https://orcid.org/0000-0003-2704-9085
Abstract
This study evaluated the effects of dietary supplementation of Iranian commercial multi-strain probiotic (IMSP) or different levels of Pediococcus acidilactici (PA), isolated from Iranian camel milk, on some biological parameters in male Ross broilers. A total of 240 1-d-old chicks were divided into four groups as follows: (1) Control-Unsupplemented, (2) supplemented with MSP, (3)106 CFU PA g−1diet, (4)108 CFU PA g−1diet (4). Groups 2 and 3 exhibited a higher total growth rate and feed conversion ratio, compared to the control and group 4 (p < .05). Group 3 showed a larger villus length/crypt-depth compared to the control (p < .05). Groups 2 and 4 displayed lower and larger count of jejunal Gram (−) bacteria and Lactobacillus spp. compared to the control, respectively (p < .05). The control group showed the lowest levels of total serum protein (p < .05). Group 3 had lower blood triglycerides compared to the control and group 2 (p < .05). These results indicated that the IMSP and 106 CFU PA g−1diet can improve growth and FCR and can be used in the poultry industry of Iran.
Supplementation diet with native multi-strain probiotic is useful. Pediococcus acidilactici isolated from Iranian camel milk can improve broilers performance and reduce blood triglycerides.
HIGHLIGHTS
Introduction
In the poultry industry, intensive poultry production conditions are unable to maintain the right balance of intestinal microbiota (Salehizadeh et al. Citation2019). Probiotics, direct-fed microbial products, are feed supplements that provide beneficial live bacteria to the host (Leyva-Jimenez et al. Citation2022) and play a crucial role in maintaining the right balance of gastrointestinal microbiota. Balanced intestinal microbiota composition enhances digestive capacity and their bio-availability, immune responses and growth performance and prevents epithelial tissue damage, ultimately leading to increased profitability (Abou-Kassem et al. Citation2021).
Probiotics play their role through different mechanisms, including nutritional competition with pathogenic microorganisms, adhesion to the surface of the gut, and secretion of anti-pathogenic compounds (Jazi et al. Citation2018; Yazhini et al. Citation2018). The effectiveness of probiotics depends on microbial strains and number, survival in the gastrointestinal system, methods of usage, and the condition of animals in terms of type, age, immune function, history of antibiotics usage, and environment (Salehizadeh et al. Citation2019). Tremendous attempts have been undertaken in recent years to find and employ more beneficial probiotic microbial strains (Zhang et al. Citation2021). Some of probiotics contain one microbial strain, while others include several microbial strains. It gained the attention of researchers and the feed industry to prepare multi-strain probiotic to obtain a synergistic interaction for better efficiency (Hussein and Selim Citation2018).
Pediococcus acidilactici (PA) is a gram-positive cocci, which belongs to class of the lactic acid bacteria (Tarabees et al. Citation2019). It is used in the production of some probiotics (Khattab et al. Citation2021). There are a few reports about the effects of PA in Iranian poultry farms. Therefore, the aim of this study was to evaluate the effects of Iranian multi- strain probiotic and different levels of PA isolated from Iranian camel milk on the performance and some biological parameters of broilers. Since it is generally recommended that probiotic products should have a minimum concentration of 106 CFU g−1diet (Shini and Bryden Citation2021), therefore, the effectiveness of 106 and 108 PA CFU g−1diet were investigated.
Materials and methods
Animals and treatment
A total of 240 one-day-old male Ross broiler chickens were weighed individually, divided into 4 groups with 6 replicates of 10 chickens for 35 d. The groups included:
Unsupplemented corn-soybean based commercial diet; supplemented with
Iranian multi-strain probiotic (IMSP),
106 CFU of P. acidilactici (PA) g−1 and
108 CFU of PA g−1diet. Water and feed were provided ad libitum. Ross broiler guideline was performed. Coccidiostat-free mash diets were formulated for starter (0–11 d), grower (12–23 d), and finisher (24–35 d) phase (Table ).
Table 1. Feed composition and ingredients of control diet.
Preparation of IMSP and PA
Based on certification by the manufacturer, each gram of IMSP under the brand name Bio-Poul (Mahan Biotherapy Company, Tehran, Iran) contains 2 × 109 CFU of Enterococcus faecium, P. acidilactici, Bacillus subtilis, Lactobacillus acidophilus, Lactobacillus Plantarum, Lactobacillus rhamnosus, Lactobacillus casei, Bifidobacterium bifidum, and Saccharomyces cerevisiae. According to the manufacturer’s suggestion, 0.2 g IMSP per kg of feed was added so that each gram of feed contained 106 CFU of probiotics according to the calculations.
PA, was previously isolated from Iranian camel milk, was obtained from the microbial collection of the Islamic Azad University, Babol Branch. PA was inoculated in the MRS agar medium and incubated under microaerophilic conditions for 24–48 h at 37° C. After determine its purity, PA was transferred to MRS broth and incubated at 37 °C under shaking conditions (120 rpm, 48 h) for increasing its number (Sarkar et al. Citation2020). Then media were centrifuged (3000 rpm x g, 10 min) and was removed the supernatant. The PA number were counted and directly, without any career, were added to the diet using spray pressure.
Growth performance
Broilerʼs live body weights were recorded on 0, 11, 23, and 35 d. Feed intake was recorded daily for each replicate, and then feed conversion ratio (FCR) was calculated.
Jejunum bacterial counts
At the end of the experiment, one chicken from each replication was killed by cervical dislocation. Sampling was immediately carried out from the jejunum and transferred to the laboratory under the cold chain condition. Subsequently, 1 cc of the contents of the jejunum was prepared after dilution and bacterial counts were measured by the pour plate method. Three types of culture media including MRS agar (Merck Company; 1.10660.0500), Eosin Metilan-Blou agar (Merck Company; 1.01347.0500) and Muller-Hinton agar (Merck Company; 1.05437.0500) were used to count the Lactobacilli, gram (−) bacteria and total aerobic microbial, respectively (Gunal et al. Citation2006; Garcia Citation2010; Zhang et al. Citation2022). The results were reported as the logarithm of CFU per gram of contents.
Intestinal histology
On day 35, 2 cm of the middle part of the jejunum of one chicken from each replicate was removed for histology study. The jejunal segment, after flushing in PBS, was fixed with 10% formalin, dehydrated, clarified, and embedded in paraffin (Tissue processor, Shadon Citadel 1000). Serial sections were cut at 0.5 mm by microtome (Leica RM2125), stained using haematoxylin and eosin, and examined by a light microscope (Olympus), equipped with a camera at a 10× magnification. The length and width of villi, as well as the depth of crypts, were measured using TCapture software. The area of the villi was calculated according to Banaszak et al. (Citation2020).
Blood biochemical parameter analyses
On day 35, 1 cc of the blood was taken from the wing vein of one chicken per pen, placed in a water bath for 60 min. The serum was collected by centrifuging (3000 rpm x g for 15 min). The levels of serum total cholesterol, triglyceride, HDL, total protein, and glucose were measured by Ra-xt autoanalyzer (Technicon-USA) as well as special diagnostic kits (Pars Azmoon made in Iran).
Statistical analysis
Data were analysed using the GLM procedure of SAS software (2003) for a completely-randomized design model. Duncan’s test was used to compare the means. The p value less than 0.05 was considered to be statistically significant. The normality of distribution was examined before analysis using the Univariate procedure.
Results and discussion
Growth parameters
Our findings (Table ) demonstrated that IMSP or PA could not affect broiler performance during the starter phase (p > .05). Some researchers reported that probiotic require time to perform an effective function in the gastrointestinal system, and this time varies depending on the microorganism strain and the circumstances of the gastrointestinal system (dela Cruz et al. Citation2019). IMSP was able to decrease total feed intake, compared to PA groups (p < .05). Probiotic, when compatible with the host intestinal microbiota in terms of kind and number available, can decrease feed intake through improving the balance of intestinal microbiota, increasing nutrients bioavailability, and improving FCR (dela Cruz et al. Citation2019; Ramlucken et al. Citation2020; Khattab et al. Citation2021). Our results indicated that different levels of PA have varying effects on broiler’s total growth and FCR (0–35 d of age); in fact, the addition of 106 CFU of PA g−1diet could improve growth and FCR compared to 108 CFU of PA g−1diet . Researchers have previously found that the dose of probiotic can affect growth performance (Bai et al. Citation2018). This result probably indicate that 106 CFU of PA g−1diet has a more important effect on maintaining a healthy balance of gastrointestinal microbiota, that is due to the fact that improved enteric microbiota balance plays an important role in dictating health and growth responses (Jazi et al. Citation2018). Most studies showed that probiotics if used correctly, can increase growth through producing vitamins and exogenous enzymes, secreting biological components, and having antagonistic effects on harmful bacteria (Ramlucken et al. Citation2020; Rivera-Pérez et al. Citation2021). The present study found that supplemented diets with 106 CFU of PA g−1diet, similar to IMSP, led to increase the total growth and improved FCR.
Table 2. Performance parameters in different groups* (mean ± SD).
Jejunum histology and bacterial counts
Intestinal morphology is an important indicator for assessing intestinal health. The length of the villi and the depth of the crypt are the main indicators of intestinal absorption and represent the maturity of intestinal tissue cells, respectively (Zhang et al. Citation2021). There are contradictory reports on the effects of probiotics on intestinal tissue. Some investigators have reported, probiotics did not affect intestinal morphology (Dela Cruz et al. Citation2019; Zhang et al. Citation2021), while others showed that probiotic increases the length of the villus and the depth of the crypts in different parts of the small intestine (Sharifi et al. Citation2012) or can increase the ratio of the villi length to crypt depth (He et al. Citation2019). Our result (Table ) indicated that, although IMSP and PA led to no effects on the length, width, and area of the villus (p > .05), the addition of 106 CFU of PA g−1diet (group 3) resulted in an increase in the ratio of villi length to crypt depth (p < .05). Furthermore, the addition of 106 CFU PA g−1diet (group 3) showed a better growth rate compared to the control group (Table ). The larger the villus, the more nutrients it can take, leading to an increased growth rate (He et al. Citation2019). Collectively, studies showed that the use of a high-quality probiotic can modulate the intestinal microbial population, increase short-chain fatty acids produced by beneficial bacteria (Sharifi et al. Citation2012), and lengthen villus, improve crypt cell proliferation (Al-Khalaifa et al. Citation2019; Khattab et al. Citation2021).
Table 3. Morphological characteristics and microbial population (cfu/g) of jejunum in different groups* (mean ± SD).
Some studies showed that probiotics suppress gram (−) bacteria. Findings from this study concluded that IMSP or 108 CFU of PA g−1 (Table ) was able to decrease gram (−) bacteria and increase Lactobacillus (p < .05). Lactic acid bacteria can prevent pathogen growth through the production of antimicrobial substances such as H2O2, alcohol, lactic acid, and other metabolites. Some researchers suggested that the secretion of pediocin peptide and the production of lactic acid by PA are important factors in disrupting the pathogens (Tarabees et al. Citation2020; Abou-Kassem et al. Citation2021). Based on our results, PA dose in the diet affects the response of the intestinal microbial population.
Biochemical parameter analyses
Blood biochemical variables are unstable biomarkers whose fluctuations are influenced by a variety of factors, such as diet, serving as indicators of animal health (Ciurescu et al. Citation2020). In the present study, PA and IMSP led to increased serum total protein, compared to the control group (p < .05). This can be evidence for increased protein uptake, because some researchers showed that beneficial bacteria, through a competitively excluded mechanism, prevented the degradation of protein and the use of nitrogen by pathogens and increase the efficiency feed protein absorption and serum total protein (Yazhini et al. Citation2018; Al-Khalaifa et al. Citation2019; Khattab et al. Citation2021). Studies showed that lactic acid bacteria can decrease cholesterol absorption through a decrease or the inhibition of Niemann-Pick C1-like1 protein expression present on the surface of enterocytes, as well as inhibition of 3-hydroxy 3-methyl-glutaryl-CoA (Yazhini et al. Citation2018). However, there are conflicting results regarding the effect of probiotics on lipid metabolites. Some studies indicated that probiotic lead to decreased blood cholesterol, triglyceride (TG), and HDL (Haque et al. Citation2017), whereas other researchers concluded that probiotics did not affect TG (Khattab et al. Citation2021). Jazi et al. (Citation2018) reported probiotics decreased TG while not affecting HDL levels. In this experiment, although blood cholesterol and HDL were not affected by IMSP or PA, it was observed that the 106 CFU of PA g−1 diet (group 3) decreased TG (Table ). There is accumulating evidence showing that beneficial bacteria increase bile salt excretion by producing the bile salt hydrolase, which in turn decreases bile salts and the efficiency of lipid uptake (Seifi et al. Citation2017; Jazi et al. Citation2018). Furthermore, beneficial bacteria can decrease or inhibit the synthesis of TG and even cholesterol in the liver by increasing short-chain fatty acids, which in turn decreases these blood metabolites (Dev et al. Citation2020).
Table 4. Biochemical parameters of the blood in different groups* (mean ± SD).
Conclusions
This study indicated that the addition of Iranian multi-strain probiotic (IMSP) or Pediococcus acidilactici (PA) of camel milk isolate in the broiler diet can inhibit and induce the count of gram (−) bacteria and Lactobacillus in the jejunum, respectively. The level of PA in the diet plays an effective role on villus length/crypt-depth, intestinal microbiota and growth performance. Supplemented diet with IMSP or 106 CFU PA g−1 diet, can improve the broilers growth rate and FCR.
Ethical approval
All chicken experiments were performed in agreement with Institutional Animal Care and Research Advisory Committee of Babol Branch, Islamic Azad University, Iran (19532).
Acknowledgements
The authors would like to announce their appreciation to Meisam Rameshgar, Ali Rezazadeh and Behzad Jalalifar for their kind assistance during sample collection.
Disclosure statement
No potential conflict of interest was reported by the author(s).
Data availability statement
The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.
Additional information
Funding
References
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