\text{NO}}_{\text{excreta }}\right)/NI],$

where NI is the nutrient intake (g), and NO_excreta is the nutrient output in excreta (g).

The retention of nutrients in CM was determined by the difference method (Fan & Sauer, 1995), with the corn-based diet used as the BD, using the following equation:

$DA=\left(DD–\left(DB\times DN\right)\right)/DCM$

where DA is the retention of a nutrient (%) in a feedstuff assay (CM),

DD is the digestibility of a nutrient (%) in the CM-containing diet,

DB is the digestibility of a nutrient (%) in the corn-based diet,

DN is the contribution of a nutrient (decimal percentage) to the assay diet from corn and,

DRB is the contribution of a nutrient (decimal percentage) in the CM-based diet from CM.

The apparent metabolizable energy (AME) content of CM was calculated according to the following equation (Woyengo et al., 2010):

$AMEofCM\left(kcal/kg\right)=[(GrossenergyretentionforCM,\%).\left(GrossenergycontentinCM,kcal/kg\right)]/100$

The AID and SID (%) of amido acids were calculated using the following formula (Nyachoti et al., 1997):

$AID(\%)=100-[100\times \left(A{A}_{digesta}\times C{r}_2{O}_{3diet}\right)/(A{A}_{diet}\times C{r}_2{O}_{3digesta})$

Where AA_diet and AA_digesta are the amino acid content (mg/kg of DM) in the diet and digesta, respectively, and Cr₂O_{3diet and} Cr₂O_3digesta are the indigestible marker contents (mg/kg de DM) in the diet and digesta, respectively.

Apparent ileal AA digestibilities were standardized using average values for basal endogenous AA losses calculated using the following formula (Nyachoti et al., 1997):

$A{A}_{EL}(g/kg)=A{A}_{digesta}\times \left(C{r}_2{O}_{3diet}/C{r}_2{O}_{3digesta}\right)$

Where AA_EL = average endogenous amino acid loss (g/kg of DM).

The SID of AA was calculated according to the following equation. as described by Opapeju et al. (2006):

$SID(\%)=\left[AI{D}_{AA}+\left(A{A}_{EL}/A{A}_{diet}\right)\right]\times 100$

Statistical analysis

The GLM procedure of SAS (Statistical Analysis System, 2014, version 9.4) was used to determine the main effects of, and the interaction between, MC and Phy. The homogeneity of variances was evaluated by the Shapiro-Wilk test (UNIVARIATE procedure). The statistical model used was:

$Yij=\mu +ai=bj+\left(ai\times bj\right)+eij,$

where Yij = variable response of broilers fed with MC and phy; μ = overall mean; ai = MC effect; bj = Phy effect; (ai × bj) = interaction between MC and Phy; eij = error contribution with average 0 and variance σ2, I = 1 … a, and j = 1 … b. Significance was accepted at p<0.05.

RESULTS

The effects of the CM diets supplemented with MC and Phy on nutrient balance and AME in broilers from 28 to 33 days of age are shown in Table 3, and the amino acids contents used to determine AID and SID are shown in Table 4 and 5.

Table 3
Apparent nutritional balance of canola meal, combined or not with enzymes, for broilers at 35 days of age(g/kg).

Item	Phy2 0		Phy 50			p-valor 4
	MC1 0	MC 200	MC 0	MC 200	SEM3	MC	Phy	MCxPhy
Dry matter	777.4	792.4	786.1	782.2	0.002	0.002	0.013	0.214
Nitrogen	749.2	758.6	769.3	779.8	0.004	0.008	0.509	0.013
AME (MJ/Kg)	13.90	13.98	14.7	14.99	0.027	0.001	0.100	0.001
Calcium	745.1	751.8	786.5	778.1	0.044	0.448	0.043	0.878
Phosphorus	568.2	625.3	601.5	615.0	0.054	0.293	0.049	0.112
NDF (%)	365.2	405.5	411.2	404.4	0.006	0.001	0.020	0.043
ME/GE	686.4	690.7	691.01	729.9	0.001	0.001	0.029	0.001

• N = 5 replicates cages. As dry matter basis. AME: apparent metabolizable energy; NDF: Neutral detergent fiber.
• ¹
  MC, multi-carboidrase level (0 and 200 mg/kg of diet), (200 mg/kg of MC contains 700 U of α-galactosidase, 2200 U of galactomannanase, 30 000 U of xylanase and 22 000 U of β-glucanase/kg of diet). ²Phy, Phytase level (0 and 50 mg/kg of diet). ³SEM: standard error of mean. ⁴Effect of MC - Multi-carboidrase, Phy - Phytase, and interaction MC x Phy, respectively.

Table 4
Apparent ileal digestibility of the amino acids of canola meal, combined or not with enzymes, for broilers at 35 days of age*.

Item	Phy² 0		Phy 50		SEM3	p-valor 4
	MC¹ 0	MC 200	MC 0	MC 200		MC	Phy	MCxPhy
Essential AA (%)
Arginine	90.14	94.13	93.97	90.64	1.636	0.787	0.001	0.962
Histidine	85.93	94.30	94.04	89.29	1.038	0.001	<0.001	0.004
Isoleucine	84.82	86.94	84.29	87.17	0.811	<0.001	0.301	0.687
Leucine	89.86	92.17	90.73	90.41	1.173	0.076	0.023	0.409
Lysine	86.68	90.78	87.41	89.40	1.251	<0.001	0.050	0.571
Methionine	92.48	95.91	96.51	95.97	1.290	0.023	0.003	0.003
Phenylalanine	89.59	92.58	92.96	89.46	1.453	0.701	0.001	0.856
Threonine	77.59	87.81	86.73	80.80	1.592	0.008	<0.001	0.153
Tryptophan	79.40	85.54	81.33	83.60	1.181	0.002	0.192	0.843
Valine	85.92	90.53	88.95	87.00	1.035	0.011	<0.001	0.587
Non-essential amino acid (%)
Alanine	83.90	93.94	93.54	84.03	1.610	0.721	<0.001	0.852
Aspartic acid	89.13	90.01	89.66	89.47	1.358	0.583	0.389	0.990
Glutamine	90.11	93.84	93.40	92.15	0.974	0.011	<0.001	0.085
Glycine	80.54	89.39	88.63	82.26	1.552	0.092	<0.001	0.497
Proline	84.76	82.02	81.49	85.81	1.233	0.172	<0.001	0.636
Serine	74.18	84.28	84.04	81.39	1.423	<0.001	<0.001	<0.001
Tyrosine	88.12	91.43	89.82	89.43	1.193	0.015	0.003	0.782

• N = 5 replicates cages. *As dry matter basis.
• ¹
  MC, multi-carboidrase level (0 and 200 mg/kg of diet), (200 mg/kg of MC contains 700 U of α-galactosidase, 2200 U of galactomannanase, 30 000 U of xylanase and 22 000 U of β-glucanase/kg of diet). ²Phy, Phytase level (0 and 50 mg/kg of diet). ³SEM: standard error of mean. ⁴Effect of MC - Multi-carboidrase, Phy - Phytase, and interaction MC x Phy, respectively.

Table 5
Standardized digestible coefficient of amino acids in canola meal, combined or not with enzymes, in broilers at 35 days of age*.

Item	Phy² 0		Phy 50		SEM3	p-value 4
	MC¹ 0	MC 200	MC 0	MC 200		MC	Phy	MCxPhy
Essential AA (%)
Arginine	94.42	96.72	96.57	94.57	1.646	0.835	0.010	0.999
Histidine	91.94	97.01	96.87	93.98	1.471	0.116	<0.001	0.170
Isoleucine	92.46	95.10	93.34	94.86	1.316	0.003	0.362	0.583
Leucine	94.16	95.83	94.66	94.67	1.171	0.127	0.131	0.536
Lysine	91.81	95.30	92.44	94.30	1.252	0.001	0.164	0.739
Methionine	98.35	96.01	97.91	99.59	0.640	0.011	0.011	0.011
Phenylalanine	93.36	95.28	95.52	93.10	1.452	0.705	0.004	0.993
Threonine	88.58	94.22	93.28	90.68	1.183	0.011	<0.001	0.289
Tryptophan	85.22	90.75	81.43	94.54	1.181	0.004	0.001	0.872
Valine	92.36	95.26	94.04	93.24	1.040	0.038	0.001	0.720
Non-essential amino acid (%)
Alanine	90.78	96.77	96.54	90.91	1.610	0.813	<0.001	0.944
Aspartic acid	94.23	94.93	94.78	95.41	1.960	0.460	0.963	0.565
Glutamine	95.51	97.79	97.51	97.40	0.973	0.024	0.014	0.050
Glycine	87.33	93.40	92.85	89.10	1.580	0.125	<0.001	0.411
Proline	89.94	89.32	88.35	91.15	1.088	0.039	0.003	0.808
Serine	88.90	95.34	95.37	95.30	1.544	0.001	0.001	0.001
Tyrosine	92.05	93.91	93.26	93.31	1.653	0.216	0.240	0.690

• N = 5 replicates cages. *As dry matter basis.
• ¹
  MC, multi-carboidrase level (0 and 200 mg/kg of diet), (200 mg/kg of MC contains 700 U of α-galactosidase, 2200 U of galactomannanase, 30 000 U of xylanase and 22 000 U of β-glucanase/kg of diet). ²Phy, Phytase level (0 and 50 mg/kg of diet). ³SEM: standard error of mean. ⁴Effect of MC - Multi-carboidrase, Phy - Phytase, and interaction MC x Phy, respectively.

The ATTD coefficients and apparent energy use of broilers fed with CM supplemented with exogenous enzymes characterize a positive interaction (p<0.05) between MC and Phy for nitrogen and energy. The isolated inclusion of Phy or carbohydrate indicated a favorable effect (p<0.05) on the digestibility of dry matter, calcium, phosphorus and fiber.

The coefficients of apparent and standardized digestibility of the AA in SM, supplemented or not with MC and Phy, are shown in Tables 4 and 5, respectively. The general results indicate that the isolated or combined supplementation of MC and Phy positively influenced (p<0.05) the AID and SID coefficients of the SM AA. The marked effect (p<0.05) of the MC and Phy combination on the AID and SID of flaxseed meal was also evident for histidine, methionine, serine, glutamine, arginine, isoleucine, lysine, phenylalanine, threonine, aspartic acid, glycine and proline amino acids. The average values of apparent and standardized digestibility of the 17 AA of FM were as follows: 85.93% and 91.85% without enzyme, 90.78% and 95.28% with MC, 89.66% and 94.66% with Phy, and 89.29% and 95.10% with MC + Phy.

Liver and pancreas weights relative to body weight are shown in Table 6. Regarding the effects of the inclusion of enzymes on the body weight of birds and the relative weights of the liver and pancreas, the differences were not significant (p>0.05).

Table 6
Relative weight of organs of broilers at 35 days of age, fed with canola meal supplement or not multi-carbohydrase and phytase.

Item	Phy2 0		Phy 50			p-value 4
	MC1 0	MC 200	MC 0	MC 200	SEM3	MC	Phy	MCxPhy
Initial BW (g)5	45	45	45	45	-	-	-	-
Final BW (g)	1561	1645	1566	1694	50.07	0.678	0.624	0.607
Liver/BW5	29.9	33.5	34.5	32.9	1.167	0.283	0.451	0.951
Pancreas/BW5	1.7	1.7	1.7	1.7	0.002	0.836	0.836	0.836

• N = 5 replicates cages.
• ¹
  MC, multi-carboidrase level (0 and 200 mg/kg of diet), (200 mg/kg of MC contains 700 U of α-galactosidase, 2200 U of galactomannanase, 30 000 U of xylanase and 22 000 U of β-glucanase/kg of diet). ²Phy, Phytase level (0 and 50 mg/kg of diet). ³SEM: standard error of mean. ⁴Effect of MC - Multi-carboidrase, Phy - Phytase, and interaction MC x Phy, respectively. ⁵BW: Body weight.

DISCUSSION

In the AID of the ingredients by the total collection of excreta, better responses were observed with the addition of exogenous enzymes, compared to not including the same enzymes. The greater efficiencies occurred with the addition of Phy alone in CM. In comparison to the enzyme-free treatment, the addition of Phy in CM led to an increase in digestibility of 1.1% of dry matter, 5.3% of calcium, 5.5% of phosphorus, and 11.2% in NDF.

In a study on broilers, Gallardo et al. (2018; 2020) reported that phytates and NSP are harmful anti-nutritional compounds in broiler feed. The negative effects of phytates and NSP are complex formation with other minerals, reduction of nutrient absorption, nutrient encapsulation, the reduction of the energy density of the feed, and improved intestinal health. The favoring of digestion by the action of carbohydrases could be associated with the reduction of intestinal viscosity and loss of nutrients (Gallardo et al., 2020), while also reducing nitrogen emission to the environment, which would be a complementary benefit of enzyme dietary supplementation (Sun & Kim, 2019). Therefore, increased nutrient and energy retention is associated with total or partial degradation of these anti-nutritional compounds. Thus, the highest numerical values observed for the inclusion of exogenous enzymes shown in Table 3 indicate the important effect of carbohydrases and phytase through their likely action on cell walls and nutrient decapsulation, favoring the effectiveness of phytase, as reported by Gallardo et al. (2017). In this case, the dietary addition of exogenous enzymes increases the digestibility and use of nutrients in plant ingredients, by reducing the viscosity of the digesta, and thus showing better absorption of minerals. The composition of amino acid and digestibility are related to the intrinsic characteristics of each ingredient, processing methods, protein fractions, crop types, and also differences in amino acid profiles (Dadalt et al., 2016). Despite the differences in chemical composition established in the literature, studies have reported positive effects on the digestibility of different plant by-products when supplementing exogenous enzymes in the diets of broiler chickens (Barekatain et al., 2014; Liu et al., 2015; Amerah et al., 2017). Some studies have indicated an increase in amino acid digestibility, apparent nitrogen balance, and energy of plant ingredients in broilers and swine that received exogenous enzymes (Gallardo et al., 2017, 2018, 2020; Trindade et al., 2020; Araujo et al., 2021, 2022).

The enzymes carbohydrase and Phy have specific mechanisms of action; therefore, their positive effects were due to the availability of a substrate for each enzyme. The action of carbohydrases ruptures cell walls, increasing nutrient digestibility (Gallardo et al., 2020). The action of carbohydrases favoring digestion could be associated with a reduction of intestinal viscosity and loss of nutrients, as well as a reduction of nitrogen emission to the environment; which would be a complementary benefit of enzyme dietary supplementation (Zijlstra et al., 2004; Gallardo et al., 2020).

The positive effect on the balance of nutrients and energy use may be directly associated with the supplementation of the enzymes Phy and carbohydrases. Our results showed that the enzymes increased (p<0.05) the AID of CM nutrients and AA. The mechanism of releasing encapsulated nutrients by breaking the cell walls of CM may explain the positive effect of enzymes such as Phy and carbohydrases, which decrease intestinal viscosity, stabilize mucus production by decreasing water absorption, and allow endogenous enzymes to interact with their respective substrates (Slominski, 2011).

The pronounced effect of the use of exogenous enzymes on calcium and phosphorus retention ratifies the increase in availability with the hydrolysis of the phytate-mineral complex (Dadalt et al., 2017). Therefore, effects on phosphorus and calcium were expected, as reported by Araujo et al. (2021), Gallardo et al. (2020) and Trindade Neto et al. (2020) when supplementing test diets for poultry and piglets, respectively, with Phy and carbohydrases. In this sense, the use of Phy can positively impact animal performance by increasing the release and absorption of phosphorus and the use of energy (Wu et al., 2015). Furthermore, given its high capacity to bind nutrients, it must be considered that Phy also reduces mineral availability in the gastrointestinal tract. Thus, the inclusion of Phy not only contributes to the release of phytic phosphorus, but also allows calcium and other minerals, energy, and nitrogen to participate in the same complex (Emiola et al., 2009; Gallardo et al., 2018). The supplementation with Phy acted in the cellular dissociation of the fiber, leading phytase to be favored for the dephosphorylation of the P-phytate complex, and thus reflecting on apparent increase in the digestibility of the CM.

This prominent effect of Phy in combination with carbohydrases suggests an increase in amino acid digestibility, which provided better results for the apparent and standardized coefficients and may be associated with greater hydrolysis of NPS in canola, as seen other vegetable ingredients used as a dietary ingredient for poultry and pigs (Gallardo et al., 2017, 2018; 2020; Trindade Neto et al., 2020; Araujo et al., 2021). The increase in standardized digestibility with the inclusion of enzymes stood out in comparison with CM without enzyme. Our results corroborated the findings of Ravindran et al. (1999), Gallardo et al. (2017, 2018, 2020), and Trindade Neto et al. (2020), who also reported positive effects on amino acid digestibility with CM. In addition to enzyme activity, the increase in amino acid digestibility depends on substrate availability, which allows the use of these and other nutrients of the tested ingredient (Emiola et al., 2009). However, the inclusion of exogenous enzymes could reduce the endogenous losses of AA, improving the digestibility of nitrogen and AA. On the other hand, studies have shown that phytate increases the production of intestinal mucin and indirectly increases losses of endogenous AA, which may affect AA digestibility (Adeola & Cowieson, 2011; Woyengo & Nyachoti, 2011; Sredanovic et al., 2012). Therefore, the effect of exogenous enzymes on the SID of AA could be associated with decreased mucin production, due to the reduction of the NSP of CM. According to Nian et al. (2011), mucin should also be considered a main source of endogenous carbohydrates in the digesta.

In the complementary assessment of liver and pancreas weights relative to body weight, the goal was to evaluate possible responses of these organs to the CM digestion process in the presence or absence of MC and Phy, since such information is scarce. Broilers fed with CM supplemented with exogenous enzymes showed numerically greater or equal liver and pancreas weights than the birds of the control group, which can be attributed to the positive effects of enzymes on nutrient absorption via fiber degradation and reduction of the secretory activity of these organs (Veldman & Vahl, 1994). Studies have found no effects of Phy and carbohydrates, alone or combination, on the weight of these organs in broiler chicks (Gallardo et al., 2017; 2018; Araujo et al., 2021). In addition, the presence of exogenous enzymes in the diet could be related to the increased availability and absorption of nutrients, decreasing endogenous enzymatic activity (Wu et al., 2015).

CONCLUSIONS

Supplementation with Phy or MC was a viable alternative to increase the ATTD of nutrients and energy use in broilers fed CM. Isolated inclusion of Phy or carbohydrate indicated resulted in higher apparent and standardized digestibility of AA from CM.

ACKNOWLEDGEMENTS

The authors thank the São Paulo State Research Foundation (FAPESP) for funding this research (2016/07352-8). They also thank Uniquimica (São Paulo, Brazil) for providing supplemental enzymes. This study was also partly financed by the Coordination for the Improvement of Higher Education Personnel - Brazil (CAPES) - Finance Code 001.

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• DECLARATION OF COMPETING INTEREST
  The authors declare no conflicts of interest regarding the publication of the manuscript and the dissemination of the results obtained.

Publication Dates

• Publication in this collection
  08 July 2024
• Date of issue
  2024

History

• Received
  18 July 2023
• Accepted
  15 Mar 2024