Special Issue:

Veterinary Medicine between Sustainable Development and Public Health to Confront Global Changes

Effect of Biocid® as a Feed Additive on Growth, Blood, Immunity and Bacteriological Indices of Chicken Broilers

Dalia Hamid Mansour1, Mohamed Elshabrawy Ghanem2, Marwa Hassan3, Yousry Ibrahim4, Ibrahim M. Hegab5*

1Department of Avian and Rabbit Diseases, Faculty of Veterinary Medicine, Suez Canal University, Ismailia, 41522, Egypt; 2Department of Theriogenology, Faculty of Veterinary Medicine, Suez Canal University, Ismailia, 41522, Egypt; 3Department of Animal Hygiene, Faculty of Veterinary Medicine, Suez Canal University, Ismailia, 41522, Egypt; 4El-Hoda Company for Mining and Agricultural Development, Cairo, Egypt; 5Department of Animal Behavior and Management, Faculty of Veterinary Medicine, Suez Canal University, Ismailia, 41522, Egypt.

Received | June 24, 2024; Accepted | August 18, 2024; Published | October 17, 2024

*Correspondence | Ibrahim M. Hegab, Department of Animal Behavior and Management, Faculty of Veterinary Medicine, Suez Canal University, Ismailia, 41522, Egypt; Email: [email protected]

Citation | Mansour DM, Ghanem ME-S, Hassan M, Ibrahim Y, Hegab IM (2024). Effect of Biocid® as a feed additive on growth, blood, immunity and bacteriological indices of chicken broilers. Adv. Anim. Vet. Sci. 12(s1): 220-231.

DOI | https://dx.doi.org/10.17582/journal.aavs/2024/12.s1.220.231

ISSN (Online) | 2307-8316; ISSN (Print) | 2309-3331

Copyright: 2024 by the authors. Licensee ResearchersLinks Ltd, England, UK.

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

Poultry farming is a major income source for small raisers in developing countries. It participates in providing the daily protein needs of humans through meat and eggs utilization (Abdou et al., 2023). A predictable need for animal protein has exceedingly increased in recent years. Therefore, during the last decade, the poultry industry has drastically expanded to be able to produce large quantities of meat to cover the high protein requirements of the increasing population worldwide (Choi et al., 2023). Rearing of broilers for commercial purposes was frequently being expanded and their meat was presented as superior economical alternative sources of animal protein (Ayalew et al., 2022; Eissawy et al., 2023). The high levels of broiler production and competent feed conversion are essential of the modern poultry industry, which to a certain extent could be achieved using specific feed additives.

One of the most common feed additives that is widely used in poultry farming is antibiotic additives which has been supplemented as growth promoters to poultry rations to stabilize the intestinal microbial flora, enhance the overall performances and preclude some particular intestinal pathology (Rafiq et al., 2022). This ultimately leads to an increase in growth and final body weight as addition of antibiotics to broilers’ diet could increase body weight gain by 5.8 % (Rahman et al., 2012). This enhancement might be explained by improved appetite and feed conversion efficiency, stimulate the immune system, and increased vitality and regulation of the intestinal microflora (Peric et al., 2009). However, the growing concern for development of antimicrobial resistance besides the consumer preference for antibiotic-free products has driven many countries to prohibit or restrict the utilization of antibiotics as feed additives (Fonseca et al., 2024).

Following the ban of antibiotics, probiotics are feed additives that have become more popular in poultry farming which are more universal and can be simply combined with other feed additives (Krysiak et al., 2021). The introduction of probiotic increases immunity of the gastrointestinal tract, more effective of microbial control, improved feeding efficiency, more antioxidant capacity, energy digestibility and are not as harmful to the environment as antibiotics which positively affect animal health as well as production performance (Al-Fatah, 2020). Yet, although fewer compared to antibiotics, probiotics usage might encompass several concerns such as their risks, handling procedures, and adaptability to the environment. For example, some probiotic strains will participate in the spread of antibiotic resistance (enterococcus) and produce toxin substances (Jha et al., 2020). Moreover, the conditions of using probiotics must be thoroughly observed as supplementation of probiotics during high temperature induced changes in the structure of fatty acids (FA) and secondary bacterial structures of the probiotics (Dianawati et al., 2013). In addition, the overdose of probiotics may cause a deterioration in the productive capacity in poultry (Haines et al., 2015).

Some dietary products such as prebiotics, yeast culture, essential oils and spices are therefore being evaluated to replace antibiotics and probiotics in broilers diets. Moreover, organic acids were also reported to improve productivity of pigs and poultry (Khan et al., 2022; Mahmoud et al., 2022). They have been previously utilized in feed preservation, shielding feed from bacterial and fungal obliteration (Karpiński and Ożarowski, 2024). They occur naturally mostly in fruits and are also manufactured in commercial quantity using hydrocarbons. The potential of organic acids to reinforce growth of monogastric animals has been proven formerly due to their antibacterial power and encouragement of villus growth (Nhara et al., 2024). They can be added to poultry rations at a concentration of 500g./ton for mold prevention and 2.5–3.0 kg/ton to decrease food pH and control Salmonella (Banupriya et al., 2016). Broilers fed on diets supplied with organic acids had significantly higher body weights and feed conversion ratio (Adil et al., 2010). Organic acids alone or their combinations are regarded safe, and execute equivalent functions like antibiotics (Khan et al., 2022).

Biocid® is a newly developed product recently manufactured by Al-Ahram Mining Company. The product comprises various ingredients including organic acids, amino acids and silica (Supplementary Certificates 1 and 2). Diet supplementation with amino acids had been previously reported to increase broilers production (Johnson et al., 2020) through upregulation of protein translation, increasing feed conversion ratio, improve intestinal integrity and immune function. Similarly, organic acids help restoration of intestinal microbiota and inflammation reduction which improve digestion of protein and increase growth and feed conversion ratio (Khan and Iqbal, 2015). Finally, silica enhances enzymatic activities, besides the protein and mineral digestion in broilers which improve growth and productive performance (Burton et al., 2020). Biocid® can be administered either in drinking water and/or diet. We hypothesize that dietary supplementation of Biocid®, as a source of organic acids, amino acid and silica, will improve the performance, intestinal histomorphology and bacteriological profile, and serum biochemistry of vaccinated and un-vaccinated broiler chicken during the rearing period.

MATERIALS AND METHODS

Birds and management

This experiment was performed at the animal educational and research farm, Faculty of Veterinary Medicine, Suez Canal University, Ismailia, Egypt. All experimental procedures were approved, prior to the commencement of work, by the animal ethics committee of the Faculty of Veterinary Medicine, Suez Canal University Approval No. (SCU-VET-REC- 2024034). This experiment was conducted on a total 105 one-day-old broilers chicks purchased from a commercial hatchery. Welfare concerns to minimize stress were followed according to Nielsen et al. (2021) where chicks were kept in floor pens supplied with dry sawdust which was periodically monitored for dryness and dampened sawdust was regularly removed with dry one. Ventilation hoods and ceiling fans were installed to ensure fresh air supply and removal of ammonia and foul air during the experiment. Optimum stocking density (7 birds/m2) was followed according to Amer et al. (2023). Temperature was pointed at 34 °C during the 1 week and regularly lowered till it reached 25 °C at the end of the experiment. The rearing environment was illuminated at 20 lux minimum, with functional areas of resting (e.g. dark brooders) offering intensities down to 0.5 lux. Chicks were provided with 23 hrs. of light up to day 3 of life with a gradual decrease of the photoperiod to 16–17 hrs. at day 7 till the end of the experiment. Chicks have ad libitum easy access to all feeders and drinkers throughout the experiment. Birds were daily checked for signs of animal‐based measures (ABMs) of welfare which included distress calls, feather and body dirtiness, huddling, panting, lethargy, total mortality, injurious pecking, hock burn, footpad dermatitis and body weight loss Nielsen et al. (2021). No apparent signs of distress were observed during the experiment.

Experimental design

Upon arrival, chicks were visually examined for any deformities and signs of health and unhealthy chicks were excluded from the experiment. Chicks were split randomly to 3 treatment groups having five replicates of seven birds each. The first group (Group A) received the Biocid® compound in both drinking water and feed and birds were vaccinated according to the regular vaccination protocol. Biocid® was mixed in diet in a concentration of 1 g/Kg diet, while in drinking water it was added according to the manufacturer protocol as from Day 1-7 in a concentration of 0.5 g/liter and from the 9th day till the end of the experiment (31 days old) as 1 g/liter. Group (B) did not receive the Biocid® compound neither in diet nor the drinking water but received the regular vaccination protocol. The final group (Group C) received the Biocid® compound only in feed (not in drinking water) as 1gm/Kg diet but did not get the regular vaccination protocol throughout the experiment. Upon arrival to the poultry house, chicks were weighed individually, and their initial body weight was recorded. The final body weight of chicks in each group was recorded at 31 days old at the end of the experiment prior to slaughter using an electronic digital balance (SF-400D; ATOM, China).

Sampling

At 31 days, ten birds from each group were slaughtered, and the blood was collected into 2 tubes; the first ones were allowed to clot and then centrifuged for 15 min at 3,000 x g. The obtained sera were transferred to Eppendorf tubes and stored at -20°C until needed for further biochemical analysis. The second tube was ethylenediaminetetraacetic acid (EDTA) tube for hematological analysis. After blood collection, birds were eviscerated and tissue samples including cecum, lymphoid organs (Thymus, Bursa and Spleen) and liver and kidney were collected from the slaughtered birds and fixed in 10% neutralized formalin for histological examination. One gram of cecal contents of five slaughtered birds in each group were collected after slaughter and transferred to a sterile test tube with 9 ml sterile Tryptic Soy broth.

Histological examination

The fixed tissue samples were transported to the pathological laboratory of the faculty of veterinary medicine, Suez Canal University, for further processing. The tissues were dehydrated with increasing concentrations of ethyl alcohol (70%, 90%, 96%, and 100%), cleared in xylene, and embedded in paraffin. The paraffin blocks were then cut using a microtome into four 5-μm-thick discontinuous paraffin-embedded sections per sample, which were then stained with hematoxylin and eosin and examined under a light microscope (Olympus C X40). The representative fields were photographed. The digital images of cecal tissues were captured and analyzed for morphometric measurements including villi length and width (µm). A computer morphometric program (Quick Photo Micro 3.2, PROMICRA, s.r.o.) was used for the histomorphometry measurements of the cecal samples.

Bacteriological examination

The mixture of the cecal contents and broth were diluted tenfold serially using a vortex (WiseMix, VM-10, Korea) and drop plate procedure described by Herigstad et al. (2001). Thirty μl of the diluted sample was plated onto various media: standard plate count agar (Plate Count Agar, 1.46365, Millipore, Germany) for total bacterial count (TBC); MacConkey Agar (100205 Millipore, Germany) and EMB agar (Lab M, LAB061, UK) for total coliform count; and lactobacillus agar (MRS AGAR), (TM medium, India) for lactobacilli count. Total aerobic bacteria (TBC) and total coliforms were grown at 37°C for 24-48 hours, Lactobacilli were incubated in a 3% CO2 atmosphere at 37°C for 48–72 h. The results were expressed as means ± standard error (in log10 cfu/g).

Carcass traits and meat quality

After slaughter, the birds bled and then weighed to obtain the weight after slaughter. Birds were de-feathered, eviscerated, and weighed again to determine the carcass weight. Weight of different organs including liver, spleen, bursa and thymus was recorded (0.01 g). The thigh and breast muscles were dissected and weighed using electronic balance (0.01 g).

Statistical analysis

SPSS 22.0 software (Armonk, NY: IBM Corp. Spss, Inc, Chicago, IL, USA) was used to analyze all data. One-way ANOVA model was used after checking the assumption of normality of distribution and homogeneity of variances of every single dependent variable using Shapiro-Wilk and levene’s test, respectively, to investigate the effect of Biocid® treatment on different parameters with a single independent variable was investigated (The effect of Biocid® treatment) while dependent variables include the growth performance and carcass traits, hematological and biochemical parameters, histo-morphometric analysis of intestine and total bacterial count of caecal contents. Duncan’s post hoc multiple comparisons test where appropriate. Dependent variables (Final body weight, Spleen, thymus and breast weights and ALT and AST serum levels) that did not follow the assumptions of the parametric One-way ANOVA were analyzed using the non-parametric Kruskal–Wallis test. The level of significance at which the null hypothesis was rejected was α = 0.05. The results were expressed as the mean ± SE.

RESULTS and Discussion

Growth performance and carcass traits

The overall influence of Biocid® addition on growth and carcass traits was listed in Figures 1 and 2. No significant mortalities were observed between treatment groups. Initial body weight was approximately similar with no significant difference amongst the treatment groups. Broilers in groups A and C received Biocid® treatment showed significantly higher final body weight (P=0.007, 2041.50±64.92 g and 1955.50±37.16 g, respectively), weight after slaughter (P=0.0095, 1934.00±60.84 g and 1866.50±35.79 g, respectively) and carcass weight (P= 0.009, 1565.50±57.84 g and 1441.50±23.44 g, respectively) by the end of the rearing period. Nevertheless, both thigh (P=0.36, Group (A): 589.50±18.23 g, (B): 557.50±20.87 g, (C): 590.50±14.60 g) and breast (P=0.51, (Group (A): 642.50±46.35 g, (B): 614.50±40.09 g, (C): 651.00±13.80g) muscles weights did not significantly differ between the treatment groups (Figure 1). Similarly, the weight of the internal organs varied between the treatment groups that the spleen (2.33±0.18 g), bursa (4.05±0.33 g) and thymus (7.88±0.61 g) in Group A were significantly heavier (P<0.0001, P<0.0001 and P=0.0007, respectively) than groups B (Spleen: 1.31±0.09g, Bursa: 2.42±0.22g, Thymus: 5.35±0.43g) and C, while the liver weight (Spleen: 1.39±0.43g, Bursa: 2.09±0.77g, Thymus: 5.24±1.16g) did not show any significant variations (P=0.91) between the experimental groups (Figure 2).

 

 

Haematological and biochemical analyses

There were non-significant variations in the erythrocytic counts between treatment groups, but the total leukocytic count was significantly (P = 0.05) lower in group (C) compared to the other groups (Table 1). Biochemical analysis of serum samples revealed that broilers in Group (A) had significant higher albumin levels (P = 0.03) compared to groups B and C while Biocid® treatment did not alter the other biochemical parameters in the treatment groups (Table 1).

Histopathological results

Histo-morphometric analysis of intestine

Groups A and C which received Biocid® showed elongated villi and increased numbers of goblet cells than group B which received a regular diet without Biocid®. Histo-morphology of intestinal villi was assessed by measuring the villus height and crypt depth. The villi length showed

 

Table 1: Haematological and Biochemical parameters (Mean±SE) among the different treatment groups.

Parameter	Group A	Group B	Group C	P value
RBCs (×106/µl)	2.98±0.25	3.67±0.50	2.37±0.31	0.10
Total leuckocytic count (/µl)	27333±4372a	28667±1764a	17333±1453b	0.05
Albumin (g/dl)	1.175±0.02a	1.08±0.05b	1.03±0.02b	0.03
Total protein (g/dl)	2.58±0.08	2.40±0.12	2.33±0.13	0.35
ALT (g/dl)	2.50±0.22	3.60±0.55	2.50±0.22	0.13
AST (g/dl)	468.00±29.30	561.80±114.10	409.80±11.56	0.37
Alkaline Phosphatase	5485.00±1228.00	7228.00±1905.00	8005.00±963.90	0.52
GGT	26.50±2.73	26.20±1.87	25.00±2.61	0.90
Urea	3.50±0.39	4.60±0.33	3.50±0.39	0.07
Creatinine (mg/dl)	0.24±0.01	0.26±0.01	0.26±0.004	0.10

 

Means with different superscript within the same row are significantly different (P˂0.05).

 

significant increase (P<0.05) in villi length in groups A (25.78%) and group C (13.63%) compared to group B and so, these groups showed a better absorptive surface of intestinal epithelium resulting in a better nutrient utilization. The crypt depth showed significant differences (P<0.05) between the treatment groups with increased depth in groups C(48.30%) and A (39.46%) compared to group B (Figure 3). The Goblet cell hyperplasia was abundant and numerous in groups A and C.

 

Lymphoid organs

The bursa of group A showed normal structure. A typical fold is covered with epithelium and contains numerous lymphoid follicles separated by thin bands of fibrous connective tissue. Group B showed mild alterations such as mild depletion in medulla with expansion of interfollicular spaces by edema. Also, group C showed normal structure without any histopathological alternations. The thymus of group A showed normal structure. Moreover, group B showed numerous Hassall ‘s corpuscle which consisted of aggregate of large epithelial cells. Within the corpuscle are vacuoles containing necrotic granulocytic leukocytes. Group C also showed normal structure. Spleen of group A showed hyperplasia of lymphoid follicle. The arterioles are surrounded by a diffuse population of T-lymphocytes. that form the periarteriolar lymphoid sheath. There was also a lymphoid follicle composed of B-lymphocytes. The spleen of groups B and C showed normal structure (Figure 4).

 

Table 2: The bacterial count (Mean±SE) of caecal content among the different treatments.

Bacterial count (log10 CFU/g)	Group A	Group B	Group C
Total bacterial count (TBC)	9.3±0.56	8.23±0.53	7.73±0.50
Total coliform count (TCC)	8.75±0.56a	7.69±0.35ab	7.02±0.22b
Total lactobacillus count (TLC)	8.27±0.47a	8.16±0.30a	6.65±0.16b
Correlation coefficient with TBC	0.855**	0.632*	0.862**

 

-Means with different superscript within the same row are significantly different (P˂0.01). *Correlation is significant at the 0.05 level (2-tailed). **. Correlation is significant at the 0.01 level (2-tailed).

 

Liver and kidney

The hepatic tissue of group A showed some mild focal leukocytic infiltration around central vein, mild disarrangement and vacuolation of hepatocytes. Group B showed moderate vacuolation of hepatocytes and desquamation epithelium. In addition, Group C showed normal hepatic plates with mild disarrangement of hepatocyte. The renal tissue of group A showed mild histopathological alterations such as infiltration of intratubular spaces by inflammatory cells and vacuolation of renal tubules. Group B also showed shrinkage of glomeruli with expansions of bowman’s capsule and massive infiltration of inflammatory cells and increase spaces between renal tubules. Finally, group C showed normal kidney structure (Figure 5).

 

The total bacterial count of caecal contents

The total bacterial count of caecal contents in the different treatment groups were presented in Table 2. The current findings showed that there was no significant difference in the total bacterial count of the caecal content (log10 CFU/g) between the three groups. Total coliform count (TCC) (log10 CFU/g) displayed a significant (P=0.02) increase in groups A (24.64%) and B (9.54%) when compared to group C,. In terms of Total lactobacillus count (log10 CFU/g), both groups A and B demonstrated a significant (p=0.001) increase (24.36% and 22.71 %, respectively) when compared to group C. Total bacterial count (TBC) was substantially correlated (P ˂ 0.01) with both Total coliform count (TCC) (log10 CFU/g) and Total lactobacillus count (log10 CFU/g) in group A, but in groups B and C, the TBC was significantly (P ˂ 0.05 and P ˂ 0.01) associated with TCC, respectively.

Data illustrated in Table 3 revealed no significant change between the three treatment groups in the caecal content of Coliform/ Lactobacillus, Lactobacillus/ Coliform, Coliform/ TBC, and Lactobacillus/ TBC ratios.

 

Table 3: Ratio between different bacterial counts among the different treatments.

Ratio	Group A	Group B	Group C
Coliform/Lactobacillus	1.06±0.05	0.95±0.04	1.05±0.02
Lactobacillus/ Coliform	0.96±0.04	1.08±0.05	0.95±0.02
Coliform/TBC	0.94±0.03	0.95±0.05	0.93±0.04
Lactobacillus/TBC	0.89±0.3	1.02±0.07	0.88±0.04

 

The previous approach of using antibiotics as feed additives has been banned due to various concerns. Recent trends of using alternative approaches such as organic acids as feed additives in gain more popularity among poultry farmers as useful replacements for antibiotics (Chattopadhyay, 2014). The presented findings examined the effect of Biocid®, a novel product containing organic and amino acids and silica, as a feed additive in poultry diet.

The current findings clearly showed that the growth performance of birds received Biocid® both in group A (diet and drinking water) and group C (diet only) was significantly higher than birds in the control group (Group B) that did not receive the Biocid® treatment. This increase in body and carcass weights could be explained via various mechanisms. First, the organic acid components in the Biocid® may have a growth-promoting effect result in better feed intake, the drop in pathogenic microbial load, and the provision of a suitable environment for the growth-enhancing bacteria (Khan et al., 2016). Chowdhury et al., (2009) reported that addition of 0.5% citric acid enhance feed intake, growth, carcass yield in broiler. Live body weight increased significantly in chicks fed a diet supplied with acetic acid, citric acid, and lactic acid (Khan et al., 2022). Different modes of action of organic acid supplementations can be explained such as they restrain the growth pathogenic bacteria and promotes pH reduction in the gastrointestinal tract of birds, improve pepsin activity, eventually enhance nutrients digestibility. Moreover, it helps counteracting the effect s of toxic substances secreted by certain bacteria and its colonization, which can reduce inflammation and competition with pathogenic microbes (Ma et al., 2021). Organic acids can work on pathogenic bacteria by infiltrating the bacterial membrane, suppressing ATP synthesis and/or interrupting the bacterial membrane and denatures their DNA. All these actions ultimately establish eubiotic intestinal environment which improves gut health and nutrient digestibility and absorption (Pirgozliev et al., 2019).

In addition, amino acids constituents of the Biocid® may increase growth, feed utilization efficiency, and carcass yields (Attia et al., 2020) which was clearly observed in groups A and C. Increasing amino acid density in broilers diet had been observed to increase the final body weight and production performance, whereas decreasing their density reduced growth and live weight (Nasr and Kheiri, 2011). Another advantage of the essential amino acid constituents of the commercial Biocid®, including arginine, threonine, tryptophan and valine, made it possible to further reduce the crude protein levels in broiler diets (Attia et al., 2022) which might be an economical prospective for poultry farmers. Also, they decrease the nitrogen loss during protein metabolism that leads to low ammonia excretion in the environment and improves growth performance of birds. Moreover, they chelated trace minerals decreasing the circulatory and intestinal levels of heat shock protein 70 (HSP70) and pro-inflammatory cytokine gene expression, optimizing broilers response to stress (Baxter et al., 2020). Feeding diets with supplementary amino acid contents can alleviate certain intestinal diseases in broilers such as necrotic enteritis augmenting the digestion and absorption. Amino acids supplementation enhance cecal butyric and total short-chain fatty acids production which increasing productivity and improve immunity which have the significant as potential immunomodulating agents to improve the immune function of broiler chickens (Xu et al., 2018). They also participate in protein synthesis, methylation reaction of DNA, elimination of reactive oxygen species (ROS) and acts as glutathione (GSH) precursor– a tripeptide, which decreases ROS and thus protects cells from oxidative stress (Alagawany et al., 2020). Silica is another compound of Biocid® which had been previously reported to have positive effects on growth performance (Burton et al., 2020) similar to the results found in groups (A and C). Silica was used as a unique feed additive owning sizeable surface area, high porosity, strong cation exchange activities, and more active sites, carrying the potential for detoxification and decontamination of feed with mycotoxins (Pappas et al., 2010). Moreover, they can execute several functions including toxin binding, improvement of the enzymatic activity in the small intestine, and control of ammonia emission into the environment. Finally, it could change GIT enzymatic secretion by raising the GIT fluids’ pH which could, in turn, reduce intestinal inflammation and improve nutrient digestibility and absorption leading to higher productive performance in broilers (Ghazalah et al., 2021).

Serum biochemical profile did not reveal any significant differences between treatment groups, except for albumin which was significantly higher in group A compared to the other treatment groups. This increase in serum concentrations of albumin could be interpreted by boosting dietary protein utilization via stimulation of digestive enzymes as protease (Tarek et al., 2022) besides improvement of liver function to synthesize albumin. These results are in partial accordance with Natsir et al. (2017) who noted that dietary organic acids mixed with broilers diet increased serum total protein and albumin. Also, total leucocytic count was significantly lower in the non-vaccinated group C which received Biocid® through feed than the other treatment groups. The increased total leucocytic count in vaccinated groups may be due to the marked lymphocytosis in response to vaccination due to the vaccine’s immunostimulatory effect as a response to antigenic stimulation caused by vaccine administration (Saleh et al., 2011). This result can be fortified by our findings that birds in group (A) showed increased weights of immune organs (Spleen, bursa and thymus) which reflects better immune status (Martínez et al., 2021) than the group (B) which received the vaccination but did not receive Biocid®. Another noticeable finding is the responses of broiler that received Biocid® (groups A and C) might not be similar. Biocid® mixing with diet and drinking water in group (A) may optimize growth, immune and productive performance in broilers rather than diet only as body and carcass weights and weights of immune organs in group A were higher than group B which may indicate better utilization of the product by increasing concentration when added to both diet and the drinking water.

In the current study, Biocid® treatment exerted beneficial effects on the gut morphology of assessed birds in both A and C groups. Birds treated with Biocid® had significantly increased villi length and width and goblet cell numbers than the control birds (group B). Also, increased villus height and width is indicative of increased epithelial cell turnover and a well-differentiated intestinal mucosa, more active cell growth and regeneration, which can promote enzyme secretion, improve digestion, and expand the surface area available for absorption (Wang et al., 2021) suggesting improved digestive and absorptive capacity which was manifested by the high productive performance of both groups in terms of body and carcass weights (Al-Garadi et al., 2022). The villus crypt is thought as the villus factory and deeper crypts reveal fast tissue turnover to allow regeneration of the villus as required in response to normal sloughing or inflammation from pathogens or their toxins and high requirements for tissue (Awad et al., 2009). Furthermore, in this study, both A and C groups showed significant increase in the number of goblet cells. Goblet cells are the main secretory cells responsible for mucin production which may contribute to several functions including lubricating intestinal surfaces, trapping and neutralizing bacteria, detoxifying heavy metal binding, interacting with the intestinal immune system, and acting as a diffusion barrier for nutrients and macromolecules (McCauley and Guasch, 2015). The obtained results are in accordance with Adewole et al., (2021) who observed that supplemented broilers diet with organic acid contribute better to the intestinal health including villi length and increased numbers of goblet cells. Normal histological conformation of liver and kidneys were also noticed in groups A and C compared to the control group (B), that did not receive the Biocid® product, which outweigh the assumption that Biocid® administration may improve the digestive and excretory functions in broilers which ultimately enhance the overall health condition of the birds. For the impacts on the lymphoid organs, the results in Figure 2 agree with those of Ghazalah et al. (2011), who found that the relative weights of primary lymphoid organs (spleen, bursa of Fabricius, and thymus) were significantly (p < 0.01) improved by supplementation of organic acids. Similarly, the presences of normal histological structure coupled with lack of any pathological alterations of the immune organs in groups (A and C) indicated that the addition of organic acids in the diet and drinking water of the broiler chicks conferred better immune response and disease resistance besides beneficial immunological advances (Al-Mutairi et al., 2020).

Biocid® administration in diet significantly increased total Coliform and Lactobacillus counts but there was no significant difference in total bacterial count between the treatment groups. The present study may display the potential effect of Biocid® on gut microbiota. From the current results, it is firstly apparent that Biocid® administration via diet only (Group C) significantly reduces the Coliform count rather than through feed and drinking water (Group A), although it is significantly reducing the Lactobacillus counts. However, comparing Coliform/Lactobacillus ratio between groups A and C revealed that the ratio was nearly 1 which means that whether dual administration via drinking water + feed or only though feed yielded the same results. Although Coliform count was significantly higher in group A, but it did not cause any pathological alterations may be due to the concurrent increase in Lactobacillus count and the Coliform/Lactobacillus ratio was nearly 1 which mean both bacteria manifest their potential effects equally. These findings are in partial agreement with those reported in Thanh et al. (2009) who showed that addition of organic acids in broiler diets decreased the colonization of pathogenic bacteria, and in agreement with Attia et al. (2023) who observed that supplementation of organic acids increased the Lactobacillus count in Japanese quails. However, another study by Ghazalah et al. (2011) showed that organic acid addition did not change Lactobacillus count, but decreased Coliforms count in broiler chickens as observed in group (C) which received Biocid® only in diet. In addition, Seifi et al. (2015) showed that Lactobacillus and Coliforms were not altered by the addition of organic acid to broiler diets. Ustundag and Ozdogan (2019) found that organic acid added to quail diets increased Coliform in male plus decreasing total bacteria counts in females, implying a gender-specific response. These discrepancies might be attributed to the route and timing of administration, the poultry species used and the experimental design. The impact of organic acids on the gut bacterial population may need further clarification, but we cannot assume that Biocid® administration improve gut health in broiler.

Nevertheless, the responses of broiler chickens to excess dietary organic acids or amino acids have shown considerable inconsistency. While some results showed positive effects, others showed no effect or even negative consequences based on several factors. Regarding organic acids, one factor could be the difference in other dietary constituents and their chemical characteristics such as buffering capacity which may affect the level of acidification that occurs with the addition of organic acids (Kim et al., 2015). Similarly, experimental settings such as the sanitation degree of the environment may clarify the dissimilar results in the literature. Organic acids may influence the microbial population in the GIT, hence, that the antimicrobial outcomes of organic acids would be more distinct when birds are subjected to less sanitary conditions (Kil et al., 2011). Moreover, feed palatability is likely affected by the sources and amounts of dietary organic acids, and consequently appears to influence the efficacy of dietary organic acids. Similarly, excess dietary amino acids to growing chickens cause toxic effects such as depressions in growth, decreases in feed intake and increase mortalities. These negative impacts may be due to amino acid imbalance, for example, additional dietary Lys causes an increase in the Arg requirement, and chicks receiving additional Lys may show signs of an Arg deficiency because Arg lessens Lys toxicity (Maynard et al., 2022).

CONCLUSIONs and Recommendations

In conclusion, Biocid® administration in diet and drinking water may be initially considered as a growth promoter in the broiler chicken as in this study they had preliminary positive results on the performance. No significant mortalities were observed between the treatment groups although group (C) did not receive the regular vaccination of broilers. Furthermore, it provides positive impact on histology of small intestine and immune organs, thereby facilitating the nutrient absorption, growth performance and immune response in broiler chicken. Further studies should be conducted to confirm the efficacy of Biocid® as a growth promoter on production in different poultry species and under different environmental circumstances.

ACKNOWLEDGMENT

The authors would like to thank Al-Ahram Mining Company and Al-Hoda Company for agricultural development for their valuable contribution to this work.

Novelty Statement

This study is the first trial to test the efficacy of Biocid®, a newly formulated compound, as a potential growth promoter in broilers. The chemical constituents of Biocid®, majorly organic acids, amino acids and other compounds, exerted a significant effect on productive and blood parameters and intestinal structures when administered through diet and drinking water or through diet only.

AUTHOR’S CONTRIBUTION

Dalia Hamid Mansour and Yousry Ibrahim conceived the presented idea. All authors (Except Yousry Ibrahim) performed the experiment. Mohamed El-Shabrawy Ghanem and Marwa Hassan verified the analytical methods. Ibrahim Hegab analyzed the findings of this work. All authors discussed the results and contributed to the final manuscript.

Conflict of interest

The authors affirm that they do not have any competing financial interests or personal relationships that may have influenced the findings presented in this paper.

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