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Current IssueEditorial BoardOnline First Articles
Asian Journal of
Dairy and Food Research
Print ISSN: 0971-4456
Online ISSN: 0976-0563
NASS Rating
5.44
SJR
0.176
CiteScore
0.357

Chief Editor:
Harjinder Singh
Massey Institute of Food Science and Technology, NEW ZEALAND
Frequency:Bi-Monthly
Indexing:
Elsevier (Scopus and Embase), AGRICOLA, Google Scholar, CrossRef, CAB Abstracting Journals, Chemical...

Full Research Article

Supplementation of Phytase and Protease Enzymes on the Performance of  KUB Chicken using the MSTN Gene Expression

Siti Rani Ayuti1,2, Mirni Lamid3,*, Sunaryo Hadi Warsito3, Mohammad Anam Al-Arif3, Widya Paramita Lokapirnasari3, Aswin Rafif Khairullah4, Ikechukwu Benjamin Moses5
1Doctoral Program of Veterinary Science, Faculty of Veterinary Medicine, Universitas Airlangga, Jl. Dr. Ir. H. Soekarno, Kampus C Mulyorejo, Surabaya 60115, East Java, Indonesia.
2Faculty of Veterinary Medicine, Universitas Syiah Kuala, Jl. Tgk. Hasan Krueng Kalee No. 4, Kopelma Darussalam, Kota Banda Aceh 23111, Aceh, Indonesia.
3Division of Animal Husbandry, Faculty of Veterinary Medicine, Universitas Airlangga, Jl. Dr. Ir. H. Soekarno, Kampus C Mulyorejo, Surabaya 60115, East Java, Indonesia.
4Research Center for Veterinary Science, National Research and Innovation Agency (BRIN), Jl. Raya Bogor Km. 46 Cibinong, Bogor 16911, West Java, Indonesia.
5Department of Applied Microbiology, Faculty of Science, Ebonyi State University, Abakaliki 480211, Nigeria.

ABSTRACT

Background: The KUB chicken industry faces several problems in early growth and active biological activity. One thing that needs to be done is the addition of phytase and protease enzymes to livestock feed. One effort to increase other livestock production is through molecular or genetic markers. The purpose of this study was to determine the effect of adding phytase and protease enzymes to the ration on the performance of KUB chickens, as well as to determine the expression of the MSTN gene on KUB chicken carcass production.

Methods: A total of 35 blood samples were taken from superior Balitnak (KUB) native chickens, consisting of 8 blood samples from groups of male chickens with the same variety, age with feed added with phytase enzyme, samples were taken from male chickens with the same body weight given feed with a mixture of protease enzymes. The following methods were used to calculate body weight increase, feed intake and feed conversion ratio (FCR). Amplification of the myostatin gene (MSTN) in KUB chickens was carried out using the Polymerase Chain Reaction (PCR) technique.

Result: Phytase and protease enzyme supplementation did not show a significant effect (P>0.05) on the relative weight of chicken internal organs, but has the potential to increase digestive efficiency and metabolic function. Enzyme activity in the digestive tract is considered an important factor that can affect gut health and nutrient digestibility. MSTN gene transcript expression was detected in all enzyme doses investigated in this study. Myostatin (MSTN) gene expression in KUB chickens was associated with muscle development, growth rate, organ composition and body weight.

INTRODUCTION


The demand for animal products is rising annually in Indonesia as people become more conscious of the value of animal protein (Yulianto et al., 2024). The yearly consumption of chicken meat from the area contributes to the rising demand for chicken (Wahyono and Utami, 2018). The Agricultural Research and Development Agency located in Ciawi (Bogor) has conducted a selection to produce superior native chickens which are named the Superior Native Chickens of the Livestock Research Center (KUB) (Tirajoh et al., 2021). KUB chickens have advantages in higher egg and meat production, more uniform growth, rapid environmental adaptation and more efficient use of rations compared to native chickens in general (Zulfan et al., 2024).
       
KUB chicken industry faces several problems in initial growth and active biological activities according to market requirements to improve the socio-economic status of poultry-oriented communities in developing countries. In order to optimize production results, one of the efforts made by livestock farmers is to provide Antibiotic Growth Promoters (AGP), but in recent years the government has regulated the prohibition of the use of AGP in supporting the improvement of livestock quality and performance (Ma et al., 2021; Okafor et al., 2019). In this case, another solution that can be done is to provide alternative feed additives to replace AGP with the aim of increasing the absorption of nutrients in the feed (Al Arif et al., 2024; Warsito et al., 2024). One thing that needs to be done is adding phytase and protease enzymes to animal feed.
       
Increasing productivity by using enzymes in feed can improve the efficiency of feed use (Alagawany et al., 2018). One type of enzyme that is often used in animal feed is the phytase and protease enzymes (Miladinovic et al., 2021). The benefits of the phytase enzyme are to break down phytic acid contained in feed and release inorganic phosphorus and inositol (Rizwanuddin et al., 2023; Ayuti et al., 2024). Phytase enzymes can also free proteins, trace minerals, amino acids and other nutrients that are attached to phytic acid (Bohn et al., 2008). Protease enzymes are useful for preventing the loss of amino acids, thereby improving production performance because nutrients will be more effectively used to improve livestock performance (Solanki et al., 2021).
       
One of the efforts to increase livestock production is through molecular or genetic markers. Genetic markers are also known as Marker Assisted Selection (MAS) (Collard and Mackill, 2008). One of the genes that is the main and determining gene in controlling growth traits and meat production is the Myostatin gene (MSTN) (Ceccobelli et al., 2022). The MSTN or Growth Differentiations Factor 8 (GDF-8) gene is a member of the Transforming Growth Factor (TGF) superfamily which functions as a regulator of skeletal muscle growth (Baig et al., 2022). The MSTN gene consists of one promoter, three exons and two introns (Khalil et al., 2017). The MSTN gene is one of the genes that is important for carcass and meat properties, one way to identify myostatin gene expression in KUB chickens is through the polymerase chain reaction (PCR) method.
               
Genetic improvement of KUB chickens for higher growth rates is an ongoing process, the nutritional needs of KUB chickens also change accordingly (Tona et al., 2022). For this reason, assessing the KUB chickens’ nutritional requirements is a continuous procedure. The performance of KUB chickens is determined by the interaction of their genetic potential and feed nutrients. In recent years, nutrigenomic approaches have increased understanding of the influence of diet on gene expression (Mierziak et al., 2021). Based on this background, it is necessary to conduct research to determine the effect of adding phytase and protease enzymes in the ration on the performance of KUB chickens, as well as to determine the expression of the MSTN gene on KUB chicken carcass production.

MATERIALS AND METHODS

Ethical approval
 
The ethical clearance committee of the Faculty of Veterinary Medicine at Universitas Airlangga, Indonesia provided consent for the use of animals with number: 1. KEH.039.03.2024.

Study area and sample collection
 
The research was conducted from April 2024 to August 2024. This study used a Completely Randomized Design (CRD) with 2 factors, namely factor A the addition of phytase enzyme with a dose of 0% (control), 0.02%, 0.04%, 0.06% and factor B the addition of protease enzyme with a dose of 0% (control), 0.03%, 0.05%, 0.07% consisting of 5 replications. This study used 35 KUB male chickens. Feeding was based on the needs of KUB chickens, feed was given 2 times a day, namely in the morning at 7.00 A.M and in the afternoon at 4.00 P.M. Preparation for the study and maintenance of KUB chickens were also carried out at the Faculty of Veterinary Medicine and sample analysis was carried out at the Institute of Tropical Disease, Airlangga University.
 
Enzyme supplementation treatment
 
A total of 20 blood samples were taken from superior Balitnak (KUB) native chickens, consisting of 8 blood samples from a group of male chickens with the same variety, age with feed supplemented with phytase enzyme, samples were taken from male chickens with the same body weight that were fed with a mixture of protease enzymes. Then after selection based on production performance during maintenance, 8 blood samples were taken again. As a control, 5 blood samples of chickens that were not given enzymes in their feed were also used. A total of 20 samples were used in this study.
 
Performance and organ sample collection
 
The following methods were used to calculate body weight increase, feed intake and feed conversion ratio (FCR). To calculate feed consumption, weekly weigh-ins and records of fed and unfed feed were made. The amount of feed supplied was subtracted from the amount of feed left to determine the amount of feed consumed. Weekly feed intake was computed and then recalculated for each chicken. The amount of feed consumed divided by the body weight gain during the 90-day period was used to calculate the FCR. In the meantime, the difference between the starting and final body weights up to 90 days of age, divided by the number of days of upbringing, was used to compute the average daily body weight growth (BWG). After reaching 90 days of age, the birds were slaughtered and feathered. The head, neck, calves and legs were cut off from the carcass.  The internal organs, including the kidneys, liver, heart, lien and gizzard, were then taken out and weighed independently to determine how much they weighed in relation to the carcass.
 
MSTN gene detection
 
RNA extraction was performed using a promega kit (Sancko-Junyaku Co., Ltd., Tokyo, Japan). The procedure is as follows: 30 μl of blood sample plus 100 μl of solution 1 (tris buffer) was stirred slowly, then 100 μl of solution 2 (guanidine thiocyanate) was added and stirred until it came out like mucus. 700 μl of solution 3 (chloroform) and 400 μl of solution 4 (Na-acetate) were added to the solution and stirred until evenly distributed, then centrifuged. The supernatant was transferred to a new tube, 60 μl of solution 5 (acetate buffer) and 600 μl of 2-propanol were added, centrifuged. The supernatant was discarded, the sediment was cleaned with 1 ml of 70% ethanol, centrifuged. The supernatant was discarded, the RNA precipitate was dried and added with 100 μl of TE (Tris-HCL EDTA) solution. The RNA concentration was calculated using 10 ng/μl prepared for PCR, namely DNA fragments were amplified with the MSTN gene marker with forward primers: 5’- AAC GGT GTT TGT GCA GAT CC -3’ and Reverse primers: 3’- CAA TCC ATC TTC ACC CGG TCC -5’ (Azhar et al., 2020). With these primers, a DNA fragment of 274 bp can be produced. To produce the MSTN gene RNA fragment, a mixture solution was made consisting of 10x buffer, 2 mM dNTP, 25 mM MgSO4, MSTN primers (forward and reverse primers, 200 pmol/μl), KOD plus polymerase enzyme and water. Amplification was carried out using the Gene Amp thermocycler, PCR system 9700, PE Applied Biosystems. The PCR conditions were programmed, namely denaturation at 94oC for 1 minute 15 seconds, annealing was made in 3 types: 10 cycles (15 seconds at 94oC, 30 seconds at 60oC, 1 minute at 68oC), 10 cycles (15 seconds at 94oC, 30 seconds at 55oC, 1 minute at 68oC) and 10 cycles (15 seconds at 94oC, 30 seconds at 50°C, 1 minute at 68oC) and elongation for 9 minutes at 68oC. After the cycle ended, it was continued with a storage temperature of 4oC. The results of the amplification of the MSTN RNA fragment were detected by segregating the prolactin promoter band pattern with an electrophoresis system on a 2% agarose gel stained with ethidium bromide and viewed with an Ultra Violet device. Then documentation is carried out by photographing the electrophoresis results with a camera.

RESULTS AND DISCUSSION

KUB chicken performance
 
The results presented in Table 1 revealed significant differences (P<0.05) in the growth performance of KUB chickens among the treatments of phytase and protease enzymes with different doses. Body Weight Gain (BWG) of phytase enzyme group during the starter phase (0-3 weeks) was significantly lower compared to other treatments which were not significantly different from each other. During the grower phase (4-6 weeks) significantly higher BWG was observed in the treatment given protease enzyme followed by the treatment given phytase enzyme addition in the feed which was statistically different and lower BWG was observed in the control treatment which was statistically. Overall BWG in the finisher phase (0-6 weeks) was significantly higher in the treatment given protease enzyme followed by the phytase treatment and control treatment which were statistically similar, while lower BWG was observed in the control treatment. The interval factors of 4-5 weeks and 0-6 weeks were significantly lower in the control treatment compared to the treatments given phytase and protease enzymes in the feed which were not significantly different from each other.

Table 1: Growth performance (mean+S.E) of chickens under different dietary enzyme phytase and protease levels.


       
The control treatment showed a much better Feed Conversion Ratio (FCR) of 4-6 weeks and 0-6 weeks compared to the treatments at 0-3 weeks which were similar to each other. The results of feed consumption showed a significant difference (P<0.05) at 6 weeks of administration. The lowest feed consumption value was found for the control treatment, which was consistent with the treatments at 0-3 weeks, namely PF2, PF4, PF6, PP3, PP5 and PP7. The highest feed consumption value was found in the treatment at 0-6 weeks, which was not significantly different from the treatment at 4-5 weeks. The results of the feed conversion ratio showed that there was an interaction (P<0.05) in the 6 weeks of administration. The lowest feed conversion ratio value was found for the PF4 treatment at 0-6 weeks, which was consistent with the PP3 treatment at 6 weeks and PF4 at 4-6 weeks. The highest feed conversion ratio value was found for the control treatment at 4-6 weeks, which was not significantly different from the PP5 and PP7 treatments at 4-6 weeks. The average performance parameters (feed consumption, feed conversion ratio, body weight gain) for the addition of phytase and protease enzymes in the feed of KUB chickens are listed in Table 1.
       
Digestive enzyme activity is thought to be a significant element that can affect gut health and the ability of nutrients to be absorbed (Zhang, 2022; Ayuti et al., 2024). Sufficient amounts of both phytase and protease in the diet are crucial for the digestive system of poultry since phytase supplements can enhance gut health and feed efficiency while proteases can influence the digesta microbiota and the activity of gut digestive enzymes (Bohn et al., 2008; Solanki et al., 2021). Protease can increase the activity of trypsin and chymotrypsin in chicks (Zheng et al., 2023). Conversely, adding phytase and protease to basal, low-energy and low-protein diets can both greatly boost the activity of chymotrypsin and trypsin in the jejunum of chicks (Jiang et al., 2020). Therefore, by boosting the digestive and absorptive capacities of KUB chickens and consequently improving their ability to gain weight, increasing the activity of trypsin and chymotrypsin through dietary supplementation with protease and phytase may have contributed to the improvement of nutrient digestibility.
       
In feed and poultry development performance, non-starch polysaccharide enzymes (mostly xylanases) and phytases can support one another to enhance protein and energy use (Junior et al., 2024). Low-energy diets can closely combine water-soluble non-starch polysaccharides and phytic acid, which facilitates the easier access of non-starch polysaccharide enzymes to the phytate surface and accelerates the rate of phytate breakdown (Gehring et al., 2013). Therefore, these roles are complementary to one another and we propose that phytate and proteases may interact to affect the activity of trypsin and chymotrypsin in the digestive tract.
 
Effect of phytase and protease enzymes on organ
 
The main results of carcass characteristics are presented in Table 2. Internal organs showed a non-significant increase (P>0.05) in chickens fed with phytase and protease enzyme supplements compared to the control group. The results of earlier studies that found that the administration of mixed enzymes did not alter relative liver weight are consistent with our conclusion (Tüzün et al., 2020). Additionally, according to Salazar-Villanea et al. (2023), the relative weight of internal organs such the liver, gizzard and heart was unaffected by the injection of phytase and protease. According to a study that found a negative interaction between amylase and protease that decreased the relative weight of the gizzard at 21 days of age but did not show this difference at 7 days, the maximum weight of these organs was reached before 9 days of age (Radhi et al., 2023). This is believed to be the result of the feed’s high insoluble fiber content, which promotes gizzard development. As a result, the feed’s composition and the chicken’s age at observation have a significant impact on how the digestive organs react to enzyme supplementation.

Table 2: Organ weight and percent (mean+S.E) of chickens under different dietary enzyme phytase and protease levels.


       
These results suggest that enzyme supplementation may hasten the development of immunological organs.However, Baloch et al., (2021) found that adding protease to male hens’ feed between the ages of 7 and 21 days considerably raised the spleen’s relative weight. They link this to enhanced nutrition absorption, better digestive processes and better metabolic hormone management, all of which can tangentially strengthen the immune system (Kaimkhani et al., 2025). The relative weight of chicken liver in this investigation was not significantly impacted by the addition of phytase and protease enzymes. These outcomes corroborate those of Abd El Latif et al. (2023), who found that adding enzymes to diet, such as phytase or mixed enzymes, had no effect on the relative weight of the liver. This implies that while feed enzymes can increase the effectiveness of digestion and nutrient absorption, their effect on liver growth is negligible, particularly when chickens are in their typical physiological state (Walk et al., 2018). Although the increase in the enzymes’ metabolic efficiency may indirectly improve the chicken’s overall performance, this lack of influence can be interpreted as meaning that the feed enzymes have no direct structural impact on the heart or cardiovascular system (Alagawany et al., 2021). There were no appreciable changes in the relative weights of the kidney and gizzard, which likewise responded similarly (Sozcu, 2019). The amount of crude fiber and the physical characteristics of the meal have a significant impact on the development of the gizzard, an organ crucial to the mechanical process of digestion. According to Maynard et al., (2023), a high insoluble fiber content can promote the development of the gizzard, though this effect was not consistently seen at all age stages.
       
The purpose of adding phytase and protease enzymes to poultry feed is to improve the efficiency of nutrient use, particularly for protein and minerals, which can affect organ function and growth directly or indirectly (Saleh et al., 2025). Increased liver metabolic performance may result from improved digestive efficiency, even if the liver’s relative weight may not usually alter much (Gautier et al., 2018). Enzyme administration also helps the kidneys and heart, particularly with regard to body homeostasis and metabolic stability (Sprigg et al., 2022). Since energy and vital nutrients like minerals and amino acids are more readily available, circulatory function in the heart can be strengthened by improved nutritional status (Hakami et al., 2022). Even though research indicates that the size of the kidneys and heart does not typically alter much, a better nutrient balance can nevertheless have a good effect on these two organs’ physiological function through the use of enzyme supplements (Huyan et al., 2022). The physical makeup and composition of the diet have a significant impact on the gizzard, a mechanical digesting organ (Ghosh et al., 2016). Although they don’t have a direct mechanical impact on the gizzard, proteases and phytoses can alter the digesta’s texture and fermentability, which can alter the gizzard’s ibility to grind feed (Hao et al., 2018). Enzyme administration has been demonstrated in multiple trials to decrease the gizzard’s relative weight because it reduces insoluble crude fiber, which increases the gizzard’s mechanical action (Ren et al., 2015). Nevertheless, the gizzard continues to function at its best physically in the digestive system and the effectiveness of nutrient absorption rises as a result of enzymes that assist in the breakdown of complicated substrates into simpler molecules that the small intestine can absorb more readily.
 
MTSN gene expression
 
The results of MSTN gene amplification in KUB chickens and the administration of phytase and protease enzymes in feed with different doses at the 4842 bp base position using a Thermal Cycler machine, obtained a PCR product with a length of 247 base pairs (bp). Based on Fig 1 and 2. the results of RNA extraction electrophoresis show that the RNA band looks quite clear and even so that there is no need for repetition. Many factors affect the quality of RNA in addition to the dose of enzyme use in feed and the difference in the amount of enzyme dose used, one of which affects the quality of RNA is the lysis process, which is a process of breaking down the cell nucleus and the concentration of RNA rehydration given. The cell nucleus can be lysed properly, so the resulting RNA has a high enough concentration so that it affects the quality and quantity of RNA (Table 3).

Fig 1: MSTN gene amplification results using PCR technique on 1.5% agarose gel.



Fig 2: MSTN gene amplification results using PCR technique on 1.5% agarose gel.



Table 3: dCT, ddCT and dddCT values of phytase enzyme treatment of MSTN genes.


       
MSTN gene expression in phytase enzyme treatment with different doses was significantly (P<0.05) upregulated in PF2 feed treatment and feed treatment without phytase enzyme with respect to PF4 and PF6 treatments (Fig 3). In protease enzyme treatment with different doses was significantly (p<0.05) upregulated in PP7 and PP3 feed treatments gene expression was significantly upregulated compared to PP5 treatment and feed treatment without protease enzyme was significantly different from each other (Fig 4). In protease enzyme treatment gene expression in PP7 and PP3 was significantly higher than other treatments, followed by phytase enzyme treatment in PF2 treatment, while the lowest expression was observed in feed treatment with phytase enzyme administration in PF6 followed by PF4 treatment. In general, fold expression was higher in PP7 protease enzyme administration treatment with different doses than phytase enzyme administration treatment (Table 4).

Fig 3: MSTN expression profile in KUB chicken at different doses of phytase enzyme under different dietary groups (mean±SEM).



Fig 4: MSTN expression profile in KUB chicken at different doses of protease enzyme under different dietary groups (mean±SEM).



Table 4: dCT, ddCT and dddCT values of protease enzyme treatment of MSTN genes.


       
MSTN gene transcript expression was detected in all enzyme doses investigated in this study. Regardless of the type of enzyme used in the feed with different doses, in relation to the reference gene, the mRNA expression levels of the target gene varied significantly at different enzyme doses. In the administration of protease enzyme, MSTN gene transcripts had relatively higher expression, namely in PP7 (1.63), PP3 (1.59), PP5 (1.28) and the treatment without protease enzyme. When compared to the expression in the feed given phytase enzyme, MSTN gene expression was regulated in the phytase enzyme treatment by PF2 (1.16), PF4 (0.86), PF6 (0.66) and the treatment without phytase enzyme. However, among all enzyme doses, those with higher and more gene expression were in the treatment with protease enzyme administration at a dose of 0.07% (PP7).
       
The Myostatin (MSTN) gene is a negative regulator that is predominantly expressed in skeletal muscle (Chen et al., 2021). Instructions for producing the protein myostatin, which is active in skeletal muscle before and after birth, are provided by this gene (Baig et al., 2022). Normally, this protein regulates muscle growth to prevent excessive muscle growth (Ramasamy et al., 2017). Treating myoblasts (muscle cells) with myostatin results in reduced cell proliferation and differentiation (McFarlane et al., 2011). Myostatin (MSTN) is the only inhibitor of skeletal muscle growth and development. Loss of myostatin function increases the diameter and amount of muscle mass (Jang et al., 2021; Ayuti et al., 2024). The potential for myostatin inhibition to increase muscle mass in KUB chickens has attracted interest in the poultry industry as a potential selection mechanism for increasing meat production. The effects of myostatin inhibition have also been investigated on egg production to determine whether myostatin could be used in the laying hen industry to increase egg production (Lee et al., 2021; Akintunde and Toye, 2024).
               
In KUB chickens, the expression of the myostatin (MSTN) gene is linked to body weight, growth rate and muscle development. Through the activation and renewal of satellite cells, myostatin expression controls the growth and development of muscle fibers (McCroskery et al., 2003). A study comparing Daweishan mini chickens (DMC) and commercial poultry broilers found that the DMC birds had a lower growth rate than the commercial poultry broilers (Dou et al., 2018). Variations in body weight in hens are linked to polymorphisms in the myostatin gene (Tanjung et al., 2019; Kannaki et al., 2017). To develop a tolerance to cold, chicks must control the expression of the myostatin gene in their leg muscles by the time they are seven days old (Ijiri et al., 2009). In freshly hatched chicks, myostatin is sensitive to the availability of nutrients and fasting lowers the amounts of MSTN mRNA in the muscle of older chickens (Saneyasu et al., 2015). The MSTN gene belongs to the superfamily of transforming growth factor beta (TGFβ), a class of proteins that aid in regulating the expansion and maturation of bodily tissues (Baig et al., 2022; Nihar et al., 2024).

CONCLUSION

The results of the study revealed significant differences (P<0.05) in the growth performance of KUB chickens between the treatments of phytase and protease enzymes with different doses. The expression of MSTN gene transcripts was detected in all enzyme doses investigated in this study.

ACKNOWLEDGMENT

The authors are grateful to the Faculty of Veterinary Medicine, Universitas Airlangga, Indonesia, for the facility supporting this study. This study was supported in part by the Program Penelitian Penelitian Skema Penelitian Dasar Unggulan (PDU) Universitas Airlangga, in the fiscal year 2024, with grant number: 1508/UN3.FKH/PT.01.03/2024.
 
Disclaimers
 
The authors are responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.
 
Informed consent
 
The ethical clearance committee of the Faculty of Veterinary Medicine at Universitas Airlangga, Indonesia provided consent for the use of animals with number: 1. KEH.039.03.2024.

CONFLICT OF INTEREST

The authors declare that there are no conflicts of interest regarding the publication of this article.

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