Endogenous Enzyme Activities, Digestibility of Nutrients and Digestive Physiology as Affected by High Inclusion of Cottonseed Meal in Broiler Chicken Diets



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Submitted Oct 18, 2023
Published Jan 17, 2024
10.24018/ejfood.2024.6.1.752

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A 3 × 3 factorial study examined the performance, endogenous enzyme activities and apparent ileal digestibility of nutrients, including energy, protein, starch, dry matter, amino acids and minerals of birds fed diets containing graded levels of cottonseed meal (CSM) with or without microbial enzymes. Nine iso-caloric and iso-nitrogenous diets were formulated mainly from wheat/sorghum/soybean meal. Three levels of CSM none (0%), low (5%, 10%, and 15%) or high (6%, 12%, and 18%) in the starter, grower, and finisher diets, respectively, were included. The diets were supplemented with three levels of a composite xylanase and beta-glucanase product at 0, 100 or 150 mg/kg diet. Each of the nine dietary treatments was randomly assigned to 6 replicates, with 10 birds per replicate. Feed intake (FI) and weight gain (WG) were recorded on day 10, 24 and 35, and feed conversion ratio (FCR) was obtained from the data, while enzyme activities and nutrient digestibility were measured on samples collected on day 10 and 24, and day 24 only, respectively. Feed intake up to day 35 decreased (P < 0.05) significantly with increasing enzyme supplementation. On the CSM-containing diets, enzyme supplementation at 100 or 150 mg/kg improved WG up to day 24, with the heaviest birds (1514 g/b) observed in the low CSM with 100 mg/kg enzyme group. Enzyme supplementation improved (P < 0.01) FCR all through the growth phases and also improved (P < 0.05) ileal crude protein and starch digestibility. High levels of CSM decreased the digestibility of starch, but this was improved (P < 0.05) by enzyme supplementation, showing an interaction between CSM and microbial enzyme on starch digestibility. The digestibility of arginine, glutamic acid and threonine improved with increased CSM inclusion, and that of methionine improved with increased enzyme supplementation. There was an interaction effect (P < 0.01) between CSM and enzyme on magnesium digestibility. Cottonseed meal inclusion improved the digestibility of copper and potassium and reduced the digestibility of phosphorus, while that of calcium and manganese was increased (P < 0.01) with enzyme supplementation. At day 10, lipase activity was increased (P < 0.05) by higher CSM levels, while on day 24, general proteolytic activity was highest (P < 0.05) when 100 mg enzyme was supplemented in the diet. These results indicate that relatively high levels of CSM in diet have no negative effects on the growth and digestive physiology of broiler chickens when supplemented with xylanase and β–glucanase.

Keywords: Broilers cottonseed meal enzyme activity nutrient digestibility

Introduction

The cotton plant (Gossypium), a genus of the Malvaceae family, is a versatile crop that produces a number of products of value to both humans and domesticated animals [1], [2]. Reference [3] reported that for each 480 kg bale of cotton fibre produced, approximately 678 kg of cotton seed is separated out in the ginning process. Cottonseed meal (CSM) is a by-product of oil extraction from cotton seeds and is a relatively rich source of protein (30% to 50%) and amino acids [4]. A review by Świątkiewicz et al. [5] concluded that CSM is an acceptable ingredient in poultry diets and can be used as a relatively rich source of protein and amino acids. However, the use of CSM as a protein supplement in poultry diets is limited due to the presence of gossypol, variation in nutrient composition and a relatively low lysine level, all of which have negative effects on the growth performance of broiler chickens compared to soybean meal [6].

The amount of CSM that can be included in the diet for birds depends largely on the amount of gossypol in the meal [7], [8]. Enhancement of the nutritive value of CSM, however, has the potential to lead to increased utilization of the meal in broiler chicken nutrition [9]. Processing methods such as cooking [10], fermentation [10], [11], expander solvent extraction [12], treatment with ferrous sulphate [13], and supplementation with lysine [7] have been used to improve the nutritive value of CSM for use in poultry nutrition, with varying results.

The use of enzyme supplementation to improve the nutritive value of feedstuffs in poultry nutrition has advanced in recent years. Most enzyme supplementation studies for the improvement of alternative feedstuffs have focused on non-starch polysaccharides and phytate [14]–[17]. However, there is scant literature on the use of microbial enzyme supplementation to improve the nutritive value and utilization of CSM in poultry nutrition. Therefore, the objective of this study was to evaluate the effects of high inclusion levels of CSM with or without a composite microbial enzyme supplement containing xylanase and beta-glucanase fed to broilers chickens on endogenous enzyme activities and apparent ileal digestibility of nutrients, including amino acids and minerals.

Materials and Methods

Dietary Treatments and Animal Husbandry

Three basal diets were formulated following breeder recommendation to satisfy the nutrient requirements of birds in each growth phase starter (day 1–10), grower (day 11–24), and finisher (day 25–35). These dietary treatments were prepared mainly from wheat, sorghum and soybean meal. Cottonseed meal was included at three levels; none, low or high. The low level had 5%, 10%, and 15%, while the high level had 6%, 12%, and 18% CSM in the starter, grower and finisher phases, respectively. The diets were supplemented with three levels of a composite enzyme (Axtra XB, Danisco Animal Nutrition, Marlborough, UK) at 0, 100 or 150 mg/kg diet (Table I). The enzyme product provided 1520 U/g of β-glucanase and 12200 U/g of xylanase per gram of added enzyme. Table II shows the analyzed nutrient composition of the tested CSM, while the calculated nutrient composition of the formulated diets is shown in Table III. Titanium dioxide (TiO2) was added to the grower diets at a rate of 5 g/kg diet as an indigestible marker to enable measurement of nutrient digestibility. The starter diets were fed in crumble form, while the grower and finisher diets were pelleted.

Ingredients Starter (CSM) Grower (CSM) Finisher (CSM)
0 5% 10% 0 10% 12% 0 15% 18%
Sorghum 110.0 90.0 80.0 110.0 97.2 102.3 200.0 200.0 150.0
Wheat 489.7 523.6 534.7 489.7 551.1 551.1 484.8 524.1 577.6
SBM 309.6 240 228.2 309.6 166.5 138.0 227.0 30.2 0.0
Canola meal 20.0 20.0 20.0 10.0 1.5 1.3 10.0 5.0 3.3
CSM 0.0 50.0 60.0 0.0 100.0 120.0 0.0 150.0 180.0
Meat & bone meal 20.0 20.0 20.0 17.0 7.5 7.5 15.0 10.0 0.0
Canola oil 22.3 21.8 22.1 32.0 32.8 33.0 39.5 37.0 38.1
Limestone 11.2 11.4 11.5 10.6 11.9 12.0 9.8 11.0 12.2
Dic. Phos. 4.2 4.2 4.2 2.8 5.1 5.2 1.4 2.6 4.7
Phytase2 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1
Salt 1.2 1.1 1.1 1.0 1.1 1.4 1.1 1.1 1.2
Na bicarb 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0
Marker (TiO2) 0.0 0.0 0.0 5.0 5.0 5.0 0.0 0.0 0.0
Vit-min. premix3 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0
Choline Cl 60% 0.6 0.6 0.6 0.5 0.5 0.5 0.5 0.5 0.5
L-lysine 2.4 4.7 5.0 3.1 5.8 6.8 3.4 8.3 9.5
DL-met 3.1 3.8 3.9 2.7 4.0 4.3 2.4 4.5 4.9
L-threonine 1.6 3.0 2.7 1.2 2.9 3.3 0.9 3.7 4.3
L-arginine 0.0 1.7 1.9 0.0 3.0 3.9 0.0 5.7 6.9
L-isoleucine 0.0 0.0 0.0 0.0 0.0 0.3 0.0 1.1 1.4
L-tryptophan 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.7
Table I. Ingredient Composition (g/kg) of Experimental Diets 1 with Different Levels of CSM
Nutrient Values
ME MJ/kg 9.7
Crude protein (%) 45.1
Crude fat (%) 1.7
Crude fibre (%) 8.6
Ash (%) 6.8
Indispensable amino acids (g/kg)
Arginine 46.7
Lysine 21
Methionine 6.9
Threonine 14.6
Tryptophan 5
Valine 21.5
Histidine 13
Leucine 26.7
Isoleucine 14.4
Phenylalanine 24.8
Minerals (g/kg)
Calcium 1.8
Total phosphorus 9.1
Sodium 0.45
Potassium 1.3
Table II. Analyzed Composition of Cottonseed Meal
Nutrient Starter Grower Finisher
0 5% 10% 0 10% 12% 0 15% 18%
ME (MJ/kg) 12.6 12.6 12.6 13.0 13.0 13.0 13.4 13.4 13.4
CP 230.0 230.0 230.0 215.0 215.0 215.0 195.0 195.0 195.0
Crude fat 42.5 41.8 42.0 51.7 51.7 51.9 59.9 57.0 56.8
Crude fibre 26.3 26.9 27.1 25.3 26.2 26.4 24.5 26.0 26.2
Arginine 14.3 13.8 13.7 13.2 12.3 12.3 11.6 10.9 10.9
Lysine 12.8 12.9 12.8 12.5 11.5 11.5 11.4 10.2 10.2
Met. 6.2 6.5 6.5 5.6 6.1 6.3 5.1 6.0 6.1
Met. + cysteine 9.5 9.5 9.5 8.7 8.7 8.7 8.0 8.0 8.0
Tryptophan 2.8 2.4 2.3 2.6 1.9 1.7 2.3 1.6 1.6
Isoleucine 9.6 8.9 8.8 9.1 7.8 7.8 8.2 7.0 7.0
Threonine 8.6 9.0 8.6 7.7 7.7 7.7 6.8 6.8 6.8
Valine 10.8 10.2 10.0 10.1 9.2 8.9 9.2 7.8 7.8
Calcium 9.6 9.6 9.6 8.7 8.7 8.7 7.8 7.8 7.8
Avail. P 4.8 4.8 4.8 4.4 4.4 4.4 3.9 3.9 3.9
Sodium 1.9 1.9 1.9 1.8 1.8 1.9 1.8 1.8 1.8
Chlorine 1.9 2.3 2.4 1.9 2.4 2.8 2.0 2.9 2.3
Choline 17.0 17.0 17.0 16.0 16.0 16.0 15.0 15.0 15.0
Table III. Calculated Nutrient Composition (g/kg) of Diets with Different Levels of CSM

A total of 540-day-old Ross 308 male chicks (40.6 ± 0.8 g) were randomly allocated to nine dietary treatments in six replicates, 10 birds per replicate. Birds were placed in wire cages (47 × 80 × 45 cm) in an environmentally controlled room at the Centre for Animal Research and Teaching, University of New England, Australia. A 24-h light period was provided on the first day and then was reduced to 18 h of light until day 35. Room temperature was set at 33 °C on day one and gradually reduced to 24 °C on day 18 and then maintained until the end of the experiment. Cages (replicates) were fitted with a feeding rack, mechanical water system using two drinking nipples, and a tray for excreta collection. Feed and water were provided ad libitum to the chickens.

Sample Collection and Processing

After pelleting and cooling, diet samples were collected, ground, and properly stored (4 °C) in order to use for dry matter (DM), crude protein (CP), gross energy (GE) and TiO2 and mineral analyses. At day 24 two birds were randomly selected from each pen, weighed and euthanized using electrical stunning and cervical dislocation techniques. The small intestine between the vitelline (Merkel’s) diverticulum and a point about 0.4 cm above the ileo-caecal junction was removed, and the ileal digesta content was flushed into plastic containers and stored immediately at −22 °C. Frozen ileal digesta were then lyophilized (Christ Alpha 1–4, B-Braun Biotech International, Melsungen, GmbH, Germany) and ground using a coffee grinder and kept in air-tight containers at 4 °C for subsequent DM, CP, GE, AA, TiO2 and starch analyses.

Nutrient Analysis

Diet samples were dried at 105 °C in a forced air convection oven (Qualtex Universal Series 2000, Watson Victor Ltd., Perth, Australia) for 24 h to determine DM content. The DM of ileal digesta was obtained directly after freeze-drying (Christ Alpha 1–4, B-Braun Biotech International, Melsungen, GmbH, Germany) at −50 °C for at least 72 h. The nitrogen content of ileal digesta and diet samples was measured using the Dumas combustion technique, following the method described by [18], by direct electronic reading on a LECO® FP-2000 automatic nitrogen analyser (Leco Corporation, St. Joseph, MI, USA). Nitrogen results were then converted to crude protein using a factor of 6.25. The gross energy of diet and ileal digesta samples was determined on an IKA®- WERKE bomb calorimeter (C7000, GMBH & Co., Staufen, Germany), and the GE value of samples was read directly as MJ/kg from the calorimeter digital system. The method developed by McCleary et al. [19] was adopted to determine the total starch of feeds and ileal digesta using a Megazyme total starch assay procedure AA/AMG (Megazyme International Ireland Limited, Bray, Co. Wicklow, Ireland). Absorbance was read at 510 nm on a spectrophotometer (Varian Cary 50 Bio UV–Visible spectrophotometer Mulgrave, Victoria, Australia) against milli-Q water.

Diets and ileal digesta samples were analyzed at the Australian Proteome Analysis Facility Ltd., (APAF, Sydney, NSW, Australia) for indispensable and dispensable amino acids. Samples underwent a 24-h liquid hydrolysis in 6 M HCl at 110 °C. After hydrolysis, all AA were analyzed using the high-sensitivity quantification technique of Waters AccQTag Ultra chemistry on a Waters ACQUITY UPLC system. The mineral contents of diets and tibia bone ash were measured according to the methods described by [20] using nitric acid, hydrochloric acid and perchloric acid as analysis substrates. Copper, Fe, Mn, Zn, Ca, mg and P were then measured under different wavelengths using inductively coupled plasma-mass spectroscopy (ICP-MS) (Vista MPX, Melbourne, Australia).

Ileal Digestibility of Nutrients

The concentration of TiO2 in the diets and ileal digesta was measured according to the calorimetric procedure of Short et al. [21]. The concentration of TiO2 was used to calculate the digestibility of nutrients according to the index method described by Moughan and Schuttert [22] and revised by [23]. The following equation was used to calculate apparent ileal digestibility of nutrients:

(1)AIDN =1−TiO2Diet×NDigestaTiO2Digesta×NDietwhere AIDN is the apparent ileal digestibility of nutrients coefficient, NDigesta is the nutrient concentration in digesta (g/kg), TiO2Digesta is the TiO2 concentration in digesta (g/kg), NDiet is nutrient concentration in diets (g/kg) and TiO2Diet is the TiO2 concentration in feeds (g/kg).

Tissue Protein Concentration and Digestive Enzyme Activities

The concentration of protein in both the jejunal and pancreatic tissue homogenate was measured using the Comassie dye-binding procedure described by [24]. The general proteolytic activity (GPA) of pancreas was determined as described by Travid [25] and Susbilla et al. [26]. Activity of trypsin in the pancreatic tissue homogenate was determined using method of Caviedes-Vidal and Karasov [27]. The Winkler and Stuckmann [28] method was adopted to determine pancreatic lipase activity. Sucrase and maltase activities in jejunum homogenate were assessed by incubation with fixed substrate concentrations according to a method developed by [29] and modified by [30] and as standardized for poultry by Iji et al. [31]. The activity of aminopeptidase was assessed as described by Maroux et al. [32] and modified by [27]. Methods described by [33] and [34] were used to determine alkaline phosphatase activity.

Animal Ethics

The experimental design and methodology were approved by the Animal Ethics Committee of the University of New England, Australia under approval no: AEC15-084. The terms and conditions of the Code of Practice for the Use of Animals for Scientific Purposes issued by the Australian Bureau of Animal Health, were followed regarding experimental animal health and husbandry.

Statistical Analysis

Data collected were analysed according to the General Linear Model (GLM) of Minitab statistical software version 17 [35]. Fisher’s protected least significant difference (LSD) method was adopted to detect significant differences between means at P ≤ 0.05.

Results

The inclusion of CSM did not have any effect on feed intake of the birds at any stage of the growth period (Table IV). The weight gain (WG) of birds improved (P < 0.05) with increasing levels of CSM in the diet at the starter and grower phases, but this was not observed during the finisher phase. Similarly, during the same periods, feed conversion ratio (FCR) improved (P < 0.05) with increasing levels of CSM, especially in the birds fed lower levels of the meal. Enzyme supplementation, especially at 100 mg enzyme/kg diet, increased (P < 0.05) WG in birds at day 10 and day 24. However, FCR was consistently improved (P < 0.05) with each level of enzyme supplementation throughout the study period.

Treatments 1–10 days 1–24 days 1–35 days
CSM levels Enzyme (mg/kg) FI WG FCR FI WG FCR FI WG FCR
None 0 352.4 325.4 1.088 1801.0 1400.5 1.286 3750.7 2547.4 1.473
100 353.9 334.0 1.060 1816.6 1464.5 1.241 3709.9 2638.1 1.407
150 341.8 326.4 1.047 1800.6 1419.7 1.269 3755.7 2617.2 1.435
Low2 0 349.5 327.5 1.067 1812.4 1460.7 1.241 3798.1 2635.5 1.442
100 353.7 337.9 1.047 1838.5 1514.1 1.214 3743.1 2697.0 1.388
150 359.8 342.9 1.048 1836.3 1473.3 1.246 3684.1 2662.0 1.385
High3 0 357.0 330.2 1.085 1862.8 1453.4 1.281 3898.2 2672.9 1.453
100 355.6 336.6 1.057 1826.3 1503.9 1.214 3766.9 2707.4 1.392
150 359.8 326.1 1.076 1781.2 1470.5 1.211 3621.7 2584.6 1.384
SEM4 1.57 1.48 0.003 10.8 8.39 0.006 20.8 14.2 0.007
Main effects
CSM levels
None 349.4 328.6b 1.065ab 1806.0 1428.2b 1.265a 3738.8 2600.9 1.438
Low 354.4 336.1ab 1.054b 1829.0 1482.7a 1.234b 3741.8 2664.8 1.405
High 354.5 331.0a 1.073a 1823.4 1475.9a 1.236b 3762.3 2655.0 1.410
0 353.0 327.7b 1.080a 1825.4 1438.2b 1.269a 3815.7a 2618.6 1.456a
100 354.4 336.1ab 1.055b 1827.1 1494.2a 1.223b 3740.0ab 2680.8 1.396b
150 350.9 331.8a 1.057b 1806.0 1454.5b 1.242b 3687.2b 2621.3 1.401b
Significance
CSM 0.308 0.077 0.029 0.685 0.010 0.011 0.871 0.127 0.094
Enzyme 0.641 0.048 0.001 0.697 0.013 0.001 0.038 0.116 0.001
CSM × Enzyme 0.171 0.148 0.255 0.604 0.998 0.103 0.225 0.384 0.883
Table IV. Feed intake (g/Bird), Body Weight Gain (g) and Feed Conversion Ratio of Broiler Chickens in Response to Dietary Cottonseed Meal Supplemented with Three Levels of Microbial Enzyme (Axtra XB) 1

The results of the effect of CSM inclusion and microbial enzyme supplementation on nutrient digestibility are presented in Table V. Digestibility of protein and gross energy was not affected (P > 0.05) by CSM inclusion in the diets. Furthermore, there was no significant difference (P > 0.05) between low and high levels of CSM inclusion in diet on the digestibility of starch, although the results obtained were significantly (P < 0.012) lower compared with birds fed the control diet. In terms of dry matter digestibility, birds fed the control diet had higher (P < 0.05) digestibility than those fed diets containing a low level of CSM. With microbial enzyme supplementation, the digestibility of protein (P < 0.05) and starch (P < 0.001) was significantly improved in birds fed diets containing CSM compared to the birds on the control diet. Again, birds fed the control diet had higher (P < 0.05) starch digestibility than those fed diets containing the lower level (100 mg/kg diet) of the enzyme. The interaction between CSM inclusion levels and enzyme supplementation significantly (P < 0.05) affected starch digestibility, resulting in better digestibility in birds fed diets with CSM and enzyme compared to those on the control diet.

CSM levels Enzyme (mg/kg) Protein Gross energy Starch DM
None 0 0.80 0.70 0.95a 0.78
100 0.82 0.70 0.97a 0.78
150 0.81 0.70 0.96a 0.79
Low1 0 0.80 0.68 0.94b 0.78
100 0.82 0.70 0.96a 0.77
150 0.81 0.69 0.96a 0.78
High2 0 0.79 0.69 0.93b 0.80
100 0.81 0.71 0.96a 0.78
150 0.81 0.71 0.96a 0.78
SEM3 0.003 0.003 0.002 0.237
Main effects
CSM levels
None 0.81 0.70 0.96a 0.79a
Low 0.81 0.70 0.95b 0.77b
High 0.80 0.69 0.95b 0.78ab
0 0.80b 0.69 0.94b 0.79a
100 0.82a 0.70 0.96a 0.78b
150 0.81ab 0.70 0.96a 0.78ab
Significance
CSM 0.579 0.532 0.012 0.033
Enzyme 0.013 0.475 0.002 0.046
CSM level × Enzyme 0.991 0.912 0.035 0.138
Table V. Effect of Cottonseed Meal Inclusion with Microbial Enzyme Supplementation (Axtra XB) in Diets on Nutrient Digestibility of the Broiler Chickens at Day 24

Table VI shows the analyzed amino acid composition of dietary treatments, while Table VII shows a summary of the results of the apparent ileal digestibility of amino acids. The digestibility of arginine (P < 0.05), glutamic acid (P < 0.05) and threonine (P < 0.001) improved with increased CSM inclusion, while methionine digestibility increased (P < 0.05) with enzyme supplementation. There was no effect of treatment on the other amino acids, nor was there any interaction between CSM inclusion and microbial enzyme supplementation on the digestibility of amino acids.

Enzyme (mg/kg) 0 100 150
CSM 0 10% 12% 0 10% 12% 0 10% 12%
Histidine 5.0 4.8 4.9 4.6 4.8 4.9 4.6 4.7 4.6
Serine 9.9 9.7 9.9 8.9 9.2 9.5 8.8 8.8 8.6
Arginine 13.0 13.0 12.9 16.0 16.9 16.6 17.5 17.9 17.3
Glycine 9.6 9.3 9.8 8.3 8.8 8.9 8.4 8.4 8.1
Aspartic acid 18.2 18.1 17.9 15.8 16.8 16.6 15.6 16.0 15.6
Glutamic acid 43.1 42.3 42.2 41.3 42.4 41.9 41.1 41.5 41.0
Threonine 8.6 8.4 8.6 9.1 9.5 9.6 9.3 9.4 9.3
Alanine 9.0 8.8 9.1 8.0 8.4 8.3 7.9 8.0 7.8
Proline 13.6 13.2 13.5 12.0 12.2 12.2 11.8 11.7 11.6
Lysine 12.1 12.1 12.2 12.3 13.6 13.3 12.4 13.9 12.9
Tyrosine 4.4 4.8 4.2 4.2 4.1 3.9 4.1 3.8 4.0
Methionine 4.1 4.9 4.1 5.0 5.6 5.3 5.2 5.6 5.6
Valine 10.4 10.1 10.2 9.4 9.7 9.6 9.2 9.4 9.2
Isoleucine 8.7 8.4 8.5 7.4 7.7 7.6 7.4 7.6 7.4
Leucine 15.5 15.0 15.3 13.6 14.1 14.0 13.2 13.4 13.1
Phenylalanine 9.9 9.6 9.7 9.3 9.6 9.6 9.2 9.4 9.1
Table VI. Analysed Amino Acid Composition (mg/g) of Dietary Treatments
Treatments Amino acids
CSM levels Enzyme (mg/kg) His Ser Arg Gly Asp Glu Thr Ala Pro Lys Try Met Val Ile Leu Phe
None 0 0.80 0.77 0.85 0.74 0.75 0.84 0.75 0.71 0.79 0.85 0.80 0.90 0.75 0.76 0.76 0.79
100 0.82 0.80 0.87 0.76 0.76 0.86 0.78 0.74 0.82 0.86 0.81 0.92 0.77 0.79 0.79 0.82
150 0.80 0.78 0.85 0.75 0.75 0.84 0.74 0.71 0.79 0.85 0.83 0.93 0.75 0.76 0.76 0.79
Low1 0 0.80 0.79 0.89 0.75 0.76 0.87 0.80 0.73 0.82 0.85 0.83 0.90 0.77 0.78 0.78 0.82
100 0.82 0.79 0.91 0.76 0.77 0.87 0.80 0.74 0.82 0.86 0.82 0.93 0.77 0.78 0.78 0.82
150 0.82 0.79 0.90 0.76 0.77 0.86 0.80 0.74 0.82 0.87 0.81 0.93 0.77 0.78 0.78 0.82
High2 0 0.82 0.79 0.91 0.76 0.77 0.87 0.80 0.74 0.82 0.86 0.83 0.93 0.77 0.78 0.79 0.83
100 0.82 0.79 0.91 0.75 0.77 0.87 0.81 0.74 0.83 0.86 0.82 0.93 0.77 0.78 0.78 0.82
150 0.80 0.78 0.89 0.74 0.76 0.86 0.79 0.73 0.81 0.86 0.79 0.93 0.75 0.77 0.76 0.81
SEM3 0.005 0.005 0.004 0.005 0.005 0.004 0.005 0.006 0.004 0.004 0.007 0.003 0.005 0.005 0.005 0.005
Main effects
CSM levels
None 0.81 0.78 0.86b 0.74 0.75 0.85b 0.76b 0.72 0.80 0.85 0.82 0.92 0.76 0.77 0.77 0.80
Low 0.81 0.79 0.89a 0.76 0.77 0.87a 0.79a 0.74 0.82 0.86 0.82 0.92 0.77 0.78 0.78 0.82
High 0.81 0.78 0.90a 0.75 0.76 0.86a 0.80a 0.73 0.81 0.86 0.81 0.93 0.76 0.78 0.78 0.82
0 0.81 0.78 0.89 0.75 0.76 0.86 0.78 0.73 0.81 0.86 0.82 0.91a 0.76 0.78 0.77 0.81
100 0.82 0.79 0.89 0.76 0.77 0.86 0.79 0.74 0.82 0.86 0.82 0.93ab 0.77 0.78 0.79 0.82
150 0.81 0.78 0.88 0.74 0.76 0.85 0.78 0.73 0.81 0.86 0.81 0.93b 0.76 0.77 0.77 0.81
Significance
CSM 0.809 0.677 0.009 0.308 0.476 0.038 0.001 0.714 0.092 0.550 0.773 0.204 0.629 0.922 0.684 0.102
Enzyme 0.328 0.438 0.238 0.280 0.826 0.467 0.265 0.613 0.337 0.756 0.766 0.046 0.487 0.492 0.401 0.369
CSM x Enzyme 0.819 0.879 0.627 0.709 0.927 0.843 0.818 0.798 0.622 0.841 0.553 0.415 0.755 0.767 0.657 0.737
Table VII. Effect of Cottonseed Meal Inclusion with Microbial Enzyme (Axtra XB) Supplementation in Diets on the Amino Acid Digestibility of Broiler Chickens at Day 24

There was an interaction effect (P < 0.01) between CSM and enzyme on magnesium digestibility. Inclusion of CSM in the diets increased (P < 0.05) the digestibility of Cu and K, while it negatively affected (P < 0.01) the digestibility of P. Enzyme supplementation improved (P < 0.01) calcium and manganese digestibility. Neither CSM inclusion nor enzyme supplementation had any effect (P > 0.05) on ileal digestibility of Zn and Fe (Table VIII).

CSM levels Enzyme (mg/kg) Mn Cu Zn Ca mg K P Fe
None 0 0.54 0.50 0.51 0.59 0.58bc 0.94 0.69 0.40
100 0.58 0.48 0.52 0.60 0.57c 0.94 0.72 0.40
150 0.55 0.49 0.51 0.60 0.58bc 0.91 0.67 0.40
Low2 0 0.50 0.54 0.50 0.58 0.60ab 0.92 0.72 0.38
100 0.55 0.53 0.53 0.62 0.56c 0.89 0.72 0.39
150 0.55 0.50 0.53 0.62 0.56c 0.87 0.72 0.39
High3 0 0.50 0.53 0.52 0.58 0.58bc 0.92 0.65 0.38
100 0.54 0.50 0.51 0.61 0.62a 0.92 0.67 0.39
150 0.55 0.54 0.54 0.61 0.60ab 0.90 0.67 0.40
SEM4 0.006 0.005 0.003 0.003 0.004 0.007 0.007 0.004
Main effects
CSM Levels
None 0.55 0.49b 0.51 0.60 0.57 0.93a 0.72a 0.40
Low 0.53 0.53a 0.52 0.61 0.57 0.89b 0.69ab 0.39
High 0.53 0.53a 0.52 0.60 0.60 0.91ab 0.66b 0.39
0 0.51b 0.52 0.51 0.58b 0.58 0.92 0.70 0.39
100 0.56a 0.51 0.52 0.61a 0.58 0.92 0.69 0.40
150 0.55a 0.51 0.53 0.61a 0.58 0.89 0.69 0.40
Significance
CSM 0.129 0.001 0.515 0.147 0.007 0.055 0.002 0.513
Enzyme 0.006 0.184 0.182 0.001 0.904 0.200 0.437 0.845
CSM × Enzyme 0.770 0.057 0.130 0.346 0.002 0.897 0.588 0.922
Table VIII. Effect of CSM and Supplemental Microbial Enzymes (Axtra XB)1 on Apparent Ileal Digestibility of Minerals

The effect of dietary CSM levels and supplemental microbial enzyme on tissue protein concentration and enzyme activities at day 10 and day 24 for pancreas and jejunum is presented in Tables IX and X, respectively. The pancreatic lipase activity at 10 d of age increased (P < 0.026) with rise in CSM inclusion. At day 24, the activities of all pancreatic enzymes were not affected (P > 0.05) by either CSM level or enzyme supplementation. Neither the inclusion level of CSM nor microbial enzyme supplementation had any effect (P > 0.05) on jejunal enzyme activities at day 10 of age. On day 24, pancreatic alkaline phosphatase activity showed an increase (P < 0.05) when 100 mg enzyme was supplemented in the diet, but no enzyme effect was noticed beyond this level. There was no interaction effect between CSM inclusion and enzyme supplementation levels on the intestinal enzyme activities of the birds.

Treatments Day 10 Day 24
CSM levels Enzyme (mg/kg) Protein ChymoT2 Trypsin GPA3 Lipase Protein ChymoT Trypsin GPA Lipase
None 0 84.4 2.97 2.93 0.60 4.46 97.1 2.23 2.54 0.44 3.28
100 87.4 2.76 2.69 0.55 4.76 105.8 2.62 2.95 0.46 4.19
150 77.5 3.13 3.11 0.58 4.68 93.1 2.35 3.22 0.40 3.93
Low4 0 78.4 3.09 2.57 0.61 5.51 116.1 2.12 3.13 0.42 3.52
100 91.1 3.30 2.90 0.58 4.70 119.4 2.60 3.40 0.48 3.43
150 82.5 2.53 3.03 0.59 4.50 94.4 2.23 3.14 0.45 3.62
High5 0 71.1 3.33 2.75 0.66 5.72 105.1 2.01 2.87 0.48 3.61
100 71.2 2.94 3.60 0.65 5.72 114.2 2.40 3.10 0.48 3.90
150 75.8 2.86 2.91 0.56 4.72 112.1 2.61 3.14 0.45 3.98
SEM6 2.31 0.118 0.103 0.011 0.124 3.55 0.124 0.172 0.001 0.092
Main effects
CSM levels
None 83.1 2.95 2.91 0.58 4.63b 98.7 2.40 2.90 0.43 3.80
Low 84.0 2.97 2.83 0.59 4.90ab 110.0 2.32 3.22 0.45 3.53
High 72.7 3.05 3.08 0.62 5.40a 110.5 2.34 3.04 0.47 3.83
0 78.0 3.13 2.75 0.62 5.23 106.1 2.12 2.85 0.45 3.74
100 83.2 3.00 3.06 0.59 5.07 113.2 2.54 3.15 0.47 3.84
150 78.6 2.84 3.01 0.58 4.63 100.0 2.40 3.17 0.43 3.84
Significance
CSM 0.097 0.949 0.605 0.188 0.026 0.327 0.964 0.777 0.135 0.329
Enzyme 0.602 0.635 0.416 0.149 0.091 0.331 0.417 0.727 0.166 0.170
CSM × Enzyme 0.719 0.590 0.351 0.316 0.190 0.734 0.940 0.980 0.568 0.493
Table IX. Effect of Dietary CSM Levels and Supplemental Microbial Enzyme (Axtra XB)1 on Pancreas Protein Concentration (mg/g Tissue) and Enzyme Activities (µmol/mg Protein/Min) AT Day 10 and Day 24
Treatments Day 10 Day 24
CSM levels Enzyme (mg/kg) Protein Maltase Sucrase AP2 APP3 Protein Maltase Sucrase AP2 APP3
None 0 60.3 0.48 0.05 0.36 3.81 108.1 0.42 0.039 0.17 3.54
100 70.5 0.48 0.05 0.33 3.97 131.9 0.41 0.041 0.22 3.47
150 60.9 0.55 0.05 0.26 4.44 113.7 0.42 0.039 0.17 3.74
Low4 0 65.7 0.47 0.04 0.35 4.82 119.6 0.47 0.037 0.17 3.17
100 65.9 0.53 0.05 0.35 5.00 95.1 0.46 0.038 0.20 3.55
150 70.1 0.52 0.06 0.32 4.42 105.8 0.45 0.041 0.18 3.40
High5 0 63.4 0.48 0.06 0.38 4.03 100.5 0.44 0.039 0.21 3.57
100 64.3 0.49 0.05 0.34 4.29 101.9 0.42 0.041 0.20 3.50
150 55.5 0.54 0.05 0.31 4.87 114.5 0.44 0.039 0.16 3.41
SEM6 1.66 0.013 0.002 0.015 0.134 3.24 0.009 0.001 0.010 0.057
Main effects
CSM levels
None 63.9 0.50 0.05 0.32 4.07 117.9 0.42 0.039 0.18 3.59
Low 67.3 0.51 0.05 0.34 4.75 107.2 0.46 0.039 0.19 3.37
High 61.1 0.51 0.05 0.34 4.40 105.9 0.43 0.040 0.19 3.49
0 63.1 0.48 0.05 0.36 4.22 109.4 0.44 0.039 0.18ab 3.43
100 66.9 0.50 0.05 0.34 4.42 110.0 0.43 0.040 0.21a 3.51
150 62.2 0.53 0.05 0.30 4.58 111.3 0.44 0.039 0.17b 3.51
Significance
CSM 0.328 0.956 0.998 0.713 0.071 0.235 0.140 0.928 0859 0.335
Enzyme 0.485 0.097 0.519 0.114 0.457 0.968 0.784 0.632 0.033 0.781
CSM × Enzyme 0.719 0.490 0.769 0.135 0.925 0.332 0.117 0.963 0.565 0.758
Table X. Effect of Dietary CSM Levels and Supplemental Microbial Enzyme (Axtra XB)1 on Protein Concentration (mg/g Tissue) and Enzyme Activities (µmol/mg Protein/Min) in Jejunum at Day 10 and Day 24

Discussion

Apparent Ileal Digestibility of Nutrients

There is little information available on how microbial enzyme supplementation of diets containing CSM affects nutrient digestibility. In the present study, the ileal digestibility of protein and gross energy was not affected by the CSM, while starch and dry matter digestibility was suppressed by CSM inclusion in the diet. One of the main concerns with the use of CSM in diets for broiler chickens is poor protein digestibility. The effect of anti-nutritional factors such as gossypol could not be excluded from the observed digestibility of protein and energy in the CSM-containing diets. Free gossypol in CSM has been found to have an inhibitory action by binding with free epsilon amino groups of lysine, thus reducing protein digestibility [1]. The fibre content of the diets containing CSM, which is higher in these diets than in the control diet (Table III), may be responsible for the reduction in starch and dry matter digestibility as a high level of fibre is known to affect the digestibility of starch and dry matter. Furthermore, in other studies involving CSM but without enzyme supplementation [36]–[38], apparent ileal digestibility of protein and energy was low compared to values for control diets and decreased with increasing CSM levels.

Adding enzymes to the dietary treatments of this study increased the digestibility of the starch and protein. It was previously reported that incorporating a xylanase and β-glucanase in a diet based on corn-and wheat increased starch digestibility [39], this may be due to the role of β-gluca-nase and xylanase in releasing more unavailable nutrients from cell walls existing in many dietary ingredients [40]. Digestibility of CP enhanced by test enzyme in this study as part of enzyme activity on reducing the negative impact of gossypol, which supports [41] result who concluded that CP and starch digestibility increased in broiler chickens fed CSM-containing diets with xylanase and β-glucanase blend.

The digestibility of arginine, glutamic acid, and threonine improved with increased CSM inclusion, while the other amino acids were unaffected. Only the digestibility of methionine improved with increased enzyme supplementation. The negative effect of gossypol content of CSM on amino acids has been reported [42], but the ileal digestibility coefficients we obtained for these amino acids were higher than have been reported by Ravindran et al. [43] and Huang et al. [44]. The variation in digestibility may be a result of processing methods, the type of CSM used or the methodology employed in the different studies.

The results obtained from this study showed an effect of CSM and microbial enzyme supplementation on the digestibility of some minor minerals. When CSM was increased, the digestibility of copper and potassium was better. It suggests that higher levels of CSM which could increase gossypol levels in diets does not influence the utilization of these minerals. However, this seems to be different for the digestibility of phosphorus. Ojewola et al. [45] reported similar finding showing that manganese, potassium and copper increased as CSM supplementation increased in broiler ration. On the contrary, Lorena-Rezende et al. [46] observed increased phosphorus digestibility when 30% CSM was supplemented with enzymes but in pigs. Increased enzyme supplementation promoted better digestibility of calcium and manganese by the birds. The enzyme may have reduced the negative impact of gossypol on mineral digestibility, suggesting that the use of enzyme in CSM-containing diets is required to increase mineral digestibility in broiler chicken diets.

Digestive Enzyme Activities

Reports on the effect of CSM inclusion in diets supplemented with microbial enzyme on endogenous enzyme activities are scanty in literature. The digestive enzymes in the gastro-intestinal tract have key functions in the breakdown and utilization of nutrients. Pancreatic lipase activity in this study was increased by higher CSM inclusion, which may derive from the observed increase in pancreas weight, while the activity of alkaline phosphatase in the jejunum increased with enzyme supplementation in the diet. The inclusion of CSM in diets for broilers may provide the ideal substrate (fat) for the activation of lipase and changes in the gut in the presence of the supplemented exogenous enzyme, and this may have enhanced the activity of these enzymes. Sun et al. [47] also observed increased digestive enzyme activity in broilers when fed diets containing fermented CSM. The activity of alkaline phosphatase is a reflection of the absorptive function of the intestine [48], hence supplementing enzymes in CSM-containing diets may be valuable for nutrient absorption.

Conclusion

The performance of broiler chickens in this study was not severely affected by high levels of CSM, and it was enhanced by enzyme supplementation. With enzyme supplementation, CSM can be included at levels up to 18% in broiler diets, especially during the finisher phase. CSM diets containing composite microbial enzyme supplements may be an effective method of reducing the negative and anti-nutritional effects of free gossypol and other anti-nutritional factors, thereby increasing the utilization of CSM.

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