Improving the Growth Performance and Nutrient Utilization in Broilers Fed Low Protein Diet: The Effect of Bacillus pumilus on Digestibility and Microflora Dynamics
Shehzad Ahmed Soomro1, Muhammad Aslam1, Shoaib Ahmed Pirzado1*, Ghulam Rasool2, Khalid Mashay Al-Anazi4, Muhammad Bilawal Arain3 and Mohammad Abul Farah4
1Department of Animal Nutrition, Sindh Agricultural University Tadojam, Pakistan, 70060
2Livestock and Dairy Development Department, Government of Balochistan. Pakistan, 70060
3Department of Veterinary Pharmacology, Sindh Agricultural University Tadojam, Pakistan, 70060
4Department of Zoology, College of Science, King Saud University, Riyadh 11451, Saudi Arabia
ABSTRACT
The main purpose of this experiment was to evaluate the effect of dietary addition of Bacillus pumilus on growth performance, nutrient digestibility and microflora of broiler chickens fed low protein diet. A total of 180 one-day-old male chicks were randomly distributed into three treatment groups having 60 birds each (six replicates of 10 birds). Experimental diets were (i) a basal (CON) diet (22% crude protein (CP) in the starter phase and 20% CP in the finisher phase), (ii) a low protein (LP) diet with 20 and 18% CP in each starter and finisher phases, respectively, and (iii) a LP-0.01 diet supplemented with Bacillus pumillus @ 0.01% of diet. The diets were offered according to a statistical design for how many days. The diets were offered using the automatic feeding and watering system. The birds were kept in an environment-controlled shed. The birds consuming the LP-0.01 diets significantly increased their live body weight (LBW) and average daily gain (ADG) whereas reduced feed conversion ratio (P<0.05) compared with control. Carcass, breast- and leg-muscle percentages were (P<0.05) higher in the birds consuming LP-0.01 than LP but not different from the control. The LP-0.01 diets had the highest availability of dry matter (DM), CP, fat and ash (P<0.05) and villus height of jejunum (P<0.05). The LP-0.01 diets reduced the count of pathogenic bacteria, Salmonella and E-Coli, while increased the Lactobacillus count (P<0.05). It is concluded that supplementation of Bacillus pumilus in low protein broiler diets counterbalances the effects arising from low protein and is equally beneficial to traditional broiler diets in terms of growth performance, nutrient digestibility, and microflora.
Article Information
Received 25 May 2024
Revised 28 July 2024
Accepted 10 August 2024
Available online 13 November 2024
(early access)
Published 13 November 2025
Authors’ Contribution
SAP and SAS conceptualized and designed the study. SAS and MA performed the data curation. MBA and MAF formal analysis and methodology. SAS and GR wrote the manuscript. SAP and KMA-A critical revision and editing of the manuscript.
Key words
Low protein, Broilers, Growth, Microflora, Bacillus pumillus, Digestibility
DOI: https://dx.doi.org/10.17582/journal.pjz/20240525073941
* Corresponding author: [email protected]
0030-9923/2025/0006-2847 $ 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
Due to the shortage and high prices of red meat, chicken meat is the alternative economical source of good quality animal protein in Pakistan, and this sector is rarely bringing the gap between supply and demand in the meat protein market. Poultry ration formulation is challenging because it uses protein and energy sources of very high quality and cost. A balance between the cost and quality of the ration ingredients forces a farmer either to reduce the content of protein in the ration or try to find cheap alternatives (Davies et al., 2010). Therefore, the use of cheap protein ingredients at optimum levels will not only reduce the cost of production but also environmental pollution by decreasing the excretion of undigested nitrogen into the environment. Variable results have been mentioned in literature regarding the use of low dietary protein levels in poultry without a certain conclusion for the reader (Rehman et al., 2017; Yang et al., 2015). Some studies reported that growth performance and carcass quality traits were negatively affected by lowering the dietary protein levels by 2 to 3%. Others reported that low protein broiler diets impair digestion, structure of intestinal villi, and protein metabolism (Pirzado et al., 2021).
Probiotics based on bacterial products have been successfully used in broiler diets leaving beneficial effects on the host. All bacterial-based probiotics are not the same in their effects, however, commonly, they are known for increasing gut and beneficial bacterial enzyme activities, gut integrity and maturation and immune function, and decreasing the unwanted bacterial enzyme activity and inflammation (Laudadio et al., 2012; Awad et al., 2017). Bacillus species are the most commonly used probiotics in chicken diets, however, other species including Bacillus pumilus, Lactobacillus, Streptococcus, Bifidobacterium and Aspergillus have also been used (Alagawany et al., 2016; Bughio et al., 2021).
Bacillus pumilus is increasingly being employed as a feed additive in fish diets. Fish and mice fed B. pumilus had higher body weights than those fed on a control diet. Supplementation of B. pumilus in fish’s diet prevents the growth of pathogenic bacteria. Although a large number of microorganisms are utilized as probiotics in chicken production, the gastrointestinal tract has a low pH value and a high bile salt concentration (Soomro et al., 2019). Bacillus species, which survives in extreme gastrointestinal environments through spore formation, is gaining more attention as a probiotic in animal feed (Aslam et al., 2024). With its longer shelf life and greater stability, Bacillus strains has a significant advantage over its competitors in the feed industry (Purba et al., 2020; Li et al., 2020). Nutritional digestibility, immune modulation, and increased nutrient absorption are just a few of the advantages of supplementing B. subtilis in chicken feed. Different types of probiotic bacteria have distinct characteristics. The effects of B. subtilis strain-specific, as well as the mechanisms by which they function, remain unknown. Research on B. subtilis strain-specific impacts on broiler performance and intestinal physiology underscores the need for further investigation on the different B. subtilis strains to better understand their mechanisms of action. Anti-clostridium spp. activity in B. subtilis is often beneficial to broiler performance. The prospect of reducing the cost of feed by using a Bacillus-based probiotic is intriguing enough to warrant further investigation. However, no research has been conducted on broilers supplemented with B. pumilus in a low-protein diet. Therefore, this study has been designed to examine whether the dietary addition of B. pumilus influences growth performance, nutrient digestibility, and intestinal microflora of broilers when fed a low-protein diet.
MATERIALS AND METHODS
Chicks and their maintenance
A total of 180 healthy day-old (Arber Acres) chicks were purchase from a local hatchery and maintained in brooders at 32°C during 1st week and it was gradually decreased by 2°C per week till it reached 22°C, the relative humidity was maintained between 55 to 65% and the light for 24 h during the whole experiment. All birds were vaccinated according to the schedule recommended by Pakistan Poultry Association (PPA).
Dietary treatments
The chicks were randomly distributed into three treatment groups having 60 birds each (six replicates of 10 birds). Experimental diets were (i) a basal (CON) diet (22% crude protein (CP) in the starter phase (0-21 days) and 20% CP in the finisher phase (22-42 days), (ii) a low protein (LP) diet with 20 and 18% CP in each starter and finisher phases, respectively, and (iii) a LP diet supplemented with Bacillus pumillus (Jinan Rentai import export co. China) 0.01% of diet (LP-0.01) as shown in Table I. The diets and water were offered ad libitum.
Sampling and feed analysis
Feed samples were collected bi-weekly and subjected to proximate analyses to determine DM, CP, crude fat and ash content following the procedures (AOAC, 2000). Live body weight (LBW) and feed intake were recorded every week. The feed conversion ratio (FCR) was calculated according to the formula FCR = Feed intake/weight gain
Carcass characteristics were determined by slaughtering two birds per replicate at the end of the experiment. The LBW and carcass weight were recorded. The dressed percentage was calculated by following formula:
Dressing percentage = (Carcass yield)/ (Live body weight) ×100
Relative weight of breast muscle, leg meat and abdominal fat were calculated by using following formula:
Relative weight of each part = (Weight of each part)/ (Carcass weight) ×100
Apparent nutrient availability
For the determination of nutrient availability, three birds per replicate were transferred to a separated pen on 39th day of the experiment for feces collection. Fecal samples were collected for 3 consecutive days and compose of each treatment. The fecal samples were then dried in an oven at 65°C and ground thereafter. The dried and ground samples were analyzed for proximate composition as described previously. The following formula was used for determination of availability of nutrients.
Table I. Feed formulation and nutrient composition of basal diets.
|
Ingredients% |
Control |
Low protein1 |
||
|
Starter |
Finisher |
Starter |
Finisher |
|
|
Corn |
57.47 |
58.98 |
63.94 |
65.73 |
|
Soybean oil |
1.50 |
4.32 |
0.52 |
3.45 |
|
Soybean |
30.96 |
25.05 |
25.51 |
19.1 |
|
CSM |
5.00 |
7.00 |
5.00 |
7.00 |
|
Salt |
0.35 |
0.35 |
0.35 |
0.35 |
|
CaPO4 |
1.53 |
1.39 |
1.50 |
1.40 |
|
Limestone |
1.54 |
1.40 |
1.61 |
1.44 |
|
Lys |
0.24 |
0.22 |
0.21 |
0.26 |
|
Meth |
0.14 |
0.15 |
0.11 |
0.14 |
|
Cyst |
0.07 |
0.04 |
0.05 |
0.02 |
|
Choline |
0.20 |
0.10 |
0.20 |
0.10 |
|
Premix |
0.50 |
0.50 |
0.50 |
0.50 |
|
Zeolite |
0.50 |
0.50 |
0.50 |
0.50 |
|
Total |
100 |
100 |
100 |
100 |
|
Nutritional value of diet |
||||
|
AME (kcal/kg) |
2950 |
3050 |
2950 |
3050 |
|
Crude protein (%) |
22.00 |
20.00 |
20..00 |
18.00 |
|
Lys (%) |
1.200 |
1.050 |
1.050 |
0.950 |
|
Meth (%) |
0.450 |
0.440 |
0.400 |
0.400 |
|
Ca (%) |
0.990 |
0.904 |
0.990 |
0.906 |
|
Total P (%) |
0.679 |
0.669 |
0.661 |
0.61 |
|
Avail P (%) |
0.450 |
0.42 |
0.450 |
0.42 |
*The pre-mixture providing (one kg of diet) VA 10000 IU, VB1 1.8 mg, VB2 40 mg VB12 0.71 mg, VB3 2000IU, VE 10IU, VK3 2.5 mg, biotin 0.12mg, folic acid 0.5mg, D-PA 11mg, cu (as copper sulfate) 8 mg, Fe (as ferrous sulfate) 18 mg, Mn (as manganese sulfate) 16 mg, Zn (as zinc sulfate) 40 mg, I (as podetium iodide) 0.35 mg and Se (as sodium selenite) 0.15 mg. 1The low protein diet was supplemented with Bacillus pumillus @ 0.01% of diet as a third treatment.
Morphometry of jejunum
The whole small intestine was dissected from the slaughtered birds carefully, the jejunum was separated and flushed with 0.9% saline to remove the content and then fixed in 10% formalin solution for further analysis. Specimens were rinsed in running tap water for a few hours after being fixed for 12 to 24 h, then dehydrated in alcohol and xylene, embedded in paraffin, cut into 2 to 5 mm thick slices, mounted on glass slides, and stained with hematoxylin and eosin. Stereoscopic microscope at a 4X was used to determine the villus height and crypt depth, using Opticam vision lite 2.1 software.
Intestinal microflora
The intestinal content from whole small intestine was taken in test tubes from slaughtered birds and stored in a deep freezer. The count of Lacto bacillus, E. coli and Salmonella was conducted in lab (Khan et al., 2022). Standard plate count method was used to figure out the total number of surface-viable bacteria cells. The number of colony-forming units per gram (cfu/g) was used to show how many bacteria were in the intestinal content.
Statistical analysis
The data were statistically analyzed according to one-way ANOVA using SPSS® 19.0 software. Duncan’s novel multiple-range test was used to compare the significant variations in treatment means. The data are presented as least square means with standard errors of the mean.
RESULTS
Significant effect of dietary treatments was found on all parameters of growth performance except FCR during starter phase (Table II). Significantly higher LBW was observed in LP-0.01 than LP but non-significant with Control (P<0.05). In starter phase, ADG and FCR were significantly better in groups LP-0.01 and Control than group LP (P<0.05). A similar trend was observed during the overall period of experiment. LBW and ADG were significantly higher in Control and LP-0.01 than LP while FCR of both groups statistically decreased than LP. During overall experimental period, highest LBW and ADG with better FCR were found in LP-0.01 (P<0.05).
Table II. Effect of dietary treatments on growth performance in broilers (g).
|
Parameters |
Control |
LP |
LP-0.01 |
P. value |
|
Starter (1-21 day) |
||||
|
LBW |
810.67±21.41ab |
755.67±25.33b |
823.67±31.09a |
0.023 |
|
ADG |
38±0.04a |
35±0.08b |
39±0.06a |
0.010 |
|
ADFI |
47±0.19a |
46±0.27a |
45±0.15a |
0.266 |
|
FCR |
1.22±0.01a |
1.31±0.01b |
1.16±0.07a |
0.001 |
|
Overall (1-42 day) |
||||
|
LBW |
2517.3±42.06a |
2296.8±35.05b |
2611.9±39.09a |
0.008 |
|
ADG |
58.897±1.56a |
53.622±1.29b |
61.155±1.05a |
0.008 |
|
ADFI |
91.587±2.20a |
87.037±2.95a |
92.460±1.76a |
0.269 |
|
FCR |
1.55±0.03b |
1.62±0.01a |
1.51±0.08b |
0.001 |
a,b,c,Superscripts with different letters in rows and columns varied significant (P<0.05). Control, basal diet; LP, low protein; LP-0.01, low protein +0.01% B. pumilus. LBW, live body weight; ADG, average daily gain; ADFI, average daily feed intake; FCR, feed conversion ratio.
Table III. Effect of dietary treatments on carcass characteristics (%) in broilers.
|
Parameters |
Control |
LP |
LP-0.01 |
P. value |
|
DP |
71.126±0.983a |
66.545±0.328b |
72.794±0.726a |
0.002 |
|
BM |
25.027±0.509a |
21.729±0.579b |
24.554±0.491a |
0.009 |
|
TM |
17.077±0.187a |
14.676±0.571b |
17.543±0.198a |
0.003 |
|
AF |
1.8479±0.096b |
2.3347±0.067a |
1.3870±0.101c |
0.001 |
For abbreviation and statistical details, see Table II. DP, dressing percentage; BM, breast muscle; TM, thigh muscle; AF, abdominal fat.
Table III shows data regarding carcass characteristics of dietary treatment. The highest dressing percentage was observed in LP-0.01 (P<0.05) followed by control. Similarly, the highest relative weight of LM was found in LP-0.01 and lowest in LP (P<0.05). While highest relative weight of BM and abdominal fat were observed in Control and LP respectively (P<0.05).
Table IV shows the significant effect of dietary treatments on nutrient availability. The highest DM, crude protein, crude fat and ash availability were recorded in LP-0.01 than LP and control respectively (P<0.05). Availability of all nutrients studied was significantly increased with supplementation of B. pumilus in LP-0.01 (P<0.05).
Table IV. Effect of dietary treatments on availability (%) broilers.
|
Parameters |
Control |
LP |
LP-0.01 |
P. value |
|
70.730±0.52b |
74.540±0.37a |
0.015 |
||
|
65.240±0.34ab |
63.810±0.29b |
0.022 |
||
|
CF |
61.278±1.20ab |
60.000±0.96b |
64.772±0.99a |
0.045 |
|
Ash |
62.073±0.55a |
59.366±0.43b |
63.123±0.33a |
0.003 |
For abbreviation and statistical details, see Table II. DM, dry matter; CP, crude protein; CF, crude fat.
Table V. Effect of dietary treatments on intestinal (jejunum) morphology (µm) broilers.
|
Parameters |
Control |
LP |
LP-0.01 |
P. value |
|
604.42± 55.65ab |
582.58± 81.80b |
837.79± 36.33a |
0.044 |
|
|
Crypt depth |
168.20± 7.40a |
149.28± 14.90a |
163.45± 6.43a |
0.451 |
For abbreviation and statistical details, see Table II.
Table V showed results of jejunum morphology, related parameters showed results on villus height and crypt depth in broilers. The highest villus height was recorded in LP-0.01 in comparison with LP, while no significant difference was observed in control (P<0.05). Moreover, no significant difference was found in crypt depth among all treatments.
Table VI showed data regarding gut microbiology that indicated pathogenic bacteria counts were decreased and Lactobacillus were increasing in LP-0.01 while lowest in Control. The highest population of Salmonella was observed in Control followed by LP and lowest in LP-0.01. A similar trend was observed for a population of E. coli.
Table VI. Effect of dietary treatments on gut microbiology (cfu/gm) broilers.
|
Parameters |
Control |
LP |
LP-0.01 |
P. value |
|
4.33±0.39a |
4.00±0.22a |
1.00±0.17b |
0.027 |
|
|
E. coli |
5.33±0.39a |
4.67±0.43a |
2.33±0.33b |
0.002 |
|
Lactobacillus |
6.53±0.66b |
5.47±0.88c |
9.00±0.57a |
0.003 |
For abbreviation and statistical details, see Table II.
DISCUSSION
Due to continuous inflation commodity prices are increasing day by day which increases the cost of production of broiler meat. Recently it has been observed that major feed ingredients like soybean meal, canola meal, sunflower meal, and maize grain prices have increased to almost double within a span of just 3 to 4 years. Protein is a major component of a broiler diet and an increase in prices of protein sources will directly impact on cost of production. Various substitution methods and feed supplementation are being tried worldwide to reduce the proportion of protein in feed like different feed additives, enzymes and probiotics. Bacillus species are helpful for the growth of lactic acid-producing bacteria (Tsai et al., 2019) and reduce the pH of intestine. Intestinal environment acidification was significantly observed to inhibit the growth of harmful bacteria e.g. Salmonella (Aron-Wisnewsky et al., 2021).
The effectiveness of probiotics is influenced by a wide range of factors, including strain selection, administration level, application method, ability of the selected strain to survive at ambient temperature, long-term storage and viability, fermentation method, and type of substrate used for probiotic microbe growth (Zhao et al., 2019). Susceptible to high temperatures and challenging storage conditions, B. pumilus thrives in these environments. As a probiotic strain for chickens, B. pumilus is universally accepted as safe (Zhang et al., 2021). Bacillus strains were shown to be beneficial to broilers, layers, and turkeys in various studies (Hassan et al., 2021). In this study, the effects of B. pumilus on broiler performance were examined. Despite extensive research on B. subtilis strains, the impact of B. pumilus on broilers has only been documented a handful of times. Probiotics have been found to have a long-term effect on growth in other studies. The BW of huge freshwater prawns and striped catfish was shown to be increased by B. pumilus (Jayaraman et al., 2013). The disparities in findings may be due to differences in strains investigated, probiotic dosage, feed mix, and rearing environment.
B. pumilus strains did not influence growth performance during the first three weeks of life (days 1–21). Even so, LBW and ADG were high in LP-0.01 groups and slightly lower in control and LP groups from day 21 to 42, while ADFI and FCR were significantly higher at day 21 in the control group and lower in the LP-0.01 groups overall, while FCR was significantly higher in the control group than the LP-0.01.
There have been limited studies on supplementation of B. pumilus in poultry. Our study provided a new way of finding an effect of B. pumilus with low protein. In our research, low dietary CP showed negative impact on LBW, ADG and FCR in broiler while B. pumilus supplementation in feed of broiler showed growth performance results similar to basal diets. Similar results revealed by (Saied et al., 2022) supplementation with reduced dietary CP thus supplementation of B. pumilus may have a positive impact on these parameters. This results also by (Hassaan et al., 2021) verified that B. pumilus addition noticeably augmented the BW, ADG, and reduced FCR in broilers. It is speculated that improvement in growth performance is possibly ascribed to triggering the secretion of numerous digestive enzyme activities and additional secreted active substances by B. pumilus probiotics. The abovementioned evidence is taken together that addition of B. pumilus in broiler diet had beneficial results on growth performance.
Dressing percentage EV, BM and LM percentage were decreased in broiler chickens fed with a low CP diet, while abdominal fat was higher in birds fed with a low CP diet line with our study this may be due to an imbalance of energy and protein. The significant effect of dietary treatment on all parameters of carcass characteristics in this study with supplementation of B. pumilus in low CP diet. The highest dressing percentage was observed in LP-0.01 followed by Control. Similarly, the highest relative weight of the leg was found in LP-0.01 and lowest in LP. While the highest relative weight of breast and abdominal fat were observed in Control and LP, respectively. Our findings agree with (Yadav et al., 2018) noted that supplementation of probiotics efficiently amplified the carcass percentage and depressed the AF rate in broilers. Furthermore, (Ghasemi-Sadabadi et al., 2019; Khan et al., 2022) noticed that dietary addition of B. subtilis (DM 03, TAM 04 and IQB 350) had a positive effect on breast muscle yield. Authors also found that poultry diet incorporation with probiotics may improve the thigh muscles weight and reduce the AF content (Tsai et al., 2019). Supplementation of B. pumilus in diet can improve digestion and absorption due to greater intestinal microflora and activity, increased nutrients intake and decreased abdominal fat content. The lower abdominal fat content indicated that B. pumilus may led to greater economic productivity and the public wants to lower fat meat.
Nutrient digestibility plays a significant role in the animal feed industry as a higher digestibility rate increases feed efficiency and overall farm economics. While DM, CP and ME digestibility compromise in broilers fed a low CP diet. B. pumilus supplementation neutralizes the negative effect of a low protein diet. In our study, highest DM availability was found with the supplementation of B. pumilus. Similarly, the highest availability of crude protein, crude fat and ash. This is because probiotics play a key role in maintaining a favorable environment in the digestive system of chicken. Our findings are well supported by the study carried out by (Pirzado et al., 2020) which proves feed suppliments can enhance the secretion of digestive enzymes of poultry birds and increase enzymatic activity. Bacillus can fragment a huge of proteins, lipids, and carbohydrates into smaller pieces (Park et al., 2016; Xia et al., 2018). The increased activity of enzymes amylase and protease may led to higher digestion and absorption of nutrients (Bonos et al., 2021).
The intestine plays a pivotal role in absorption of basic nutrients, and improvement in digestibility plays an important role in morphology of intestine. The villus structure plays a key role as villus is the basic unit to absorb nutrients. Whereas, crypt depth is considered a villus factory, and deep crypt indicates rapid tissue turnover and high demand for new tissues (Yang et al., 2016; Mushtaq et al., 2023).
Our study results showed a significant effect with supplementation of B. pumilus on villus height and crypt depth in broilers. The highest villus height with numerically lowest crypt depth was observed through supplementation of B. pumilus. The positive effects of some dietary Bacillus spp. probiotics on intestinal morphology of broilers have been previously documented and it has been reported that Bacillus probiotic supplementation can interfere positively with influence on morphology of intestine of broilers, enhancing feed efficiency and growth (Xiao et al., 2018). Our results are well supported by research conducted which indicates that villus height, villus width and muscle thickness of fish fed on probiotic supplemented diets were significantly higher than that of the fish without treatment. (Aron-Wisnewsky et al., 2021) disclosed that Nile tilapia kept in the cage fed B. amyloliquefaciens showed increase in the number of goblet cells per villus in comparison with the control.
Microorganisms play a key role in GIT as these create favorable and unfavorable environments in the intestine for digestion, secretion, and absorption. Bacillus and Lactobacillus species play a favorable role in the gut of the chickens. Our results based on the experiment show that counts of pathogenic bacteria were decreased, and Lactobacillus were increasing through supplementation of B. pumilus. The highest population of Salmonella was observed in Control followed by LP and lowest in LP-0.01. A similar trend was observed for a population of E. coli. However, the population of Lactobacillus was lowest in Control (6.33 cfu/g) and highest in LP-0.01. Supplementing broiler feed with B. pumilus improves growth of birds, modulates intestinal microflora, improves morphometry of intestine, and improves meat-oxidative stability. Qin et al. (2024) reported that positive effects of B. pumilus addition are co-related to the alterations in the intestinal microflora mechanism and nutrients absorbed more efficiently in the gut. Hence, it may be suggested that B. pumilus has probiotic potential in chicken feed and can be tried in large-scale studies.
It is concluded that supplementation of Bacillus pumilus in low-protein broiler diets counter-balance the effects arising from low protein and is equally beneficial to the traditional broiler diets in terms of growth performance, nutrient digestibility and intestinal microflora. It is further added that dietary protein levels of boiler diets could be reduced if B. pumilus is included in feed at commercial levels.
Declarations
Acknowledgement
We are highly grateful to the staff of Central Veterinary Diagnostic Laboratory (CVDL) and Department of Veterinary Pathology for providing us with a supportive environment and valuable assistance in microflora and histology analysis.
Funding
The authors would like to extend their sincere gratitude to the research supporting project (no: RSPD2024R694), King Saud University, Riyadh, Saudi Arabia.
IRB approval
The research study was approved by Departmental Board of Studies, Department of Animal Nutrition (approval code: No. NUTR-245-2023), Faculty of Animal Husbandry and Veterinary Sciences, Sindh Agriculture University, Tandojam.
Ethical statement
All the methods and experimental protocols were approved by the Animal Care and Ethics Committee (AEC-FAHVS-59-2023) of Sindh Agriculture University, Tandojam.
Statement of conflict of interest
We certify that there is no conflict of interest with any financial, personal, or other relationships with other people or organization related to the material discussed in the manuscript.
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