Effect of Phytase, Protease, and their Combination on the Nutrients Bioavailability and Performance Indices of Broilers Fed a Sorghum-Based Diet Under Local Climatic Conditions of Khyber Pakhtunkhwa Pakistan

Aaqil Muhammad1*, Asad Sultan1, Sarzamin Khan1 and Umer Sadique2

1Department of Poultry Science, Faculty of Animal Husbandry and Veterinary Sciences, The University of Agriculture Peshawar, Pakistan.

2College of Veterinary Sciences, Faculty of Animal Husbandry and Veterinary Sciences, The University of Agriculture Peshawar, Pakistan

ABSTRACT

The present study evaluated the effect of protease and phytase in a sorghum-based diet on nutrient bioavailability and performance indices of broilers. A total of 160-day-old broiler chicks were purchased from the local hatchery and assigned to four treatment groups replicated four times with 10 birds per replicate in a completely randomized design. There were four dietary treatments designated as control contained 100% maize-based diets without sorghum and enzyme, HDV-100+protease (T1) had 100% sorghum without any maize and supplemented with protease enzyme; HDV-100+phytase (T2) had 100% sorghum without maize and supplemented with phytase enzyme; HDV-100+protease+phytase (T3) had 100% sorghum without maize and supplemented with the combination of protease and phytase enzyme. The result showed improved body weight gain and feed conversion ratio during the overall experimental period in T1, T2, and T3 diet groups than in the control group. During the entire experimental period feed intake and livability remind unchanged. On days, 21 and 28 of the experimental period, the litter moisture content was not effected but at day 35 of the experimental period, the litter moisture content was calculated significantly higher in the T2 and T3 diet groups than in the control and T1 diet groups. Relative organ weight was not effected throughout the experimental period. The crude protein and apparent ileal metabolizable energy were calculated significantly higher in the T3 diet groups as compared to the remaining groups. Overall, the digestibility of essential amino and non-essential amino acid were recorded (P<0.05) higher in the T1, T2, and T3 diet group as compared to the control group. In conclusion, the replacement of maize with sorghum in combination with exogenous enzyme protease and phytase can effectively improve the growth performance and nutrient digestibility in the broiler.

Article Information

Received 02 March 2023

Revised 18 April 2023

Accepted 12 May 2023

Available online 26 July 2023

(early access)

Published 08 January 2025

Authors’ Contribution

AM conducted experiment and data collection. AS design experiment, statistical analysis, manuscript writing and reviewing. SK and US data manipulation and manuscript reviewing.

Key words

Maize, Sorghum, Exogenous enzymes, Nutrient digestibility, Broiler

DOI: https://dx.doi.org/10.17582/journal.pjz/20230302130319

* Corresponding author: [email protected]

0030-9923/2025/0001-0167 $ 9.00/00

Copyright 2025 by the authors. Licensee Zoological Society of Pakistan.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

INTRODUCTION

The poultry industry for feed production mostly relies on highly digestible feed for energy and protein, such as soybean meal and maize (Zhu et al., 2014). In tropical countries, maize is mostly used for bioethanol production and human consumption (Morgan and Choct, 2016). Due to higher metabolizable energy in compression to other cereal grains, it is commonly used in poultry feed as an energy source (Bhuiyan et al., 2013). Maize is used to feeding poultry in Pakistan, but it is also a principal food for humans. Maize production in some parts of Asia is hampered by low yields caused by drought (Wang et al., 2018), making it an unusual and expensive product, mainly for poultry production. The inadequate quantity and high cost of maize have posed a risk to the poultry industry because maize prices are high and unpredictable, using substitute feedstuffs may be the best approach in formulating feeds for profitable poultry production. Sorghum has long been used as a substitute feed component for maize in broiler diets due to its lower production cost. However, most poultry studies have focused on the effects of maize or sorghum-based diets and have not considered the incomplete replacement of the dietary cereal grain (Tancharoenrat et al., 2014). The response of exogenous enzyme supplementation has different results due to the composition of the cereal grains. Moreover, the phytate arabinoxylan and crude protein may affect the efficacy of exogenous enzymes like phytase and protease of the sorghum and maize grains-based diet in broilers (Choct, 2015). Although sorghums have higher protein content, the digestibility of amino acids is lower as compared to maize (Rostagno et al., 2011). The main protein in sorghum is kafirin, which has a low digestibility (Li et al., 2018). Kafirin, content in sorghum contains tannin and phytate, both of which are anti-nutrients that can impair energy and nutrient utilization (Pasquali et al., 2017). Tannin may bind with other nutrients rendering them inaccessible, while phytate binds to minerals and reduced its availability (Woyengo and Nyachoti, 2013). Exogenous enzymes such as phytase and protease have been widely studied in the broiler to reduce the negative effects of these anti-nutritional factors. As a result, the first goal of this study was designed to evaluate the effect of exogenous enzymes in broiler fed with sorghum-based diets.

MATERIALS AND METHODS

Bird’s husbandry and experimental diets

A total of 160 days old broiler chicks were purchased from the local hatchery and the birds were assigned to four treatment groups replicated four times having 10 birds per replicate in a completely randomized design. Before the experiment, the experimental house and all other equipment were washed and disinfected. Chicks were reared in a small brooding area where the temperature was kept at 34oC for the first week and gradually decreased, as the birds grew older, and continued at 22oC for the rest of the experimental period. All commercial and experimental diets were provided ad libitum, and birds had 24-h access to water and feed. Individually installed feeders and drinkers in each pen were used. Every day, the feeders and drinkers were cleaned. The birds in all groups were vaccinated according to the standard schedule used in the area. Four experimental diets were formulated fulfilling all the nutritional requirements of the broiler. In experimental diets, the sorghum replacement level was 0%, and 100% with diets formulated to be iso-nutritive and isoenergetic. There were four dietary treatments designated as control contained 100% maize-based diets without sorghum and enzyme, HDV-100+protease (T1) had only 100% sorghum without any maize and supplemented with protease enzyme; HDV-100+phytase (T2) had only 100% sorghum with without any maize and supplemented with phytase enzyme; HDV-100 (T3) had only 100% sorghum with without any maize and supplemented with the combination of protease and phytase enzyme. Protease, phytase, and their combination were added at the rate of 0.01% in the final diet after mixing. The dietary composition and calculated analysis of both the starter and finisher experimental diets are shown in Table I.

 

Table I. Dietary composition and calculated nutrient content of the experimental diets used in the study.

Ingredients (g)	Control	Diets
		T1	T2	T3
Maize corn	566	0.00	0.00	0.00
Sorghum grain	0.00	566	566	566
Soybean meal	346	346	346	346
Poultry oil	50.2	50.2	50.2	50.2
Di-calcium phosphate	17.5	17.0	17.0	17.0
Limestone	9.06	8.16	8.16	8.16
Salt	4.46	4.13	4.13	4.13
Lysine-HCL	2.18	2.20	2.20	2.20
Threonine	1.26	1.24	1.24	1.24
DL- Methionine	2.64	2.80	2.80	2.80
Choline and Coban 90	2.25	2.22	2.22	2.22
Vitamin premix٭	0.25	0.25	0.25	0.25

 

٭Vitamin mineral premix (each kg contained): retinol, 200,000 IU; tocopherol, 1072 IU; ascorbic acid, 26000 IU; cholecalciferol, 80,000 IU; thiamine, 11666 IU; menadione, 11,333 IU; pyridoxine, 33333 IU; niacin, 5,36,000 IU; folic acid, 13600 IU; riboflavin, 54,000 IU; methylcobalamin, 223 IU; biotin, 1340 IU; Ca, 195 g; Mg, 6 g; Fe, 2,000 mg; Na, 18 g; Zn, 1,200 mg; K, 70 g; Mn, 1,200 mg; Cu, 400 mg; I, 40 mg, Co, 20 mg and Se, 8 mg.

 

Growth performance parameters

Body weight gain and feed intake were calculated on weekly basis. Total body weight gain was calculated by subtracting initial weight from final body weight. The refused feed was subtracted from the offered feed to calculate feed intake. Data obtained from total body weight gain and feed intake were used to calculate the feed conversion ratio (FCR) (Shuaib et al., 2022). At 21 and 35 d of the experimental period, two birds of average body weight were randomly selected from each experimental unit and sacrificed after fasting for 2 h. The relative weight of the organs was determined concerning the body weight. On the days 21 and 35 of the experimental period litter samples were collected from four different places in each cage from surface to bottom from the area near drinkers and feeders. The collected litter samples were stored were mixed and stored in plastic bags. From each group, 70 grams were taken and weighed into trays. The samples were dried in a forced air oven at 65 °C for 24 h. The moisture content in the litter was determined according to the methods described by (AOAC, 2000).

Hematological analysis and nutrient digestibility

On day 35 of the experimental period, blood samples were collected after fasting the birds for 12 h before sampling. From representative birds, blood samples were collected in tubes containing EDTA as an anticoagulant. Samples were transported in ice packs to the laboratory to prevent deterioration. Blood samples were analyzed as described by Ritchie et al. (1994). On day 36 of the experimental period, five birds of uniform body weight and size were randomly selected and transferred to metabolic cages for the digestibility assy. The birds in the experimental groups were fed with experimental diets from day 37 to day 42 of age. Diets were provided in a mesh form and birds have free excess to water and feed all the time. During the last four days, excreta were collected and dried in a forced air oven and stored until further analysis. Feed and digesta proximate analysis was performed as outlined in the AOAC (2005) procedure. For the determination of indigestible contents, a UV absorption spectrophotometer was used (Williams et al., 1962). An adiabatic bomb calorimeter was used to determine the gross energy of feed and fecal samples. For the determination of apparent metabolizable energy (AME), the digestive marker method was used. The final AME values were calculated by using the formula described by (Adejumo et al., 2021). For ileal amino acid digestibility, the test ingredient and ileal digesta samples were oxidized with a hydrogen peroxide-formic acid-phenol solution, and the excess oxidation reagent was decomposed with sodium disulfite. After oxidation, samples were hydrolyzed for 24 hs in 6M HCl. The pH of the hydrolysate was adjusted to 2.20 before it was centrifuged, filtered, and then injected into an amino acid analyzer for the determination of the amino acid profile and then the apparent ileal amino acid digestibility was determined as described by (Ullah et al., 2016).

Statistical analysis

The data was analyzed by completely randomized design through SAS software employing one-way ANOVA. The mean difference was compared using the Least Significant Difference (Steel, 1997) test.

RESULTS

The results regarding the effect of the complete replacement of maize with sorghum and supplementation of protease and phytase enzymes individually and in combination on feed intake, weight gain, FCR, livability, and litter moisture are presented in Table II. The supplementation of enzymes alone or in combination did not effect the growth performance parameters like BWG, feed intake, and FCR during the starter phase from day 1-21. However from day 22-35 and day 1-35 higher (p<0.05) body weight gain and best FCR was recorded for the T3 diet group as compared to the T1, T2, and control groups. During the entire experimental period feed intake and livability remind unchanged. On days, 21 and 28 of the experimental period, the litter moisture content was not effected but on day 35 of the experimental period, the litter moisture content was calculated significantly higher in the T2 and T3 diet groups than in the control and T1 diet groups. Relative organ weight was not effected throughout the experimental period. The result of the hematological parameters and nutrient digestibility are presented in Table III. All the dietary treatments had no significant (p>0.05) effect on all the blood parameters. It is evident from the data that enzyme supplementation did not affect the dry matter and crude fat digestibility compared with a maize-based diet without enzyme supplementation. However, crude protein and apparent ileal metabolizable energy were calculated significantly (P<0.05) higher T3 diet groups as compared to the remaining group. Overall, the digestibility of essential amino and non-essential amino acid were recorded (P < 0.05) higher in the T1, T2, and T3 diet group as compared to the control group.

DISCUSSION

In the present study, the use of an exogenous enzyme in poultry improved the production performance parameters which is attributed to the enzyme’s ability to cause non-starch polysaccharide (NSP) degradation in the cell wall matrix of feed ingredients. This resulted in the release of encapsulated nutrients, as well as lower viscosity of digesta caused by soluble NSP and an improved rate of diffusion between enzyme and digestion end products (Selle et al., 2010; Gidado et al., 2020). Exogenous enzymes, by stimulating intestinal motility in broiler chicken, expose the locked nutrient in the intestine to endogenous enzymes for proper unlocking of the nutrients. The activity of endogenous enzymes like amylase, trypsin, and lipase increase with age (Sakomura et al., 2004). The exogenous protease perfected the action of endogenous proteolytic enzymes in the GIT thus improving the growth performance by improving nutrient digestibility (Pasquali et al., 2017). The relative weight of the organs remained unaffected in the present study. Similarly, Thomas and Ravindran (2008) found that sorghum-based diets did not affect the relative weight of the different organs. Gizzard and small intestine

 

Table II. Effect of enzyme supplementation on broiler production performance, relative organ weight and liter moisture content fed with sorghum-based diet.

Parameters		Control	Treatments			P-value
			T1	T2	T3
Starter phase (day 1-21)	BWG (g)	633.9±0.08	646.4±0.090	629.2±0.03	733.4±0.96	0.061
	FI (g)	1140±0.021	1110±0.021	1105±0.031	1120±0.022	0.097
	FCR	1.79±0.023	1.71±0.03	1.75±0.04	1.52±0.05	0.102
	Livability (%)	96.23±0.49	95.60±0.56	96.82±0.44	96.20±0.52	0.234
Finisher phase (day 22-35)	BWG (g)	1198.8±0.97b	1196.8±0.61b	1189.7±0.18b	1220.4±0.24a	0.045
	FI (g)	2160±0.92	2159.7±0.94	2155.2±0.73	2151±0.45	0.098
	FCR	1.80a±0.03	1.80a±0.04	1.81a±0.03	1.76b±0.03	0.041
	Livability (%)	96.31±0.39	95.60±0.46	96.93±0.37	96.0±0.61	0.122
Overall period (day 1-35)	BWG (g)	1832.7±0.21b	1843.2±0.75b	1818.9±0.50b	1953.8±0.16a	0.000
	FI (g)	3300.2±0.36	3259.2±0.74	3260.2±0.94	3270±0.53	0.378
	FCR	1.80±0.03a	1.76±0.04a	1.79±0.04a	1.67±0.02b	0.000
	Livability (%)	96.31±0.12	95.71±0.53	96.82±0.74	96.02±0.75	0.000
Relative organ weight (%)	Gizzard	1.18±0.04	1.19± 0.05	1.20 ± 0.06	1.25±0.05	0.152
	Pancreas	0.24±0.02	0.26±0.03	0.25±0.02	0.24±0.03	0.228
	Liver	1.75±0.03	1.74±0.02	1.78±0.04	1.76±0.03	0.457
	Cecum	0.54±0.05	0.58±0.02	0.55±0.01	0.59±0.09	0.256
Intestinal segment weight (%)	Duodenum	1.47±0.18	1.48±0.30	1.45±0.70	1.44±0.51	0.674
	Jejunum	1.76±0.11	1.76±0.18	1.76±0.28	1.74±0.01	0.951
	Ileum	2.47±0.01	2.46±0.50	2.47±0.01	2.46±0.03	0.070
Intestinal segment length (cm)	Duodenum	30.00±0.36	30.53±0.56	30.65±0.42	30.34±0.49	0.344
	Jejunum	80.35±0.42	81.03±0.54	79.93±0.44	77.68±0.56	0.342
	Ileum	87.16±0.70	88.50±0.76	89.16±0.61	85.66±0.42	0.895
Moisture content (%)	Day 21	21.10±0.25	21.50±0.34	21.83±0.30	21.50±0.56	0.518
	Day 28	24.62±0.41	24.52±0.41	24.32±0.53	24.31±0.21	0.078
	Day 35	27.36±0.21b	27.00±0.57b	28.26±0.42a	28.16±0.30a	0.048

 

Means in rows having different superscripts are significantly different. BWG, body weight gain; FI, feed intake; FCR, feed conversion ratio.

 

weight were decreased numerically. It may be due to the presence of NSP in diets that harm access of digestive enzymes to their substrates, thus, adapting the structure and functions of digestive organs. The supplementation of exogenous enzymes reduced the reactions in organs of the digestive thus, reducing the size of the gizzard and small intestine (Zhu et al., 2014). Litter moisture content was increased by the supplementation of exogenous enzymes into the broiler diet at 35 of the experimental period which is in agreement with the findings of Pos et al. (2003), and Farahat et al. (2013) who observed an increase in litter moisture content in turkeys and broiler, respectively. This effect could also be attributed to an increase in digesta osmolarity in broilers fed with phytase-containing diets, which is due to the release of more cations than anions in the early stages of phytate degradation, because 6-phytase acts cyclically, gradually releasing cations and anions in unequal proportions (Letourneau et al., 2011). There was significant interaction among the supplementation of different exogenous enzymes individually in broilers when maize was replaced by sorghum in the diet. Enzyme supplementation significantly increased the crude protein digestibility and AME of the sorghum-based diet compared with the maize-based diet. These results of the present study are in agreement with the findings of Gidado et al. (2020), and Pasquali et al. (2017) who observed a significant effect of exogenous enzymes on the crude digestibility sorghum-based diet. The exogenous enzymes can form a chelate with minerals and thus increase the crude protein digestibility. Moreover, phytate binds with protein through complexes

 

Table III. Effect of enzyme supplementation on the hematology and nutrient digestibility of broiler fed with sorghum-based diet.

Parameters	Control	Treatments			P-value
		T1	T2	T3
Hematological parameters
PCV (%)	27.12±0.04	27.25±0.05	27.27±0.06	27.46±0.07	0.484
Hb (mg/dl)	9.58±0.20	9.82±0.13	9.48±0.25	9.52±0.29	0.787
RBC (cells x106/mm3)	2.63±0.02	2.54±0.04	2.61±0.03	2.60±0.03	0.981
WBC (cells x103/mm3)	10.67±0.03	10.38±0.04	10.47±0.06	10.47±0.01	0.898
Neutrophils (%)	11.47±0.04	11.54±0.01	10.35±0.01	12.36±0.06	0.186
Heterophils/Lymphocyte (%)	88.45±0.03	90.46±0.09	91.47±0.02	91.42±0.03	0.073
Nutrient composition
Dry matter (%)	77.30±0.02	78.26±0.01	79.90±0.02	81.90±0.01	0.070
Crude protein (%)	79.73±0.50c	79.78±0.31c	80.61±0.20b	82.70±0.33a	0.001
Crude fat (%)	82.20±0.33	83.60±0.33	84.32±0.23	85.45±0.23	0.078
AME (Kcal kg-1)	2721.7±0.39b	2799.2±0.18b	2796.2±0.40b	2888.2±0.41a	0.000
Essential amino acid (%)
Arginine	82±0.24	84±0.11	84±0.13	85±0.17	0.066
Histidine	77±0.37	77±0.31	77±0.32	78±0.15	0.001
Leucine	85±0.24b	86±0.14b	88±0.21a	89±0.23a	0.000
Lysine	81±0.24c	85±0.14a	84±0.27b	86±0.21a	0.016
Methionine	81 ±0.17c	83±0.21b	84 ±0.15b	85±0.23a	0.014
Phenylalanine	84±0.24c	86±0.13b	87±0.17a	88±0.37a	0.012
Threonine	70±0.12b	74±0.13b	74±0.11b	76±0.27a	0.013
Valine	80±0.12c	83±0.14b	83±0.34b	85±0.21a	0.011
Non-essential amino acid (%)
Alanine	85±0.32c	87±0.12b	88±0.37b	89±0.15a	0.012
Aspartic acid	80 ±0.33c	84±0.31b	83±0.15b	85±0.13a	0.010
Glutamine	84±0.37c	88±0.15b	87±0.24b	89±0.13a	0.011
Glycine	73±0.31c	77±0.37b	77±0.24b	79 ±0.39a	0.011
Serine	78±0.36c	81±0.21b	81±0.25b	83±0.35a	0.013
Tyrosine	76±0.32b	80±0.11a	81±0.36a	82±0.27a	0.014

 

Means in rows having different superscripts are significantly different. PCV, packed cell volume; Hb, hemoglobin; RBC, red blood cells; WBC, white blood cells.

 

and binds with starch directly or indirectly through starch granule-associated protein thus improving the protein digestibility and apparent metabolizable energy. The AME values were significantly higher in the sorghum-based diet compared to the maize-based diet when supplemented with enzymes and this effect can be attributed to longer feed retention time, which is evidenced by a longer transit time when sorghum-based diets are used (Mateos et al., 1982). In addition, sorghum may have lower starch digestion coefficients than maize, and feedstuffs with low starch digestion tend to stay longer in the small intestine of birds (Weurding et al., 2001). Ileal amino acid digestibility was improved by the supplementation of exogenous enzymes, phytase, protease, and their combination. The digestibility of some essential amino acids like leucine, lysine, methionine, phenylalanine, threonine, and valine was significantly improved by the supplementation of the exogenous enzyme. The maximum increase in the digestibility of these amino acids was observed in the group supplemented with a combination of protease and phytase enzymes compare with the individual use and control group. Complete replacement of sorghum may compromise the bird’s performance, nutrient utilization, and amino acid digestibility. However, enzymes could be the capability to restore the compromised bird performance and increase the availability of nutrients (Wu et al., 2017). The exogenous enzymes such as phytase and protease when added in combination can further enhance the nutrient availability of birds.

CONCLUSION

The current study found that adding dietary enzymes could improve the nutrient digestibility of a sorghum-based diet in the broiler. Furthermore, supplementation of exogenous enzymes in a sorghum-based diet has a synergistic effect on nutrient digestion when used in combination.

ACKNOWLEDGEMENT

We acknowledge the Department of Poultry Science, Faculty of Animal Husbandry and Veterinary Sciences, The University of Agriculture, Peshawar, Pakistan for the provision of an experimental shed and technical facility.

Funding

No funding was provided/ received for this experimental work.

IRB approval

The experimental work was approved by the Board of Studies meeting (No. 458/PS/UAP dated 04/01/2021), The University of Agriculture Peshawar, KP, Pakistan.

Ethical statement

Before the start of the experiment, approval was taken from the Ethical Review Committee of the Faculty of Animal Husbandry and Veterinary Sciences, The University of Agriculture, Peshawar, Pakistan, and all the measures and tools were considered to minimize the pain and discomfort of birds during the conduction of this experiment.

Statement of conflict of interest

The authors have declared no conflict of interest.

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