Evaluation of Growth Performance, Carcass Characteristics, Digestibility and Ileum Histomorphology of Broiler Chickens fed Two Forms of Diets Supplemented with Exogenous Proteases

ABSTRACT

The aim of this study was to evaluate the effects of diets supplemented with exogenous proteases on the performance, carcass characteristics, digestibility and histomorphology of broilers. A total of 1,536 male broilers (one day old) were used for the study. All broilers were divided into 96 floor pens with 16 chicks each. The dietary treatments were as follows: A= basal diet; B= basal diet + 0.02% RelePro (alkaline, acidic, and neutral proteases), C= basal diet + 0.03% Kemzyme (multiproteases), and D=basal diet + 0.02% ProAct (protease-derived Bacillus licheniformis). They were fed in two forms of diets (mash or pelleted) in a 4x2 factorial design. Growth performance and carcass traits were evaluated. The digestibility of dry matter and crude protein were measured, as well as histomorphological parameters. The results showed that birds in the C and D groups in pelleted form had a higher body weight, production and performance index, and better feed conversion ratio. Chickens fed a pelleted diet with protease C, followed by proteases B and D, and chickens fed a diet mashed with protease C had a higher digestibility of dry matter and crude protein. Animals in the group receiving protease B in the pelleted diet had a higher crypt depth. Chickens fed pelleted diet with protease C had higher villus height to crypt depth ratio and goblet cells. The live weight and relative weight of the breast increased, and the relative weights of the wings decreased with pelleted diet. In conclusion, the use of protease had positive effects on growth performance, dry matter and crude protein digestibility, and ileum histomorphological parameters without negatively affecting carcass characteristics, which was more evident when feeding a pelleted diet to broilers.

Keywords: Proteases; diets forms; performance; digestibility; histomorphology; broilers

INTRODUCTION

The increasing interest in the use of enzymes in chicken feed is a consequence of the rising cost of feed ingredients (Huyan et al., 2022). However, in an effort to reduce feed costs, additives have been continuously introduced to improve the efficiency of protein utilization in broiler diets (Vieira et al., 2023). Although the use of exogenous enzymes in broiler diets has been extensively studied, the commercial use of enzymes is still relatively new. Phytases are becoming essential in modern feed formulations to digest phytate, which can bind vital nutrients such as phosphorus, calcium, and amino acids (Abbasi et al., 2019). Likewise, proteases have been introduced to the feed industry to liberate amino acids from undigested proteins (Lee et al., 2020). The practical use of exogenous proteases in chicken diets is well established and there is a growing interest in exploring the use of enzymes targeting additional substrates for feed protein utilization (Walk et al., 2018). Fewer products have only one substrate specificity, while the majority of commercial enzyme products now on the market have multiple enzyme activities (Mahmood et al., 2017). Endogenous proteases are synthesized and released in the intestine of chickens, which is generally considered sufficient to optimize the utilization of feed protein (Zheng et al., 2023). However, there are differences in the digestibility of proteins and amino acids in feed, and large amounts of proteins pass through the gastrointestinal tract undigested (Hu et al., 2020; Zhao & Liu, 2023). This undigested protein represents an opportunity for the addition of specific exogenous proteases (Angel et al., 2011). Studies suggest that the addition of exogenous proteases improves protein digestion, feed conversion and gut health in broilers fed normal or low-protein diets (Stefanello et al., 2016; Law et al., 2018; Ndazigaruye et al., 2019). However, for these enzymes to be commercially viable, they must remain active after the high heat treatments used in feed processing (Ravindran, 2013). Feed processing, such as pelleting, promotes performance, resulting in improved weight gain and feed efficiency compared to unprocessed mash feeds (Serrano et al., 2012; Massuquetto et al., 2019). Pelleted feeds provide more energy and nutrients per mass of feed intake (Lemons et al., 2021).

Microbial proteases appear to be more beneficial than animal or plant sources due to lower production costs and higher efficiency (Erdaw et al., 2018). Research by Zheng et al. (2023) confirms this and shows that different types of exogenous proteases (including alkaline, acidic, and neutral proteases) improve weight gain, feed intake and protein digestibility in broiler chickens. Furthermore, dietary supplementation with exogenous protease has been reported to have a positive effect on growth performance and intestine morphology of broilers (Cowieson et al., 2016; Xu et al., 2017; Huyan et al., 2022). Cowieson et al. (2017) suggested that the benefits of exogenous proteases may be due to improved protein and energy digestibility, as well as positive effects on gut morphological characteristics.

The addition of exogenous proteases to the diet of broiler chickens could be influenced by gastrointestinal pH, age, dosage and feed formulation (Zheng et al., 2023). The variety of feed ingredients and the various proteases, which are not usually well characterized, as well as methodological differences, are other factors that often lead to contradictory and varying results in studies (Freitas et al., 2011). Moreover, enzyme addition (full matrix or ontop), interaction with phytase, and feed processing may be other factors affecting the repeatability of protease use. To our knowledge, there is limited research on applying the full matrix of proteases in broiler diets containing phytase formulated with the full matrix and prepared in two feed forms. Therefore, this study aimed to examine the inclusion of various commercial proteases in pelleted and mash feeds containing phytase on growth performance, carcass characteristics, nutrient digestibility, and ileum histomorphology of broiler chickens.

MATERIALS AND METHODS

Study design, birds, and housing

A total of 1,536 one-day-old broiler chicks (Ross 308) were obtained from a local hatchery. All chicks were sexed with feathers, weighed into groups, and randomly as-signed to 96 floor pens (100 cm2) as experimental units using a completely randomized block design (rooms as blocking factor). Four diets (A, B, C, D) were fed in 2 dietary forms (mash or pelleted) in a 4x2 factorial design, resulting in 8 dietary treatments (see Table 1). Each treatment was replicated 12 times (6 in each room), with 16 chicks per floor pen (half males and half females).

Table 1
Dietary treatments for broiler chickens.

Dietary treatments	Feed Form	Protease	Description
A	Mash	None	Basal diet in the mash form without any additives
B	Mash	RelePro® 1	Basal diet in the mash form + add 0.02% RelePro
C	Mash	Kemzyme® 2	Basal diet in the mash form + add 0.03% Kemzyme
D	Mash	ProAct® 3	Basal diet in the mash form + add 0.02% ProAct
A	Pellets	None	Basal diet in the pellets form without any additives
B	Pellets	RelePro®	Basal diet in the pellets form + add 0.02% RelePro
C	Pellets	Kemzyme®	Basal diet in the pellets form + add 0.03% Kemzyme
D	Pellets	ProAct®	Basal diet in the pellets form + add 0.02% ProAct

• ¹
  RelePro® Protease contains alkaline, acidic and neutral proteases, making it stable and effective throughout the gastrointestinal tract.
• ²
  Kemzyme® Protease contains multiproteases.
• ³
  ProAct® Protease is a serine protease preparation derived from a genetically modified strain of Bacillus licheniformis

Exogenous phytase (OptiPhos® Plus) was used to release phytate-bound phosphorus, calcium, and amino acids in feed ingredients, and multicarbohydrase (Smart non-starch polysaccharide enzyme; NSPase) was used to improve viscosity problems associated with certain NSPs and for its the ability to degrade cell wall polysaccharides. Both were used at a standard dose of 0.02% for each dietary treatment (A-D). The full matrix values of phytase were applied in all diet formulations.

RelePro® (Addican, Miscouche, Canada), Kemzyme® (Kemin Industries, Inc., USA) and RonzymeProAct® (DSM Nutritional Products Ltd, Basel, Switzerland) were used in the feeds with full matrix values to degrade proteins and proteinaceous anti-nutritional factors in commercial feeds (Landy and Toghyani, 2018) at a dosage of 0.02% (B), 0.03% (C) and 0.02% (D) per kg of diet, respectively. The dietary treatments were formulated according to the nutrient requirements of the Ross 308 Management Guide recommendations (Aviagen, 2022) at two feeding stages: starter stage (1 to 15 days of age) and grower stage (15 to 30 days of age). The feed ingredients with enzymes and nutrient content of the dietary treatments are listed in Table 2. Each treatment during the feeding stages were analyzed in the Biovet feed analysis laboratory (Huvepharma, Inc., Peachtree City, GA) for chemical compositions such as dry matter, crude protein, crude fat, crude fiber, crude ash, starch, and total sugars, according to the methods of the Association of Official Analytical Chemists (AOAC, 2012).

Table 2
Feed ingredients and nutrient content of dietary treatments (%).

Feed Ingredients (%)	Broiler Starter (1-15 d)				Broiler Grower (15-30 d)
	A	B	C	D	A	B	C	D
Corn	61.79	62.22	62.37	62.20	66.56	66.94	66.93	66.90
Soybean meal (48%)	24.61	22.89	22.63	22.87	21.06	19.31	19.20	19.28
Corn DDGS	6.00	6.00	6.00	6.00	8.00	8.00	8.00	8.00
Wheat bran	3.82	5.10	5.10	5.10	0.95	2.34	2.42	2.38
Monocalcium phosphate	0.48	0.48	0.48	0.48	0.37	0.37	0.37	0.37
Limestone	1.39	1.40	1.40	1.40	1.30	1.31	1.31	1.31
DL-Methionine	0.40	0.38	0.38	0.38	0.34	0.33	0.33	0.33
L-Lysine HCL	0.43	0.43	0.43	0.43	0.38	0.39	0.39	0.39
L-Threonine	0.17	0.15	0.17	0.17	0.13	0.11	0.13	0.13
L-Valine	0.09	0.08	0.09	0.08	0.06	0.05	0.06	0.05
T-Binder	0.10	0.10	0.10	0.10	0.10	0.10	0.10	0.10
Common salt	0.33	0.36	0.43	0.37	0.35	0.34	0.34	0.34
Vit. and Min. premix a	0.20	0.20	0.20	0.20	0.20	0.20	0.20	0.20
Smart NSPase	0.02	0.02	0.02	0.02	0.02	0.02	0.02	0.02
Turbo Grow b	0.10	0.10	0.10	0.10	0.10	0.10	0.10	0.10
Lipidol c	0.05	0.05	0.05	0.05	0.05	0.05	0.05	0.05
OptiPhos® Plus	0.02	0.02	0.02	0.02	0.02	0.02	0.02	0.02
RelePro®	0.00	0.02	0.00	0.00	0.00	0.02	0.00	0.00
KEMZYME®	0.00	0.00	0.03	0.00	0.00	0.00	0.03	0.00
ProAct®	0.00	0.00	0.00	0.02	0.00	0.00	0.00	0.02
TOTAL	100.00	100.00	100.00	100.00	100.00	100.00	100.00	100.00
Analyzed nutrient
M.E.	3015	3015	3015	3015	3075	3075	3075	3075
Dry matter	88.75	88.75	88.65	88.40	88.85	88.55	88.85	88.50
Crude protein	21.15	20.90	20.45	20.45	19.15	18.55	18.80	19.05
Crude fat	3.35	3.30	3.25	3.20	3.45	3.30	3.40	3.50
Crude fiber	1.95	2.10	2.15	2.05	2.25	2.15	1.95	2.00
Crude ash	5.25	5.25	5.15	5.30	4.80	4.65	4.60	4.70
Starch	41.50	41.30	41.75	41.75	44.25	44.75	44.85	43.85
Total sugar	4.30	4.35	4.25	4.15	3.70	3.75	3.95	3.80
Ca	0.90	0.90	0.90	0.90	0.84	0.84	0.84	0.84
Total P	0.51	0.51	0.51	0.51	0.45	0.46	0.46	0.46
Calculated amino acids
d Lysine	1.25	1.25	1.25	1.25	1.13	1.13	1.13	1.13
d Methionine	0.68	0.68	0.68	0.68	0.62	0.62	0.62	0.62
d Threonine	0.84	0.84	0.83	0.84	0.75	0.76	0.76	0.76
d Arginine	1.10	1.10	1.10	1.10	1.00	1.00	1.00	1.00

• ^a
  Contains in kg of premix: Vit. A = 2,400,000 IU; Vit. D = 1,000,000 IU; Vit. E = 16,000 IU; Vit. K = 800 mg; Vit. B1 = 600 mg; Vit. B2 = 1600 mg; Vit. B6 = 1000 mg; Vit. B12 = 6 mg; Biotin = 40 mg; Folic acid = 400 mg; Niacin; 8000 mg; Pantothenic acid = 3000 mg; Cobalt = 80 mg; Copper = 2000 mg; Iodine = 400 mg; Iron = 1200 mg; Manganese = 18,000 mg; selenium = 60 mg; zinc = 14,000 mg.
• ^b
  Contains in kg Turbo Grow: yeast = 435,000 mg; betaine hydrochloride = 60,000 mg; calcium propionate = 50,000 mg; calcium format = 30,000 mg; citric acid = 20,000 mg; diatomaceous earth = 100,000 mg.
• ^c
  Lipidol helps to emulsify fats during digestion.

All optimal environmental conditions were maintained according to the guidelines of the Ross strain. The room temperature and humidity were set at 35°C and 40% when the chicks arrived (on the first day), and then were gradually lowered by 2°C every three days until a constant temperature of 22°C and 50% was reached after 25 days of age. The lighting program was offered for 24 hours (30-40 lux) at the age of 1-7 days and for 18 hours (at least 20 lux) at the age of 8-30 days. Feed and water were offered ad libitum to the birds throughout the experimental period. All chicks were vaccinated against Newcastle disease at the hatchery according to the manufacturer’s instructions (Fort Dodge Animal Health-USA). This study was approved by the Scientific Ethics Committee of King Saud University (SEC-KSU-20), which complies with the ARRIVE guidelines for in vivo trials.

Growth performance parameters

The performance parameters of the broilers were evaluated in the starter (1 to 15 days of age) and grower stages (15 to 30 days of age). The live body weight and amount of feed were recorded for each replicate pen per treatment on days 1, 15 and 30. Weight gain (the difference between the final and initial body weight) and feed intake (the difference between the rejected and offered feed) were calculated according to Diler et al. (2021). Furthermore, feed conversion ratio was determined based on the feed intake of the offered feed divided by the weight gain during the feeding stage (Dai et al., 2021). In addition, the production efficiency index and performance index were evaluated as parameters for the economic, productive, and welfare status of broiler chickens during the feeding stage (Huff et al., 2013; Zhang et al., 2023).

Production efficiency index = (livability multiplied by live weight) divided by (age in days multiplied by feed conversion ratio), all multiplied by 100.

Performance index = body weight gain divided by feed conversion ratio, all multiplied by 100.

The cumulative mortality rate for each dietary treatment was expressed as a percentage during the feeding stages (Itafa et al., 2021).

Cumulative mortality rate= (total mortality in the feeding stage per pen) / (number of chickens in the pen) X 100.

Nutrient digestibility

At 31 days of age, 2 chickens per pen (1 male and 1 female, 24 per treatment) were separated and reared in battery cages intended for digestion experiments, with 12 cages per treatment with 2 chickens each. The chickens had 3 days to acclimatize to the feed. Fecal samples were then collected and stored (-20°C) for 4 days to determine the digestibility of dry matter and crude protein using the direct method (Raza et al., 2023). The feed and fecal samples were oven-dried at 105°C for 24 hours to calculate dry matter, while crude protein was determined using the Kjeldahl method (AOAC, 2012). The digestibility of dry matter and crude protein was calculated using the following equations: Digestibility % = 100 minus [100 multiplied by (fecal extracted divided by feed intake for each dry matter and crude protein)].

Ileum histomorphology parameters

At 31 days of age, 12 chickens per dietary treatment were randomly selected to collect tissue samples from the ileum. A 2 cm long mid ileum segment was excised and fixed in a 10% formalin buffer solution for 72 hours before histomorphology measurement. All necessary steps were performed automatically, including dehydration, embedding in paraffin, and subsequent sectioning at a thickness of 4 μm on microscope slides using a microtome (Leica Biosystems, Germany). The slides were stained with hematoxylin, eosin, and Alcian blue, and then examined with a light microscope (Nikon, Tokyo, Japan) to measure all histomorphology parameters. The villus height (VH), villus width (VW), crypt depth (CD) and goblet cell count (GC) were measured on ten villi per tissue section (Lee et al., 2023). The villus surface area (SA=2π multiplied by (villus width at the bottom edge of the tissue surface divided by 2) multiplied by the villus height) and the ratio of villus height to crypt height (VH:CD) were calculated according to Huyan et al. (2022).

Carcass characteristics

At 31 days of age, 4 birds per pen (2 males and 2 females, 48 birds per treatment, 384 in total) were randomly selected and weighed for slaughter by decapitation after being deprived of feed for 10 hours to ensure that the digestive tract was empty. After slaughter, the carcass weight (CW) and body parts such as wings, breast (major and minor), saddle (thigh and drumstick) and back were weighed separately. Dressing yield (DY) and body parts were calculated and expressed as a percentage of live body weight.

DY = carcass weight divided by live weight, all multiplied by 100 (Alkhulaifi et al., 2022).

The percentage of body parts was calculated as body part divided by live weight, all multiplied by 100 (Thema et al., 2022).

Statistical Analysis

The average of pens was used as the experimental unit for all parameters studied, based on a completely randomized block design (room was block effect). Data were statistically analyzed based on a 4 x 2 factorial analysis of variance (4 diets x 2 feed form) using the GLM of SAS software (SAS, 2008). The statistical model used was as follows:

Observed (Yijb) = general mean (μ) + proteases (Pi, i = A, B, C, D) + feed form (Fj, j=mash, pellets) + protease * feed form ij + block (Bb) + the random error (eijb).

The data were tested for normality using the skewness and boxplot methods before statistical analysis. Tukey’s test (p < 0.05) was used to detect significant differences between means. All relative data were transformed (data/100) to check for significant differences at p<0.05. Finally, all values were expressed as mean ± standard error of the means (SEM) for each parameter.

RESULTS

Growth performance parameters

During the feeding phase, there was no mortality in any of the dietary treatments, so no such data were shown in this study. The effects of the dietary treatments (proteases and feed form) on the growth parameters of broiler chickens are shown in Table 3. The results showed that the chickens fed proteases C (0.03% protease Kemzyme®) and D (0.02% protease ProAct®) had lower live body weight and weight gain than the control group (A) and protease B birds (p<0.05) at the starter stage (1 to 15 days of age). At the grower stage (15 to 30 days of age) and overall stage (1 to 30 days of age), the chickens fed protease C (0.03% protease Kemzyme®) had higher live weight and weight gain than the control group (A) and protease B (p<0.05), but did not differ significantly from protease D. Regarding the feed form, the chickens fed with feed pellets had higher live weight and weight gain during the feeding stages than those fed with feed mash (p<0.05). The interaction between the dietary proteases and the feed form showed a significant effect on live weight and weight gain in the starter and grower stages (p<0.05). Live weight and weight gain were lower in chickens fed mash diet compared to the pellet form proteases, while proteases C (0.03% protease Kemzyme®) and D (0.02% protease ProAct®) were lower in the starter stage and higher in the grower stage compared to the other proteases in pellet-fed chickens.

Table 3
Effect of dietary treatments (proteases and feed form) on growth parameters of broiler chickens.

Item		Live body weight			Weight gain
		1 d	15d	30d	1-15 d	15-30 d	1-30 d
Proteases 1
A		42.57	487.5a	1667.1c	444.9a	1179.6c	1624.5c
B		42.56	490.6a	1706.5bc	448.0a	1216.0bc	1664.0bc
C		42.54	470.9b	1770.0a	428.4b	1299.1a	1727.5a
D		42.53	471.9b	1723.3ab	429.4b	1251.3ab	1680.7ab
SEM 2		0.02	3.89	13.15	3.89	11.91	13.19
Feed Form
Mash		42.55	418.1b	1534.5b	375.6b	1116.3b	1491.9b
Pellets		42.55	542.3a	1899.0a	499.7a	1356.7a	1856.4a
SEM 2		0.013	2.815	9.508	2.815	8.41	9.506
Mash	A	42.57	417.8cd	1461.4d	375.3cd	1043.5d	1418.9d
	B	42.54	431.0c	1576.4c	388.4c	1145.4c	1533.8c
	C	42.54	402.8d	1564.9c	360.3d	1162.1c	1522.4c
	D	42.53	420.9cd	1535.2cd	378.3cd	1114.3cd	1492.7cd
Pellets	A	42.57	557.0a	1872.7b	514.4a	1315.7b	1830.1b
	B	42.56	550.2a	1836.7b	507.6a	1286.5b	1794.1b
	C	42.55	538.9b	1975.1a	496.4b	1436.2a	1932.6a
	D	42.53	522.9b	1911.3a	480.4b	1388.3a	1868.8a
SEM 2		0.03	5.51	18.59	5.51	16.46	18.59
Source of variance (p-value)
Proteases		0.403	0.003	<.0001	0.0003	<0.0001	<0.0001
Feed Form		0.857	<0.0001	<.0001	<0.0001	<0.0001	<0.0001
Proteases * Feed Form		0.938	0.003	0.0003	0.004	0.0001	0.0003

• ^{a- d}
  Superscript numbers above the mean values for each parameter within a column indicate a significant difference (p < 0.05).
• ¹
  Protease additions: A= chickens fed basal diet without protease; B= chickens fed basal diet with 0.02% protease RelePro®; C= chickens fed basal diet with 0.03% protease Kemzyme®; D= chickens fed basal diet with 0.02% protease ProAct®.
• ²
  SEM= standard error of the mean values.

The effects of the dietary treatments (proteases and feed form) on the feed efficiency of broiler chickens are shown in Table 4. The results of the study show that feed intake in the starter stage was not affected by the protease additions (p>0.05). In the grower stage, protease D (grower and overall stage) reduced feed intake compared to the control group (p<0.05), but was not significantly different from the other proteases (B and C). In terms of feed form, the pellet-fed chickens consumed more feed than the mash-fed ones (p<0.05). In contrast, feed intake during the feeding stage was not affected by the interaction between proteases and feed forms (p>0.05). Feed conversion ratio was lower in proteases A (basal diet without protease) and B (0.02% protease RelePro®) than in protease C (p<0.05), but they were not significantly different from protease D (0.02% protease ProAct®) at the starter stage. In contrast, proteases C and D (grower and overall stage) were lower compared to the other proteases (p<0.05). As with feed form, feeding feed pellets improved feed conversion ratio compared to feed mash during the feeding stages (p<0.05). The interaction between feed treatments and feed form showed a significant effect on feed conversion ratio during the feeding stage (p<0.05). Chickens fed a pelleted diet with protease additions had lower feed conversion ratios than proteases A (basal diet without protease) and C (0.03% protease Kemzyme®) with mash diets at starter stage, while proteases C (0.03% protease Kemzyme®) and D (0.02% protease ProAct®) with pelleted diets were lower than the other proteases with both diet forms at the grower and overall stages.

Table 4
Effect of dietary treatments (proteases and feed form) on the feed efficiency of broiler chickens.

Item		Feed intake			Feed conversion ratio
		1-15 d	15-30 d	1-30 d	1-15 d	15-30 d	1-30 d
Proteases 1
A		581.3	1820.5a	2401.8a	1.31b	1.55a	1.48a
B		578.5	1794.9ab	2373.4ab	1.29b	1.47b	1.42b
C		575.9	1771.7ab	2347.7ab	1.35a	1.41c	1.38c
D		568.0	1753.7b	2321.7b	1.32ab	1.37c	1.36c
SEM 2		5.58	14.66	18.12	0.01	0.01	0.01
Feed Form
Mash		509.3b	1630.8b	2140.1b	1.36a	1.46a	1.44a
Pellets		642.6a	1939.6a	2582.2a	1.29b	1.43b	1.39b
SEM 2		3.859	10.14	12.96	0.01	0.01	0.01
Mash	A	511.1	1665.9	2177.0	1.36ab	1.59a	1.53a
	B	503.5	1621.4	2124.9	1.29cd	1.41d	1.38d
	C	510.5	1619.0	2129.5	1.41a	1.39d	1.40cd
	D	512.0	1616.9	2128.8	1.35bc	1.45cd	1.42bcd
Pellets	A	651.4	1975.1	2626.5	1.27d	1.50bc	1.43bc
	B	653.5	1968.4	2621.9	1.29d	1.53b	1.46b
	C	641.5	1924.4	2565.9	1.29d	1.34f	1.32f
	D	624.1	1890.6	2514.6	1.30cd	1.36f	1.34f
SEM 2		7.71	20.28	25.34	0.01	0.01	0.01
Source of variance (p-value)
Proteases		0.255	0.009	0.016	0.005	<0.0001	<0.0001
Feed Form		<0.0001	<0.0001	<0.0001	<0.0001	0.002	<0.0001
Proteases * Feed Form		0.099	0.36	0.197	0.0002	<0.0001	<0.0001

• ^{a- f}
  Superscript numbers above the mean values for each parameter within a column indicate a significant difference (p < 0.05).
• ¹
  Protease additions: A= chickens fed basal diet without protease; B= chickens fed basal diet with 0.02% protease RelePro®; C= chickens fed basal diet with 0.03% protease Kemzyme®; D= chickens fed basal diet with 0.02% protease ProAct®.
• ²
  SEM= standard error of the mean values.

The effects of dietary treatments (proteases and feed form) on the production index and economic indicators of broiler chickens are shown in Table 5. The results show that the chickens fed proteases A (basal diet without protease) and B (0.02% protease RelePro®) had a higher production efficiency index and performance index at the starter stage (1 to 15 days of age) than the other protease additions (p<0.05). At the grower and overall stages, chickens fed proteases C (0.03% protease Kemzyme®) and D (0.02% protease ProAct®) had a higher production efficiency index and performance index than the other proteases (p<0.05). As for the feed form, chickens fed with pelleted feeds had a higher production efficiency index and performance index during the feeding stages than those fed with feed mash (p<0.05). The interaction between proteases and feed form showed a significant effect on the production efficiency index and performance index during the feeding stage (p<0.05). Chickens fed pelleted diets with protease additions had higher production efficiency index and performance index than those fed mashed diets with protease additions at the starter stage, while proteases C (0.03% protease Kemzyme®) and D (0.02% protease ProAct®) with pelleted diets had higher results than the other proteases with both diet forms at the grower and overall stages.

Table 5
Effect of dietary treatments (proteases and feed form) on production index and economic indicators of broiler chickens.

Item		Production efficiency index			Performance index
		1-15 d	15-30 d	1-30 d	1-15 d	15-30 d	1-30 d
Proteases 1
A		249.2a	360.7d	376.4d	34.1a	76.6d	110.1d
B		253.6a	385.9c	399.3c	34.7a	82.5c	116.7c
C		234.0b	433.3a	434.7a	32.0a	95.4a	127.3a
D		238.0b	410.4b	416.3b	32.5b	89.4b	121.8b
SEM 2		2.91	4.82	4.34	0.43	1.26	1.29
Feed Form
Mash		206.1b	351.2b	357.4b	27.8b	76.7b	104.2b
Pellets		281.4a	443.9a	455.9a	38.9a	95.3a	133.7a
SEM 2		2.05	3.49	3.07	0.30	0.87	0.93
Mash	A	204.9cd	305.6e	317.8d	27.6cd	65.5d	92.5d
	B	222.2c	371.4cd	379.4c	30.0c	80.9bc	110.7c
	C	189.7d	375.1cd	373.4c	25.5d	83.6bc	108.9c
	D	207.5cd	352.9d	358.9c	28.0cd	76.8c	104.7c
Pellets	A	293.4a	415.9b	435.1b	40.6a	87.7b	127.5b
	B	284.9ab	400.4bc	419.1b	39.4ab	84.1bc	122.8b
	C	278.4ab	491.5a	496.1a	38.5ab	107.2a	145.6a
	D	268.6b	467.9a	473.6a	37.0b	101.9a	138.9a
SEM 2		4.12	6.82	6.13	0.59	1.74	1.82
Source of variance (p-value)
Proteases		<0.0001	<0.0001	<0.0001	<0.0001	<0.0001	<0.0001
Feed Form		<0.0001	<0.0001	<0.0001	<0.0001	<0.0001	<0.0001
Proteases * Feed Form		0.0003	<0.0001	<0.0001	0.0004	<0.0001	<0.0001

• ^{a- d}
  Superscript numbers above the mean values for each parameter within a column indicate a significant difference (p < 0.05).
• ¹
  Protease additions: A= chickens fed basal diet without protease; B= chickens fed basal diet with 0.02% protease RelePro®; C= chickens fed basal diet with 0.03% protease Kemzyme®; D= chickens fed basal diet with 0.02% protease ProAct®.
• ²
  SEM= standard error of the mean values.

Nutrient digestibility

The effects of the dietary treatments (proteases and feed form) on the digestibility of dry matter and crude protein in broilers are shown in Table 6. The results show that chickens fed diets with protease C (0.03% protease Kemzyme®), followed by protease D (0.02% protease ProAct®), had higher digestibility of dry matter and crude protein than the other proteases (p<0.05). In terms of feed form, the digestibility of dry matter and crude protein was higher in chickens fed feed pellets than in those fed feed mash (p<0.05). The interaction between feed proteases and feed form showed a significant effect on the digestibility of dry matter and crude protein (p<0.05). Chickens fed pelleted and C (0.03% protease Kemzyme®) followed by proteases B and D and chickens fed mashed diet with protease C had higher digestibility of dry matter and crude protein than those fed the other proteases.

Table 6
Effect of dietary treatments (proteases and feed form) on dry matter and crude protein digestibility of broiler chickens.

Item		Dry matter digestibility	Crude protein digestibility
Proteases 1
A		74.94c	65.64d
B		75.76bc	67.39c
C		77.05a	71.61a
D		75.91b	69.37b
SEM 2		0.233	0.308
Feed Form
Mash		75.42b	67.61b
Pellets		76.41a	69.39a
SEM 2		0.16	0.20
Mash	A	74.66c	65.59d
	B	75.14bc	66.20d
	C	76.29b	70.38b
	D	75.58bc	68.29c
Pellets	A	75.22bc	65.70d
	B	76.37ab	68.58bc
	C	77.80a	72.85a
	D	76.25b	70.45b
SEM 2		0.35	0.46
Source of variance (p-value)
Proteases		<.0001	<0.0001
Feed Form		0.0002	<0.0001
Proteases * Feed Form		0.401	0.033

• ^{a- d}
  Superscript numbers above the mean values for each parameter within a column indicate a significant difference (p < 0.05).
• ¹
  Protease additions: A= chickens fed basal diet without protease; B= chickens fed basal diet with 0.02% protease RelePro®; C= chickens fed basal diet with 0.03% protease Kemzyme®; D= chickens fed basal diet with 0.02% protease ProAct®.
• ²
  SEM= standard error of the mean values.

Histomorphology parameters

The effects of dietary treatments (proteases and feed form) on the histomorphology parameters of the ileum of broiler chickens are shown in Table 7. The results show that chickens fed protease C (0.03% protease Kemzyme®) had higher ileum histomorphology parameters such as VH, VW, SA, VH:CD and CG than those fed the other proteases (p<0.05). In terms of feed form, chickens fed a pellet diet had higher VH, VH:CD and CG and lower W than those fed mash (p<0.05). The interaction between protease additions and feed form had a significant effect on CD, VH:CD and CG (p<0.05), while L, W and SA were not affected (p>0.05). Chickens fed pelleted diets and protease B had higher CD than protease D (0.02% protease ProAct®). In addition, chickens fed a pelleted diet with protease C had higher VH:CD and CG than those fed the other proteases.

Table 7
Effect of dietary treatments (proteases and feed form) on ileum histomorphology parameters of broiler chickens.

Item		VH	VW	SA	CD	VH:CD	GC
		(μm)	(μm)	(mm2)	(μm)	(μm: μm)	(NO.)
Proteases 1
A		769b	174b	0.42b	115	6.78b	87b
B		786b	182b	0.45b	119	6.68b	82c
C		836a	199a	0.52a	112	7.49a	92a
D		806b	175b	0.44b	112	7.18b	88b
SEM 2		11.18	4.56	0.01	2.70	0.19	1.12
Feed Form
Mash		754b	198a	0.47	114	6.69b	82b
Pellets		844a	167b	0.44	115	7.42a	92a
SEM 2		7.90	3.22	0.01	1.91	0.13	0.79
Mash	A	740	182	0.42	113ab	6.72c	82c
	B	735	198	0.45	111ab	6.66c	80c
	C	778	220	0.53	115ab	6.79c	8,4c
	D	764	192	0.46	117ab	6.60c	83c
Pellets	A	797	167	0.42	117ab	6.85c	92b
	B	838	166	0.44	126a	6.70c	84c
	C	893	179	0.50	110ab	8.19a	99a
	D	847	158	0.42	107b	7.96b	93b
SEM 2		15.81	6.45	0.02	3.82	0.27	1.58
Source of variance (P-value)
Proteases		0.005	0.005	<.0001	0.240	0.009	<0.0001
Feed Form		<0.0001	<0.0001	0.059	0.757	0.003	<0.0001
Proteases * Feed Form		0.274	0.230	0.718	0.007	0.013	0.002

• ^{a- c}
  Superscript numbers above the mean values for each parameter within a column indicate a significant difference (p < 0.05).
• ¹
  Protease additions: A= chickens fed basal diet without protease; B= chickens fed basal diet with 0.02% protease RelePro®; C= chickens fed basal diet with 0.03% protease Kemzyme®; D= chickens fed basal diet with 0.02% protease ProAct®.
• ²
  SEM= standard error of the mean values.

Carcass characteristics

The effects of dietary treatments (proteases and feed form) on carcass characteristics and body components of broiler chickens are shown in Table 8. The current results show that the dietary treatments or their interaction effect had no effect on all parameters of carcass traits such as live weight, CW and DY, and body components such as relative weight of wings, breast, saddle and back (p>0.05). Regarding the effects of feed form, live weight and relative weights of breast were increased, and the relative weights of wings were decreased in chickens fed feed pellets (p<0.05), while other parameters were not affected when compared with mash feeds (p>0.05).

Table 8
Effect of dietary treatments (proteases and feed form) on carcass characteristics and body components of broiler chickenss.

Item		Carcass characteristics				Body components
		Live weight (g)	CW (g)	DY (%)	Wings (%)	Breast (%)	Saddle (%)	Back (%)
Proteases 1
A		1647	1237	76.52	7.25	25.92	26.56	5.24
B		1644	1066	64.81	7.26	25.32	26.91	5.12
C		1659	1076	64.86	7.10	25.45	26.71	5.24
D		1643	1061	64.54	7.26	25.11	27.28	5.37
SEM 2		18.15	94.08	6.49	0.06	0.22	0.25	0.09
Feed Form
Mash		1477b	1037	70.39	7.42a	25.07b	27.05	5.34
Pellets		1820a	1182	64.98	7.01b	25.83a	26.67	5.15
SEM 2		14.59	75.6	4.93	0.05	0.17	0.20	0.07
Mash	A	1472	1290	88.15	7.46	25.72	26.46	5.32
	B	1488	961	64.62	7.41	24.77	27.12	5.23
	C	1475	954	64.71	7.28	25.16	26.91	5.29
	D	1472	943	64.09	7.55	24.62	27.72	5.52
Pellets	A	1823	1183	64.89	7.04	26.11	26.66	5.16
	B	1800	1171	65.01	7.11	25.86	26.70	5.01
	C	1843	1198	65.01	6.91	25.74	26.50	5.19
	D	1813	1178	65.00	6.97	25.60	26.84	5.23
SEM 2		25.67	133.0	9.18	0.09	0.31	0.36	0.13
Source of variance (p-value)
Proteases		0.943	0.523	0.517	0.441	0.145	0.369	0.460
Feed Form		<.0001	0.167	0.353	<.0001	0.003	0.245	0.072
Proteases * Feed Form		0.800	0.5261	0.507	0.626	0.695	0.585	0.911

• ^{a- b}
  Superscript numbers above the mean values for each parameter within a column indicate a significant difference (p < 0.05).
• ¹
  Protease additions: A= chickens fed basal diet without protease; B= chickens fed basal diet with 0.02% protease RelePro®; C= chickens fed basal diet with 0.03% protease Kemzyme®; D= chickens fed basal diet with 0.02% protease ProAct®.
• ²
  SEM= standard error of the mean values.

DISCUSSION

Some studies suggest that the addition of exogenous proteases improves the growth performance, nutrient utilization, and intestinal morphology of broilers (Law et al., 2018; Ndazigaruye et al., 2019; Amiri et al., 2021), while others show minimal impacts (Ghazi et al., 2002; Freitas et al., 2011). Factors such as broiler age, protease type, and overall feed formulation likely influence the results (Zheng et al., 2023). In this study, 0.03% protease Kemzyme® and 0.02% protease ProAct® led to lower body weight, weight gain, production index and economic indicators, as well as higher FCR than the other protease additions (starter stage). This suggests that these proteases might have negatively impacted early growth. No significant differences in feed intake were observed between different protease additions (A-D) during the starter phase. This suggests that during the initial growth phase, chicks might not have been sensitive to the tested enzyme supplements in terms of feed intake. Dietary supplementation with a protease enzyme derived from Bacillus subtilis improved the growth performance and nutrient digestibility in broiler chickens during the starter stage (Jabbar et al., 2021). The gastrointestinal tract of broiler chickens is still developing in the first week, so their production of digestive enzymes may be limited (Alam et al., 2020). However, in the grower and overall stages, 0.03% protease Kemzyme® followed by 0.02% protease ProAct® led to higher live weight and weight gain, thus improving FCR compared to the control and protease B. This indicates that 0.03% protease Kemzyme®, which contained a multiprotease, might have promoted growth later during the grower stage. Therefore, the results on growth performance in response to supplemental protease enzyme could be due to the improvement of protein digestibility in broiler chickens (Kamel et al., 2020). Research suggests that protease supplementation enhances nutrient digestion and subsequent growth in broiler chickens, with a greater impact observed during the growing phase compared to the finishing phase (Doskovic et al., 2013). In the current study, birds fed with enzyme showed lower FCR compared to the control group without enzyme. These findings have practical economic implications for poultry producers, since an improved FCR could be achieved with the use of a protease enzyme. Improved production and performance indices lead to better economic outcomes for broiler producers. Higher live weight gain and feed efficiency have a direct impact on profitability. In addition, the higher growth performance parameters in broiler chickens fed feed pellets compared to feed mash is consistent with previous research demonstrating the influence of feed form on the growth of broiler chickens (Zhang et al., 2023). Denser pellets encourage higher feed intake due to ease of consumption and potentially reduced satiety when compared to mash. These findings suggest that protease additions and feed form have a significant impact on feed intake and feed conversion ratio in broiler chickens. The results are consistent with previous research that has also demonstrated the influence of protease additions and feed form on feed efficiency in broiler chickens (Lemme et al., 2004; Gracia et al., 2009). The protease enzyme derived from Bacillus subtilis in broilers diet (pellet form) improved the growth performance and nutrient digestibility from 15 to 28 days of age (Jabbar et al., 2021).

The results of the study showed that chickens fed diets with 0.03% protease Kemzyme® followed by 0.02% protease ProAct® had higher digestibility of dry matter and crude protein. The study revealed a significant interaction between protease additions and feed form, suggesting their combined effects on digestibility. Chickens fed a pelleted diet with protease C, followed by proteases B and D, and those fed mashed diet with protease C had the highest digestibility of dry matter and crude protein. This suggests a potential synergistic effect of specific proteases with pelleted feed in maximizing nutrient utilization. The purpose of adding exogenous proteases to feed is to enhance protein digestibility and inactivate protein-based antinutritional factors present in the diet (Law et al., 2018). The addition of a commercial protease derived from Bacillus licheniformis (ProAct) in pigs’ diets improved growth performance, nutrient digestibility, and intestinal morphology (Park et al., 2020). In the current study, CP digestibility was increased by protease supplementation irrespective of the levels of protein in the diet (Jabbar et al., 2021). Similar findings were also reported by Freitas et al. (2011). Angel et al. (2011) reported a 6.1% increase in protein digestibility in response to protease supplementation in diets consisting of variable levels of protein. Feeding pelleted feeds led to higher dry matter and crude protein digestibility compared to mash feeds. This aligns with the benefits of pelleted feed in improving nutrient utilization and reducing waste, leading to better nutrient absorption. Pelleting improved digestibility compared to the mash form, likely due to the heat and pressure during processing. This disrupts cell walls, making nutrients more available for absorption (Jiménez-Moreno et al., 2009).

Several studies have reported that exogenous proteases have positive effects on intestinal histomorphology parameters such as VH, SA, VH:CD and GC (Cowieson et al., 2017; Huyan et al., 2022). The small intestine plays a crucial role in the digestion and absorption of nutrients. Therefore, measurements of the histomorphology of the ileum such as VH, VH:CD, SA and GC are considered important indicators of the functioning of the intestinal mucosa and its function (Emami et al., 2012; Kim et al., 2017). However, chickens fed 0.03% protease Kemzyme® showed significantly improved ileum morphology, including higher VH, VW, SA and VH:CD, along with greater GC. This suggests that 0.03% Protease Kemzyme® positively influences intestinal development and function. Feeding pelleted feeds resulted in an increase in VH, VH:CD and GC compared to mash feeds. This is consistent with the improved nutrient utilization and digestibility associated with pelleted feed, potentially leading to better intestinal health. The study found a significant interaction between protease additions and feed form, suggesting that their combined effects influence ileal morphology. Chickens fed a pelleted diet with protease B had a higher CD than those fed 0.02% protease Pro-Act®. This suggests a possible interaction between 0.02% protease RelePro® and pelleted feed on crypt development. Conversely, chickens fed pelleted diet with protease C had the highest VH:CD and GC. This highlights the synergistic effect of 0.03% protease Kemzyme® and the pelleted feed on these important intestinal parameters. The addition of an exogenous protease enzyme to the basal diet can have a positive effect on the performance and intestinal morphology of broilers (Amiri et al., 2021).

The current results show that the dietary treatments or their interaction effect had no effect on any parameters of carcass characteristics such as live weight, CW and DY, nor on body components such as relative weight of wings, breast, saddle and back. On the other hand, live weight and the relative weights of the breast were increased, and the relative weights of the wings were decreased in chickens fed with feed pellets. This is consistent with Freitas et al. (2011) and Rehman et al. (2017), who reported that no effects of protease were observed on the characteristics of the carcasses and body components. In contrast, another study reported that supplementing the diet with exogenous protease increased the purification rate of the carcass and breast meat (Abudabos, 2012). This may be attributed to protease promoting protein deposition in the body’s muscles (Lauri et al., 2003).

CONCLUSION

In conclusion, the results provide valuable insights on the factors influencing broiler production and provide valuable information for the development of effective feeding strategies related to protease supplementation in broilers. The present results provide useful evidence that the use of Kemzyme® protease containing a multiprotease has positive effect on growth performance, dry matter and crude protein digestibility, and ileum histomorphology parameters without negatively affecting carcass characteristics when compared with the control group, which was more evident when feeding broiler chickens with feed pellets.

ACKNOWLEDGEMENTS

The authors thank King Saud University, Riyadh, Saudi Arabia, for funding this work through the Research Project Group (RSPD2024R581).

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• FUNDING
  This research was financially supported by King Saud University, located in Riyadh, Saudi Arabia. The specific grant came from the Research Project Group (RSPD2024R581).
• DATA AVAILABILITY STATEMENT
  The data that support the findings of this study are available from the corresponding author (H.H.A.) upon reasonable request.
• DISCLAIMER/PUBLISHER’S NOTE
  The published papers’ statements, opinions, and data are those of the individual author(s) and contributor(s). The editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions, or products referred to in the content.

Edited by

• Section Editor:
  Maria Fernanda Burbarelli

Data availability

The data that support the findings of this study are available from the corresponding author (H.H.A.) upon reasonable request.

Publication Dates

• Publication in this collection
  13 Jan 2025
• Date of issue
  2024

History

• Received
  08 July 2024
• Accepted
  20 Oct 2024