Introduction
The increasing demand for protein has led to the need to
enhance the production of protein sources. Japanese quails (Coturnix japonica) are being raised as a
potential alternative broiler protein source. Quails are disease-resistant and
offer a distinct meat taste (Priti and Satish
2014; Mnisi et al. 2021).
Animal feed supplements such as enzymatic products (phytase) and living
microorganisms (probiotics) enhance animal performance. Phytase is naturally
found in seeds of legumes and their by-products and other feedstuffs that serve
as microbial sources (Rabie and Abo El-Maaty
2015). The stomach plays a crucial role in utilizing supplemental
phytase as an enzyme due to its acidic environment and high resistance to
pepsin. The phytase enzyme hydrolyzes phytate into inositol and inorganic
phosphate (Alam et al. 2020). Microbial phytase enhances the digestibility
of dietary phytate phosphorus and positively influences the digestion of other
nutrients in Coturnix japonica. However, there is a lack of evidence on
the impact of phytase on bone growth and development in quails (Mansoori et al.
2012; Ahiwe et al. 2021).
Phytase
supplementation in poultry diets has become increasingly common due to public
concerns regarding phosphorus pollution and its ability to increase non-phytate
phosphorus consumption. However, it also decreases trypsin, pepsin, and
α-amylase activity. The addition of dietary phytase leads to increased
bioavailability and digestibility of phytate-bound minerals such as zinc,
copper, calcium and phosphorus. Furthermore, microbial phytase improves the
digestibility of crude protein and amino acids while reducing nitrogen and
phosphorus excretion (Alagawany et al. 2020).
Probiotics, living microorganisms, are critical in enhancing the
bioavailability and digestibility of nutrient content in the gastrointestinal
tract (GIT) (Gul and Alsayeqh 2023).
These microorganisms are derived from various sources such as yeast, fungi and
bacteria, including Lactobacillus, Bifido bacterium, Cerus bacillus and Saccharomyces
(Hossain and Momu 2022; Ramaiyulis et al. 2023). Probiotics enhance
the absorptive area of gastrointestinal tract, weight gain, reduce infection
rate and stimulate immune system. Additionally, these microorganisms play a
crucial role in lowering serum cholesterol levels. This decrease in cholesterol
levels could be due to the assimilation or uptake of cholesterol by live
microbial cells such as Lactobacillus
or through the co-precipitation of cholesterol with de-conjugated bile salts (Abd El-Moneim et al. 2019; Ramaiyulis et al. 2023).
Several
studies have demonstrated that probiotics and phytase alone positively affect
the health of poultry animals (Dim et al. 2022). While a large
amount of data is available on the separate use of these supplements, there is
limited literature on their combined use, particularly in Japanese quails. This
research aimed to investigate the impact of probiotics and microbial phytase,
individually and in combination, on the biochemical profiles and physiological
processes in C. japonica.
Materials
and Methods
The Avian Research and Training Center at the University
of Veterinary and Animal Sciences in Lahore provided 200 day-old quails (Coturnix japonica) for an experiment.
The quails were kept in experimental sheds for 8-10 days and separated into
four groups of five replicates and ten birds per replicate. For 28 days, the
quails were fed a commercial corn-based basal diet (BD) supplemented with
probiotics and phytase and given free access to water. Group A acted as a
control and received a regular diet, whereas groups B, C and D received BD +
0.01% Microbial phytase, BD + 0.1% Bacillus
cereus and BD + 0.01% phytase + 0.1% probiotics, respectively. The temperature
and humidity were kept at appropriate levels during the experimental course. At
the end of the trial, blood specimens of two birds from every replicate were
collected for serum analysis and liver and kidney samples were taken and
preserved at -40ºC. In addition to serum total protein, albumin, globulin,
urea, creatinine, uric acid, alanine aminotransferase (ALT), aspartate
aminotransferase (AST), triiodothyronine (T3), thyroxin (T4), glucose, oxidant
and antioxidant levels and lipid profile (TC, TG, HDL, and LDL), oxidant and
antioxidant levels were also measured in homogenized muscles and tissues (liver
and kidney).
The recorded
data were analyzed with Statistical Package for Social Sciences (SPSS) in
one-way analysis of variance (ANOVA). The findings were reported as means,
standard error of the means (SEM). The means were compared using Duncan's
multiple range tests, with the significance level set at P<0.05.
Results
The results revealed that
serum proteins, group D showed a critical rise (P<0.05) in total protein,
while groups C and D exhibited a rise (P<0.05) in albumin concentration when
linked to other groups (Table 1). Renal function tests (RFT) (urea, uric acid
and creatinine) were non-significant (P<0.05) results in all treated groups
(Table 1). When compared to the control group, AST levels were significantly
reduced (P<0.05) in groups C and D, but serum ALT levels were
non-significant (P<0.05) in all supplemented groups (Table 1). Thyroid
hormones showed that T3 levels were significantly reduced in groups C and D,
while T4 was non-significant in all groups (Table 1). When contrasted with the
untreated group, all supplemented groups demonstrated a significant increase
(P<0.05) in serum glucose (Table 1).
The lipid profile revealed that total cholesterol concentrations in
treatment groups were not significantly different (P>0.05) from the control
group (Fig. 1A). However, when contrasted with the untreated group,
triglyceride levels were significantly lower (P<0.05) in all treatment
groups. In contrast, serum high-density lipoprotein (HDL) levels were
significantly higher (P<0.05) in supplemented groups C and D (Fig. 1B).
Oxidative status results showed that the activity of serum and muscle
Malondialdehyde (MDA) was considerably lower (P<0.05) in all treatment
groups when contrasted to the control group (Fig. 1C, E). Group D was found to
decrease liver MDA levels when contrasted to groups B and C (Fig. 1D). The
serum catalase activity was found to be considerably higher (P<0.05) in all
treatment groups (Fig. 1F), while all treatments had a non-significant effect
(P<0.05) on muscle catalase activity (Fig. 1H). The concentration of
catalase in the liver was estimated to be significantly higher (P<0.05) in
group D when contrasted with control and other supplemented groups (Fig. 1G).
These findings are essential in understanding the effect of probiotics
and phytase supplementation on quail health and may have implications for
developing dietary strategies to enhance quail health and productivity.
Discussion
The
present study evaluated the potential positive effects of probiotics (B. cereus) and Phytase
Supplementation on the health of Japanese Quails. The
probiotic B. cereus is a vital food pathogen most commonly used in
livestock and humans. Probiotics have been shown
to improve the antioxidant status in birds by reducing the production of free radicals.
They enhance the absorptive area of the gut, improve feed conversion ratio,
support weight gain, reduce mortality and disease infection, and stimulate the
immune
Table 1:
Mean and SEM of different parameters in quails supplemented with basal
diet, phytase, and combination
|
Parameters |
Experimental Groups |
||||
|
Control |
Microbial phytase (0.1 g/kg) |
Bacillus cereus (1 g/kg) |
Microbial phytase + B. cereus |
P - value |
|
|
Serum
proteins |
3.86±0.49c |
4.12±0.58c |
8.58±1.17b |
14.00±1.04a |
0.000 |
|
Serum
albumins |
2.28±0.16b |
2.78±0.45b |
3.60±0.19a |
3.13±0.17a |
0.006 |
|
Serum
urea |
30.24±0.68 |
32.63±0.94 |
34.64±2.52 |
30.50±2.52 |
0.302 |
|
Serum
uric acid |
12.78±0.22 |
13.64±0.49 |
13.53±0.41 |
13.22±0.34 |
0.406 |
|
Serum
creatinine |
6.49±1.42 |
5.09±1.27 |
5.01±1.01 |
8.83±1.01 |
0.074 |
|
Serum AST |
37.12±3.21a |
26.20±3.01a |
3.27±0.63b |
8.73±6.55b |
0.000 |
|
Serum
ALT |
2.62±0.43 |
4.80±1.27 |
3.27±1.09 |
2.91±0.46 |
0.294 |
|
Triiodothyronine (T3) |
329±16.81a |
330±10.09a |
289±10.25b |
269±12.54b |
0.003 |
|
Thyroxine (T4) |
2.36±0.18 |
2.17±0.35 |
1.83±0.10 |
1.90±0.16 |
0.299 |
|
Serum
glucose |
293.5±25.5c |
397.8±71.7ab |
336.3±56.8bc |
393.4±48.3a |
0.001 |
Values
represent the Mean ± SEM of experimental groups of quails
Different
superscripts a-c indicates an essential difference between groups (P
< 0.05)
%20312-316_files/image002.png)
Fig. 1: Effects of phytase and probiotics supplementation
on quail serum, liver, and muscle oxidative stress biomarkers and serum
catalase activity. Quails in Group A served as the control (Control), Group B
received BD + 0.01% Microbial phytase (Phytase), Group C received BD +0.1%
Bacillus cereus probiotic), and Group D received BD + 0.01% phytase + 0.1%
probiotics (comb). (A) Serum triglycerides concentration, (B) Serum HDL
concentration, (C) Serum MDA concentration, (D) Liver MDA concentration, (E)
Muscle MDA concentration, (F) Serum catalase concentration, (G) Liver catalase
concentration, and (H) Muscle catalase concentration are presented as Mean ±
SEM. Different superscripts a-c represent significant differences (P<0.05)
among the groups
system (Ramlucken et al. 2020). This study
found that Group D had significantly higher serum total proteins and albumin
levels. In addition, the probiotic-supplemented group (Group C) showed a
significant increase in albumin levels because the probiotic-supplemented group
was given B. cereus (Fig. 1C–D). These findings are consistent with
previous research where concentrations of albumin and serum proteins were
similar to other bird species, such as ostriches, peregrine falcons, amazons
and pigeons (Yalçin et al. 2008; Scholtz et al.
2009). Our research findings indicated no
significant changes in RFT, including urea, uric acid and creatinine, in the
serum of supplemented quails. These results are partially consistent with
previous studies where globulin, total serum proteins, albumin, urea and
creatinine, in addition to ALT, AST activities had no significant (p<0.05)
differences among control and feed additive supplemented groups (Algedawy et al. 2011; Owosibo et al. 2013).
Results showed that serum AST levels were significantly lower in groups C
and D of quails, whereas all groups had no significant effect on serum ALT
levels. These results are consistent with the findings of (Sarangi et al.
2016). The probiotic supplementation appears to have a nurturing effect on
the liver by lowering the activity of liver enzymes and mitigating the impact
of stress due to decreased concentration of AST. Our results demonstrated a significant decrease in serum T3 levels in
birds group C and group D. Non-significant results were found for T4 levels in
all supplemented groups (Fig. 1C–D). These findings have been consistent with
(Zhan et
al. 2007). The blood glucose level was high in all supplemented
groups, and the results agreed with (Shata et
al. 2017). However, phytase responses against blood glucose are
still unknown because 80% of dietary glucose is absorbed through sodium active
transport systems; due to this, phytase indirectly increases blood glucose
levels through effects
on sodium (Cowieson et al. 2004).
The current
study revealed that the total cholesterol concentration in serum was not
considerably (P<0.05) exaggerated by treatments. These results are supported
by the study conducted by (Rajput et al. 2013). This decrease in cholesterol levels could be due to
the assimilation or uptake of cholesterol by live microbial cells such as Lactobacillus or through the
co-precipitation of cholesterol with de-conjugated bile salts. The serum
triglyceride (TG) was significantly decreased in group C with probiotics alone,
supported by a study conducted by (Rajput et al. 2013; Behrouz et al. 2020).
The consequences of our study designated that the
addition of phytase led to a significant increase (P<0.05) in HDL
cholesterol compared to probiotic supplementation alone. However, the
combination of probiotic and phytase appeared to have a synergistic effect on
serum HDL cholesterol, leading to a significant increase (P < 0.05). The
results of (Priyodip et al. 2017)
research showed similar results. Our study demonstrated that the novel
combination of phytase and probiotic as a feed supplement resulted in a
significant (P<0.05) increase in HDL and a decrease in TG (P<0.05) but
had no critical effect on TC. These findings align with our expectations, as
both phytase and probiotics are known to reduce bad cholesterol, and the
combination of the two was more effective in achieving this.
The present study on quails showed an effective decrease in the activity
of liver MDA in all supplemented groups, which corroborated the research
results of (Arendt and Allard 2011; Chalasani et al. 2012; Rozman 2014). The observed decrease in liver MDA's
activity is due to decreased oxidative stress, and decreased liver MDA's
activity is beneficial because MDA is an indicator of lipid peroxidation. In
the present study, serum catalase activity was higher in all treated groups in
broiler chicks (Aluwong et al. 2012). Our study found that the liver level of
catalase was considerably higher (P<0.05) in group D contrasted with the
untreated group and other supplemented groups. As a novel feed
supplement, the combination of phytase and probiotics significantly (P<0.05)
boosted the catalase activity and reduced (P<0.05) MDA in serum and liver
tissue in all the supplemented groups. These findings are according to our
expectations, as both phytase and probiotics are responsible for decreasing
oxidative stress.
Conclusion
Probiotics and phytase, alone and in combination, have
positive effects on the biochemical profiles and physiological processes of
Japanese quails. The supplementation of probiotics and phytase improved liver
health and thyroid hormones, preventing energy loss by storing glucose in
glycogen. The combination of probiotics and phytase was more effective than
either supplementation alone. However, additional study is required to
determine the mechanisms underlying these impacts and evaluate the long-term
impact of supplementation on other physiological and immunological parameters.
Acknowledgment
All authors are grateful to their representative
universities.
Author
Contributions
CN, MB and MA performed material preparation, data
collection, and analysis. The original draft of the manuscript was written by
CN & MA. RS, AH, UM, and HM completed the revision and editing of the
manuscript. MA reviewed, edited and approves the final version of manuscript.
SAF provided valuable supervision.
Conflict
of Interest
The authors declare no conflict of interest among them.
Data Availability
The datasets generated and analyzed during this study
are included in this published article.
Ethics Approval
All the procedures adopted to perform this experiment
were adopted by the ethical review committee of the University of Veterinary
and Animal Sciences, Pakistan
Funding
Source
No specific funding was acquired for this work.
References
Abd El-Moneim AEME, EM Sabic, AM Abu-Taleb (2019). Influence
of dietary supplementation of irradiated or non-irradiated olive pulp on
biochemical profile, antioxidant status and immune response of Japanese quails.
Biol Rhythm Res 53:519‒534
Ahiwe EU, TT Tedeschi Dos Santos, H Graham, PA Iji (2021).
Can probiotic or prebiotic yeast (Saccharomyces cerevisiae) serve as
alternatives to in-feed antibiotics for healthy or disease-challenged broiler
chickens?: A review. J Appl Poult Res 30:100164
Alagawany M, SS Elnesr, MR Farag, R Tiwari, MI Yatoo, K
Karthik, I Michalak, K Dhama (2020). Nutritional significance of amino acids,
vitamins and minerals as nutraceuticals in poultry production and health – a
comprehensive review. Vet Quat 41:1‒29
Alam S, S Masood, H Zaneb, I Rabbani, RU Khan, M Shah, S
Ashraf, IA Alhidary (2020). Effect of Bacillus cereus and phytase on the
expression of musculoskeletal strength and gut health in Japanese quail (Coturnix
japonica). J Poult Sci 57:200‒204
Algedawy SA, MK Soliman, IM Elashmawy (2011). Effect of
commercial probiotic and exogenous enzymes on the growth performance, immune
response, and hemato-biochemical parameters of broiler chickens. In: Proc 4th Sci Conf Anim Wealth
Res Middle East North Africa, Foreign Agric Relations (FAR), pp:430‒445.
Cairo, Egypt
Aluwong T, F Hassan, T Dzenda, M Kawu, J Ayo (2012). Effect
of different levels of supplemental yeast on body weight, thyroid hormone
metabolism and lipid profile of broiler chickens. J Vet Med Sci 75:291‒298
Arendt BM, JP Allard (2011). Effect of atorvastatin, vitamin
E and C on nonalcoholic fatty liver disease: Is the combination required. Amer
J Gastroenterol 106:78‒80
Behrouz V, N Aryaeian, MJ Zahedi, S Jazayeri (2020). Effects
of probiotic and prebiotic supplementation on metabolic parameters, liver
aminotransferases, and systemic inflammation in nonalcoholic fatty liver
disease: A randomized clinical trial. J Food Sci 85:3611‒3617
Chalasani N, Z Younossi, JE Lavine, S Jazayeri (2012). The
diagnosis and management of non-alcoholic fatty liver disease: Practice
guideline by the American Association for the Study of Liver Diseases, American
College of Gastroenterology and the American Gastroenterological Association. Hepatology
55:2005‒2023
Cowieson AJ, T Acamovic, MR Bedford (2004). The effects of
phytase and phytic acid on the loss of endogenous amino acids and minerals from
broiler chickens. Brit Poult Sci 45:101‒108
Dim EC, EA Akuru, FN Mgbor, CE Oyeagu, AB Falowo, FB Lewu, AE
Onyimonyi (2022). Study of supplementation of various levels of biochar on
health and production performance of growing local turkey (Meleagris
gallopova) poults. Intl J Vet Sci 11:315‒320
Gul ST, AF Alsayeqh (2023). Probiotics improve physiological
parameters and meat production in broiler chicks. Intl J Vet Sci 12:182‒191
Hossain MA, JM Momu (2022). Yogurt as probiotic: Comparative
effect on growth performance of broiler Japanese quail (Coturnix Japonica).
Turk J Agric - Food Sci Technol 10:987‒991
Mansoori B, MR Fatemi, M Modirsanei, J Honarzad (2012).
Influence of phytase on tibia bone characteristics of broiler quail fed on
corn-soybean meal diets. Iran J Vet Med 6:149‒154
Mnisi CM, M Marareni, F Manyeula, MJ Madibana (2021). A way
forward for the South African quail sector as a potential contributor to food
and nutrition security following the aftermath of COVID-19: A review. Agric
Food Secur 10:48
Owosibo AO, OM Odetola, OO Odunsi, OO Adejinmi, OO
Lawrence-Azua (2013). Growth, haematology and serum biochemistry of broilers
fed probiotics based diets. Afr J Agric Res 8:5076‒5081
Priti M, S Satish (2014). Quail farming: An introduction. Intl
J Life Sci 2:190‒193
Priyodip P, PY Prakash, S Balaji (2017). Phytases of
probiotic bacteria: Characteristics and beneficial aspects. Ind J Microbiol
57:148‒154
Rabie MH, HMA Abo El-Maaty (2015). Growth performance of
Japanese quail as affected by dietary protein level and enzyme supplementation.
Asian J Anim Vet Adv 10:74‒85
Rajput IR, LY Li, X Xin, BB Wu, ZL Juan, ZW Cui, DY Yu, WF Li
(2013). Effect of Saccharomyces boulardii and Bacillus subtilis
B10 on intestinal ultrastructure modulation and mucosal immunity development mechanism
in broiler chickens. Poult Sci 92:956‒965
Ramaiyulis, Mairizal, Salvia, N Fati, T Malvin (2023).
Effects of dietary catechin Uncaria gambir extract on growth
performance, carcass characteristics, plasma lipids, antioxidant activity and
nutrient digestibility in broiler chickens. Intl J Vet Sci 12:169‒174
Ramlucken U, R Lalloo, Y Roets, G Moonsamy (2020). Advantages
of Bacillus-based probiotics in poultry production. Livest Sci
241:104215
Rozman D (2014). From nonalcoholic fatty liver disease to
hepatocellular carcinoma: A systems understanding. Dig Dis Sci 59:238‒241
Sarangi NR, LK Babu, A Kumar, CR Pradhan, PK Pati, JP Mishra
(2016). Effect of dietary supplementation of prebiotic, probiotic, and
synbiotic on growth performance and carcass characteristics of broiler
chickens. Vet World 9:313‒319
Scholtz N, I Halle, G Flachowsky, H Sauerwein (2009). Serum
chemistry reference values in adult Japanese quail (Coturnix coturnix
japonica) including sex-related differences. Poult Sci 88:1186‒1190
Shata RFH, K Elkloub, M El Moustafa (2017). Effect of dietary
sumac seed powder as an antioxidant and growth promoter on growth performance
of Japanese quill. Egypt J Nutr Feed 20:105‒113
Yalçin S, H Erol, B Özsoy, I Onbaşılar, S
Yalçın (2008). Effects of the usage of dried brewing yeast in the diets on
the performance, egg traits and blood parameters in quails. Animal
2:1780‒1785
Zhan XA, M Wang, H Ren, RQ Zhao, JX Li,
ZL Tan (2007). Effect of early feed restriction on metabolic programming and
compensatory growth in broiler chickens. Poult Sci 86:654‒660