Impact of buffered sodium butyrate as a partial or total dietary alternative to lincomycin on performance, IGF-1 and TLR4 genes expression, serum indices, intestinal histomorphometry, Clostridia, and litter hygiene of broiler chickens

Background Sodium butyrate (SB) is a short-chain fatty acid and a safe antibiotic alternative. During 35 days, this study compared the impact of coated SB (Butirex C4) and lincomycin (Lincomix) on broiler growth, gut health, and litter hygiene in 1200 one-day-old Ross-308 broiler chicks that were randomly assigned into 5-dietary groups with 5-replications each. Groups divided as follows: T1: Basal diet (control), T2: Basal diet with buffered SB (1 kg/ton starter feed, 0.5 kg/ton grower-finisher feeds), T3: Basal diet with 100 g/ton lincomycin, T4: Basal diet with buffered SB (0.5 kg/ton starter feed, 0.25 kg/ton grower-finisher feeds) + 50 g/ton lincomycin, and T5: Basal diet with buffered SB (1 kg/ton starter feed, 0.5 kg/ton grower-finisher feeds) + 50 g/ton lincomycin. Birds were housed in a semi-closed deep litter house, where feed and water were available ad libitum. Results were statistically analyzed using ANOVA and Tukey’s post hoc tests. Results Combined dietary supplementation with SB and lincomycin (T4 and T5) significantly enhanced body weights, weight gains, feed conversion ratio, and profitability index. Also, carcasses in T4 and T5 exhibited the highest dressing, breast, thigh, and liver yields. T5 revealed the best blood biochemical indices, while T3 showed significantly elevated liver and kidney function indices. T4 and T5 exhibited the highest expression levels of IGF-1 and TLR4 genes, the greatest villi length of the intestinal mucosa, and the lowest levels of litter moisture and nitrogen. Clostridia perfringens type A alpha-toxin gene was confirmed in birds’ caeca, with the lowest clostridial counts defined in T4. Conclusions Replacing half the dose of lincomycin (50 g/ton) with 0.5 or 1 kg/ton coated SB as a dietary supplement mixture showed the most efficient privileges concerning birds’ performance and health.


Background
Over the past decades, using antibiotics in subtherapeutic doses as feed additives for animal and poultry diets has achieved multiple benefits of promoting growth, minimizing mortalities, and reducing production costs although, numerous safety worries emerged due to that practice, as the persistent inclusion of antibiotics in the food animal industry caused injury to animal's intestines, suppression of gut health, contamination of animal products with antibiotic residues, and polluting of the environment with antibiotic-resistant bacteria [1,2].All these drawbacks challenged animal producers to find efficient growth-promoting substitutes for antibiotics.
The gut microbiome is a critical determinant of a bird's performance and productivity [3], and recent investigations reported the disturbance effects of antibiotics on intestinal microbiota [1,4].Tang et al. [1] administered lincomycin (1 g/kg feed) to weaned piglets (21 days of age) for one week and documented adverse health effects due to the lowered level of intestinal beneficial bacteria confronted by the rise in potential pathogens and decreased body weights.The same findings, in addition to immunosuppression, were reported by Zhang et al. [5] following the administration of lincomycin to young mice for one week in drinking water (1 g/L water).Lincomycin did not influence feed efficiency, broiler immunity, or intestinal microbial counts in the Azeem trial [6].
Lincomycin is a natural lincosamide antibiotic that originates from Streptomyces lincolnensis and is usually used against intestinal gram-positive bacterial infections.Lincomycin absorption through the gastrointestinal tract (GIT) was reported to be very weak, and its primary side effect is gastrointestinal inflammation, especially with long-term usage [5,7].The lowered body weight gain of animals that received lincomycin was attributed to its negative impact on intestinal permeability, wall thickness, and nutrient absorbability [6].
Short-chain fatty acids (SCFAs) are vital metabolites in the intestinal microbiome and are essential as antiinflammatory agents and for supporting gut health.In the Romick et al. [8] and Zhang et al. [5] studies, lincomycin was reported to destroy numerous intestinal SCFAproducing bacteria and lower the production of SCFAs and butyrate.SCFA and their salts are generally considered safe alternatives to antibiotics for animals.Poultry producers widely applied butyric acid in broiler chicken diets as an alternative feed additive to antibiotic growth promotors (AGP).SB is the sodium salt of butyric acid characterized by its stability, non-odorous, and ease of handling during feed manufacturing [9,10].SB is supplemented in either free form to encourage the upper GIT development or buffered (coated) to prevent its dissociation in the upper part of GIT and enhance its bioavailability in lower GIT; due to its slow release [11].
The buffered SB promotes intestinal mucosa modulation, regulates gene expression, augments SCFAs production, and improves protein synthesis [12][13][14].SB activates the expression of Toll-like receptors 4 (TLR4) and insulin-like growth factor-1 (IGF-1) but decreases DNA fragmentation induced by pathogenic bacteria like Clostridium perfringens [15][16][17].SB has an antimicrobial effect by increasing the synthesis of mucin and antimicrobial peptides and decreasing intestinal epithelial permeability [18].Moreover, it favours intestinal health by increasing the viable counts of Lactobacillus and Bifidobacterium beneficial bacteria [19].SB is an energy source for enterocytes resulting in increased intestinal villi development and absorptive surface [12,20,21].SB possesses anti-inflammatory, antioxidant and immuneenhancing properties [22][23][24][25], as it enhances protein digestibility and serves as a substrate in intermediate metabolism, thus improving the broiler's overall health and performance [26].
Previous studies indicated the application of coated SB at a rate of 600 to 1000 mg/kg of feed could enhance broiler chicken weights and feed conversion rates [2,10].Further investigations reported that no significant body weight differences were obtained [13].Yet, most of the experiments proved the modulating effect of coated SB long-term administration on gut microbiota concerning promoting the beneficial microbes [2,13].
The current study examined the efficiency of dietary buffered SB 54% (Butirex C4®) supplement to replace lincomycin (Lincomix® 50) addition to broiler feed entirely or partly.The experimental design tested varying dosage regimens of both products.The evaluation included measuring a range of indices involving the weekly and overall performance parameters, carcass traits, blood biochemistry, intestinal histology and microbial counts, tissue gene expressions (IGF-1 and TLR4), and litter microbial and chemical conditions.

Experimental design, diets, and housing
The Institutional Animal Care and Use Committee, Faculty of Veterinary Medicine, Cairo University, Egypt, approved the experimental design (Vet CU 12/10/2021/347).The trial was conducted at the Animal and Poultry Research Center, Faculty of Veterinary Medicine, Cairo University, Giza, Egypt.
The house with a semi-closed ventilation system was subdivided into 25 identical floor pens of 2.9 m × 1.6 m dimensions.The floor type was concrete covered with 7-10 cm wood shaving litter.Birds received similar managemental and hygienic conditions.After the chicks had arrived at the poultry house, they received 24 h of light for the first three days, then maintained under 23 L:1D for the remainder of the experiment.The temperature of the house was 32 ± 1 °C for the first 3-days, then gradually reduced by 0.5 °C per day until it declined to 24 °C [28].The humidity ranged between 55 and 60% throughout the experimental period [29].Clean water was available ad libitum in bell-shaped drinkers (4-litre capacity) and replaced with 8-litre drinkers in older age [30].Feed was available ad libitum via round plastic feeders throughout the experimental period [31].

Growth parameters
Body weights (BW) of broilers were recorded weekly for each replicate on a pen basis at 1, 7, 14, 21, 28, and 35 days of age.The weekly body weight gain (BWG), average weekly feed intake (FI) and feed conversion ratio (FCR) for each week and the overall period of the experiment (from 1 to 35 days of age) was measured, as described previously [32].Daily bird mortalities were recorded for each group.After adjusting mortality, FCR was calculated by dividing weekly feed intake by weekly weight gain.At the end of the experiment, EPEF (European Production Efficiency Factors) was calculated according to the following formula [29]: EPEF = (livability (%) × live body weight (kg) / (age in days × FCR) × 100.

Carcass characteristics and immune organs
At the end of the experiment (day 35), 25 birds (5 birds/ treatment) were slaughtered, defeathered, and eviscerated after 12 h of fasting [33].Carcasses were weighted after removing the head, neck, and legs.Then, carcasses were dissected to measure the relative weights of breast, thigh, and drumstick muscles [34].Moreover, the giblet weights (gizzard, liver (without gall bladder) and heart) and immune organ weights (spleen and bursa of Fabricius) were recorded and expressed as a percentage of live weight [35].

Blood biochemical analysis
At the end of the experiment (day 35), five blood samples per bird group were collected from the jugular vein after slaughter.After centrifuging for 15 min at 3000 rpm, sera were separated and stored at -20 °C until analysis.In serum samples, different biochemical parameters were measured by spectrophotometer (UV-2100 Spectrophotometer, USA) using spectrum diagnostics kits (Spectrum Diagnostics Egyptian Company for Biotechnology).Serum albumin [36], total protein [37], triglycerides [38], cholesterol [39], uric acid [40], alanine transaminase (ALT), and aspartate transaminase (AST) [41] were determined according to the manufacturer's instructions.

NDV vaccinal antibody titers
Haemagglutination inhibition test was performed to assess NDV antibody titers, following [42] guidance.Two-fold serial dilutions of 25 µL of each serum sample were conducted in 99-V-bottomed microwell plates.To each well, 25 µL of four haemagglutination units of ND-Lasota commercial antigen were added, and plates were incubated at room temperature for 20 min.To each well, 25 µL of 1% chicken-RBC suspension was added.Antibody titers were reported as mean log 2 haemagglutination inhibition titers.

Gene expression analysis
About 100 mg of breast and thigh muscles and liver tissue were dissected from five birds per group.The samples were disrupted in a lysis buffer solution using a tissue homogenizer.Samples were processed for total RNA extraction according to the protocol of the easy-spin Total RNA Extraction Kit (Cat.No.17,221; iNtRON Biotechnology DR).The quantity and purity of RNA were assessed using Nanodrop [43].The cDNA synthesis was performed using M-MLV Reverse Transcriptase (enzynomics Cat.# RT001S).The transcript level of IGF-1 (growth-related gene) in both breast and thigh muscles and TLR4 (immune response-related gene) in the liver were evaluated at the mRNA level by qRT-PCR using RealMOD™ Green W² 2x qPCR mix (Cat.No 25,350) according to the manufacturer's instructions.Each RT-PCR was performed in triplicate [44].Real-time quantitation of mRNAs was normalized to an endogenous reference of the β-actin gene [45].The fold change was calculated by the comparative threshold cycle (C T ) method (2 −ΔΔCt ) [46].The primers used in Real-time PCR were designed using Primer 3 program (https://primer3.ut.ee/) [47][48][49].The primers' sequences were shown in Table 2.

Microbiological examination of caecal content and deep litter
After birds were slaughtered at day 35, five caecal contents and five deep-litter samples were collected from each group for microbiological examination.The upper 7 cm of deep litter were scraped from 3 different spots of each replicate pen and placed in sterile plastic bags [50,51].All samples were maintained at 4 °C until examination.
For microbial examination of caecal content, a 10 − 1 dilution was prepared by diluting and homogenizing 1 g of each sample in 9 ml sterile saline solution, followed by 10-fold serial dilutions till the 10 − 10 dilution [52].For microbial examination of litter samples, 3 g of each homogenized litter sample were transferred to tubes containing 27 mL sterile saline solution (10 − 1 dilution).Samples were kept at room temperature for 30-60 min and frequently shacked to allow the litter to mix well with the diluent [50,51], followed by 10-fold serial dilutions till the 10 − 14 dilution.Then, 100 µl were taken from the last 3-dilutions and spread onto Nutrient Agar (Hi-Media Laboratories, India) plates to enumerate total aerobes and incubated for 24-48 h at 37 °C.Another 100 µL were spread onto Reinforced Clostridial Agar (Oxoid Ltd, Basingstoke, Hants, UK) plates to enumerate total Clostridia and incubated for 24-48 h at 37 °C under anaerobic conditions.Finally, the counts of bacterial colonies were reported as mean 10-logarithm colony-forming units (log 10 CFU) for each gram of litter and caecal content.

Confirmation and toxin typing of Clostridium perfringens
Genomic DNA of suspected C. perfringens isolates was extracted using an extraction kit (QIA amp mini kit, Qiagen, Hilden, Germany).The multiplex PCR assay was used to detect the presence of genes encoding alphatoxin (cpa), beta-toxin (cpb), epsilon-toxin (etx), iotatoxin (iap) and CPE (cpe) [53].Primer sequences were published previously [53].The PCR reaction mixtures were analyzed by electrophoresis on a 1.5% (w/v) agarose gel in the presence of a 100 bp DNA ladder (Fermentas Life Science, USA).

Physical and chemical examinations of litter
The upper 7 cm of deep litter were scraped from 3 different spots of each replicate pen and placed in sterile plastic bags [50,51].Litter moisture was estimated by drying 10 g of litter samples in the hot air oven at 100 ± 5 °C for 24-48 h [50].Moisture % was calculated by subtracting dry weights from the initial weights.Additionally, the

IGF1
Insulin Like Growth Factor 1 total nitrogen content of litter samples was determined as total Kjeldahl nitrogen [54].

Histomorphometric analysis
The duodenum, jejunum, and ileum from five birds per group were collected and flushed with saline solution (0.9% NaCl) to remove contents, then fixed in 10% neutral buffered formalin for 48 h for histological examination.After fixation, samples were dehydrated in ascending grades of ethyl alcohol, cleared in xylene, and embedded in paraffin wax.Sections of 3-4 μm in thickness were obtained by rotatory microtome, deparaffinized, and stained with haematoxylin and eosin (H&E) stain for examination under the light microscope [55].H&E stained sections were used for the histomorphometry.Approximately five intestinal tissue sections were measured by a high-power lens (X 40).Parameters measured include villus height from the tip of the villus to the crypt and crypt depth from the base of the villi to the submucosa.A computerized microscopic image analyzer, attached to a full HD microscopic camera (Leica Microsystems, Germany), was used to determine the histomorphometric parameters using statistical analysis.

Statistical analysis
Data were checked for normality by the Shapiro-Wilk test.Then, the mean differences were compared between groups via analysis of variance (ANOVA) and Tukey's post hoc tests using PASW Statistics 18.0 software (SPSS Inc., Chicago, IL, USA).Results were reported as means and standard error of the mean (SEM).Charts were generated with R (Version 3.6.1,R Foundation for Statistical Computing) using ggplot2 [56], ggpubr [57], tidyverse [58], and rstatix [59] packages.The significance was considered at P < 0.05.

Performance parameters
The results of the growth performance analysis (Table 3) revealed that no significant difference was noted between the mean values of BW, BWG, and FCR among the groups during the starter period (0-14 d).However, there was a significant decrease (P < 0.05) in day 14's FI of T5 (1 kg/ton buffered SB combined with a half dose of lincomycin) compared to the control (T1).In addition, there was a significant increase (P < 0.05) in BW at the grower (15-28 d) and finisher (29-35 d) periods in T5, followed by T4.Moreover, during the finisher period, also there was an improvement in BW in T2.However, the lincomycin-supplemented group (T3) and control (T1) showed the lowest final BW.On day 35, and for the overall period, there was an improvement (P < 0.05) in BWG and FCR in T5, T4, and T2.Weekly FI of different groups at different periods did not differ statistically   (P > 0.05) from each other.The highest cumulative feed intake was noted in T3, while the lowest was noted in T5.
The EPEF (Table 4) was significantly (P < 0.05) the highest in all SB-supplemented groups (T2, T4, and T5) and lowered in lincomycin supplemented group (T3) and control (T1).The lowest mortality rate was recorded for T2, while the highest was in T3.However, mortality was not significantly (P > 0.05) different between bird groups.

Carcass characteristics and immune organs
The inclusion of dietary buffered SB supplement with reducing lincomycin to half its dose (T4 and T5) showed a significant increase (P < 0.05) in dressing yield (Table 5).T4 exhibited the highest dressing yield, while the control (T1) showed the lowest value.The relative weight of drumstick muscle showed a significant decrease (P < 0.05) in control (T1) compared with other groups.T4 and T5 reported the highest breast %, while T5 reported the highest thigh% compared to other groups.Although, no significant differences (P > 0.05) were indicated in breast and thigh muscle percentages among all groups.
There was a significant increase (P < 0.05) in the relative weight of the liver in groups that received SB and lincomycin combination (T5, T4), whereas; T3 reported significantly (P < 0.05) lowered liver %.No significant differences (P > 0.05) were observed in the relative weight of the gizzard, heart, and immune organs weights (spleen and Bursa of Fabricius) between different groups.

Blood biochemical parameters
The highest serum total protein and albumin concentrations were shown in T5 (P < 0.05), as presented in Table 6) T2 reported the lowest serum cholesterol level but was not significantly different from other groups.The TAG concentration significantly decreased in all supplemented groups compared to control T1 (P < 0.05).ALT activity significantly dropped in T5 compared to other groups (P < 0.05).AST activity significantly decreased in T2 and T5 (P < 0.05).The uric acid concentration reported the   T1: Control -basal diet; T2: Butirex C4 1 kg/ton in starter feed (0-15d) and then 0.5 kg/ton in grower and finisher feed; T3: Lincomix recommended dose (100 g/ton); T4: Lincomix half the recommended dose (50 g/ton) + Butirex C4 0.5 kg/ton at 0-15d and then 0.25 kg/ton; T5: Lincomix half the recommended dose (50 g/ton) + Butirex C4 1 kg/ton at 0-15d and then 0.5 kg/ton 1 SEM: Standard error of mean Number of sampled birds (N) = 5 birds/group lowest value in T5 (P < 0.05) and the highest in T1 and T3.In general, T5 showed the most satisfactory blood biochemical parameters, while T3 reported the worst liver and kidney function indices.

IGF-1 and TLR4 genes expression
Dietary SB, lincomycin, and their combined supplementation significantly (P < 0.05) increased the transcript level of the IGF-1 gene (growth-related gene) in the breast muscle compared to the control (Fig. 1).The transcript level of the IGF-1 gene in thigh muscle increased significantly in T2, T4, and T5 compared to control (T1).
The transcript level of the TLR4 gene (immune responserelated gene) in the liver was significantly higher in T3, T4, and T5 than in T1 (Fig. 2).IGF-1 and TLR4 genes exhibited the highest expression levels in T4 and T5.

Microbiological examination of caecal content
The bacterial counts per gram of caecal content were the lowest in the T4 bird group, with an average of 9.07 ± 0.24 log 10 CFU of total aerobic bacteria (P = 0.045) and 9.32 ± 0.53 log 10 CFU of total Clostridia.The T3 and T5 groups did not demonstrate improvement from the control group (T1) but had bacterial counts higher by nearly 1.19 log 10 CFU/g (Fig. 3).

Confirmation and toxin typing of Clostridium perfringens
The amplification of the C. perfringens type A alpha-toxin gene at 324 kb was confirmed in the suspected C. perfringens isolates of all groups.C. perfringens isolates from the control group (T1) displayed both C. perfringens type A alpha toxin and C. perfringens enterotoxin genes.

Litter chemical and microbiological parameters
Regarding the microbial quality of deep litter at day 35 (Fig. 4), there were no significant differences between the experimental groups (P > 0.05

Histomorphometry of small intestine
In the duodenum, the intestinal villi and crypt depth in T2, T3, T4 and T5 were significantly greater than in control group T1 (P < 0.05).There were significantly higher increases in the length of duodenal villi and crypt depth in T4 and T5 compared to T2 and T3.In the jejunum, T5 showed the highest villi length, followed by T4, while T3 exhibited the lowest values ((P < 0.05).Jejunal crypt depth was the largest in T5.In the ileum, T5 recorded the highest villi length and crypt depth, followed by T4 (P < 0.05) (Table 7; Fig. 5).

Discussion
In the current study, performance data during the starter period revealed that BW, BWG, and FCR were not affected by either the dietary supplementation of SB or lincomycin, which, according to an earlier study [60], may be explained by the immature digestive functionality of young birds at the starting phase.Birds are still developing their gut microbiota during the first week of life since it has not yet stabilized.As a result, large concentrations of some additives can disrupt the natural development of microflora, thus impacting bird performance [61].Similarly, some studies documented that dietary supplementation of SB at increasing doses of 500, 1000, or 2000 mg/ kg had no effect on broiler chicken growth performance during the starter phase [13,62].Other studies reported that AGP did not influence BW, FCR, and BWG during the starter period of broilers [63,64].Additional studies have shown that providing lincomycin as AGP to broilers benefited their growth rate [65][66][67].However, in the current study, no significant differences were noticed in growth performance and feed efficiency between lincomycin and the control, as formerly observed [68].This result may be due to increased feed intake, so it doesn't imply greater feed conversion efficiency.The combined supplementation of SB and lincomycin in T5 significantly reduced weekly FI at day 14.Other researchers have published similar findings; regarding the strong tendency of combined butyrate supplements to reduce feed intake and improve feed efficiency [69].In grower and finisher periods, the partial or total SB dietary substitution to lincomycin in T5, T4, and T2 displayed an improvement in BW, BWG, FCR and the production profitability, which was indicated through the significant increase of EPEF as reported previously [70,71].This improvement could be due to the improved nutritional digestibility by SB that enhanced intestinal structure, as it augmented both crypt depth and villus height of the three intestinal segments, so raised intestinal absorption as indicated in our histomorphometric results.Butyrate has numerous beneficial effects on intestinal tissues and gut health as it is an essential energy source for the development and proliferation of the gastrointestinal epithelium [72,73].SB promotes nutrient digestibility and absorption due to the modification of gut microstructure and increased digestive enzymes' activity, hence improving the productive efficiency of broilers [74,75].SB positively impacts protein and mineral digestibility, which boosts weight gain and FCR [76,77].Interestingly, SB beneficially modulates Toll-like receptor 4 (TLR4) activation by lowering the activation of mitogen-activated protein kinase and nuclear factor B (NF-B) pathways, as well as the generation of proinflammatory cytokines [15], so improves immune status and growth rate.Cumulative mortality was not significantly affected by SB or lincomycin supplementations.This result complies with previous research that showed no influence on the whole mortality rates by the graded levels of encapsulated butyric acids [63,78] or lincomycin [79].
In the present study, SB and lincomycin supplements enhanced dressing yield, especially in T4.Similar studies showed that organic acids and antibiotic combination supplements exhibited higher carcass yields [80].
Furthermore, previous research [81] demonstrated that birds fed diets supplemented with butyric acid gained higher dressing yields when compared to other groups.Contrarily, in a recent study [82], no significant effects were observed on carcass traits through butyric acid supplementation compared to the control and antibiotic-supplemented groups.These findings may be attributed to elevated expression levels of insulin-like growth factors (IGF-1), which promote growth and enhances feed utilization efficiency in broilers [83], resulting in enhanced final live body weights and dressing yields.
According to our results, no significant differences were observed in breast and thigh muscles between different groups.However, the percentage of the economically valuable parts, like drumstick muscle, was increased in SB and lincomycin-supplemented groups compared to the control, which could indicate faster maturity due to rapid growth.Like our findings, drumstick meat yields increased by organic acid supplementation compared to the control group [84].On the other hand, dietary butyric acid did not affect breast and thigh yields [85].
Combining dietary SB with lincomycin in T4 and T5 enhanced birds' liver weights better than those supplemented with lincomycin (T3).That may be related to enhanced liver function indices via decreased ALT and AST levels in our biochemical results, as increased serum levels of AST demonstrated possible hepatocyte and liver dysfunction [86].Similar studies reported a slight increase in the relative liver weight in birds fed organic acid-supplemented diets compared to Enramycin-supplemented diets [87].No significant differences were observed in relative gizzard, heart, and immune organs weights.Likewise, heart and immune organs weights were not affected by the coated organic acids or antibiotic growth promoters [87,88].
In this study, the mixture of SB and lincomycin in T5 significantly improved serum total protein concentrations, which indicated increased dietary protein utilization [32].The increased proteolytic enzyme activity stimulates nutrient digestibility and elevates the absorption rate caused by increased intestinal villi length [89].For the lipid profile, supplementary SB and lincomycin significantly lowered serum TAG in all treated groups.Similar trials suggested that butyrate influences gene expression, which regulates the catabolism of lipids [11].The intake of animal meat products with low lipid content is beneficial for human health [90].Also, SB supplementation in T2 and its combination with lincomycin in T4 and T5 improved the liver and kidney function indices.Previous findings [10] suggested that SB supplementation significantly decreased ALT and AST levels compared with the control group.Alike, serum uric acid levels were reduced in the butyric acid-supplemented groups [82].Another study [91] reported that SB supplementation didn't affect the serum levels of total protein, protein fractions, ALT, AST, and uric acid while decreasing the total and LDL cholesterol.
Haemagglutination inhibition antibody titer is a marker of birds' humoral immunity.In this study, the SB and lincomycin supplements did not enhance the humoral immunity of broilers at 35 days of age against NDV compared to the control.An earlier study [10] indicated that SB supplement at application rates of 0.3, 0.6, and 1.2 g/kg of feed at different ages (14, 21, and 28 days) could raise the humoral immunity of broilers compared to control.Another study [67] reported that lincomycin induced non-significant increases in the immunity parameters of broilers.
The IGF-1 gene is protein-encoded and produced by the liver under the stimulatory effect of growth hormone.It stimulates systemic body growth and has anabolic effects on different cells in the body, especially skeletal muscle, cartilage, bone, liver, kidney, and lung cells.In addition to the insulin-like activity, the IGF-1 gene can regulate cellular DNA synthesis [92].In this study, a combination of SB and lincomycin achieved the highest upregulation of IGF-1 m-RNA in the breast muscle of T4 and T5 birds and the highest upregulation in the thigh muscle of the T5 group.These results come per the current results of breast and thigh relative weights.Formerly, the supplementation of broilers with SB was reported to increase the expression level of the IGF-1 gene [17].The TLR4 gene is encoded by the TLR4 protein (a member of the toll-like receptor family).Its activation leads to an intracellular signaling pathway and inflammatory cytokine production, which stimulates the innate immune system [93].In our study, the transcript level of the TLR4 gene in liver tissue was increased in T3, T4, and T5, while it was the highest in the T5 group.Smith et al. [94] reported the upregulation of TLR4 m-RNA in birds fed butyrate in their diet.
In the current study, the combination of SB and lincomycin at half their recommended doses significantly lowered the caecal bacterial and clostridial counts.These findings did not apply to litter.These results agreed with previous work that displayed that micro-encapsulated butyrate diminished the pathogen colonization in caeca [95].Notably, in a former feeding trial, 0.1% butyrate reduced caecal bacteria better than 0.2% butyrate, and they attributed that to the higher transcription level of the AvBD9 gene in the caeca and caecal tonsil when supplementing birds with 0.1% butyrate compared to 0.2% butyrate [96].They recommended the careful investigation of the optimum butyrate dosage for each animal species, as higher application rates of butyrate could be cytotoxic [74,97,98].Weak organic acids diffuse into the cytoplasm of the bacterial cell, where they dissociate and rapidly drop the cytoplasm pH, causing bacterial cell death [99].
The supplementation of dietary butyrate derivatives boosted butyrate concentration in the large intestine and the number of neutrophils in the colonic lamina propria, which indicated that butyrate is a powerful promoter of neutrophil activity during infection [100].Likewise, lincomycin disrupts the elongation of the peptide chain and genetic coding of the bacterial cells [101].But antibiotics reduce the butyrate-producing bacteria in the colon, which may impair the epithelial barrier and increase susceptibility to pathogens [102].Guinan et al. [102] documented an antibiotic-induced decline in SCFA levels in mice caeca accompanied by enriched growth and colonization of C. albicans.Those reports could interpret the significantly higher caecal bacterial and clostridial counts in lincomycin-supplemented birds T3 compared to T4, where birds received a diet with partial substitution of lincomycin with SB.
Regarding litter quality parameters, the combined dietary supplementation with SB and lincomycin lowered the litter's moisture and nitrogen contents in T4 and  T5.Previous studies pointed out that SB reduced intestinal pH and increased the activity of digestive enzymes yet increasing the digestibility and absorption of protein and minerals [74][75][76][77].Dietary supplementation of broiler rations with SB and lincomycin could improve the birds' ability to utilize nutrients and reduce their levels of excreta hence reducing ammonia emissions in the environment.Butyrate was indirectly associated with improving urea recycling and nitrogen retention based on the augmented expression of urea transporter in the rumen epithelia of steers delivered a rumen butyrate-enhancing diet [103].
In the current study, SB supplementation significantly increased the villi length and crypt depth of the duodenum and villi length of the ileum.These findings correlated with earlier experiments that reported improved villus length and crypt depth in the duodenum by 0.2, 0.4, and 0.6% dietary butyrate concentrations [81].Similar results reported higher crypt depth in the duodenum of broiler chicks fed 0.2% butyrate [72].Contrariwise, previous research [104] stated that coated SB supplementation resulted in a significant increase in villi height of jejunum compared to the control group.Similarly, another investigation [70] documented that supplementation of SB in broilers improved jejunal and duodenal histomorphometrics compared to the control.Those results suggested the increased intestinal absorption area due to the encouraging villus height growth induced by organic acid supplementation [19].In the current work, the combination of SB and lincomycin supplementation resulted in a significant increase in villi length and crypt depth of the three intestinal regions compared to the control The Lincomix (100 g) fed group revealing significant increase in the length of villi (red lines) compared to control group.D: The Lincomix (50 g) plus Butirex (0.5 kg then 0.250 kg) fed group showing increase in the length of intestinal villi but not significant compared to groups 1, 2, and 3. E: The Lincomix (50 g) plus Butirex (1 kg then 0.5 kg) fed group exhibiting increase in the length of intestinal villi but not significant compared to groups 1, 2, 3, and 4. Number of sampled birds (N) = 5 birds/group group, leading to high intestinal absorption and muscular weight gain.The increased villi height and villus/crypt ratios indicated the increased turnover of intestinal epithelial cells and the stimulated blood circulation of the intestine [9,105].On the contrary, our results disagreed with [106], who stated that lincomycin-supplemented broilers revealed necrosis of intestinal villi tips and massive inflammatory cell infiltration in intestinal propria and submucosa.

Conclusion
Combined dietary supplementation with buffered SB and lincomycin (T4 and T5) significantly enhanced body weights, weight gains, FCR, profitability index, and carcass yields.SB supplementation (in T4 and T5) mitigated the antibiotic-induced adverse effects of lincomycin on the intestine, liver, and kidney, which appeared in lowered caecal bacterial counts, improved intestinal histomorphology, and enhanced blood biochemistry indices compared to T3.IGF-1 and TLR4 genes exhibited the highest expression levels in SB + lincomycinsupplemented groups (T4 and T5).Caecal bacterial and clostridial counts were the lowest in T4.Litter hygiene became more satisfactory in T4 and T5 than in other groups.Dietary SB + lincomycin (T4 and T5) increased the villi length of the intestinal mucosa.Hence, supplementing broilers' diets by SB with lincomycin in half their doses (0.5 kg and 50 g per ton of feed, respectively) positively impacted birds' performance and functional indices.

a, b Fig. 1
Fig. 1 Influence of dietary sodium butyrate and lincomycin on mRNA relative expression level of IGF gene (growth-related gene) in both breast and thigh muscles of broiler chickens.Data are represented as mean ± SEM.Groups having different letters are significantly different from each other at P ≤ 0.05.Groups having similar letters are non-significantly different from each other at P ≤ 0.05.T1: Control -basal diet; T2: Butirex C4 1 kg/ ton in starter feed (0-15 d) and then 0.5 kg/ton in grower and finisher feed; T3: Lincomix recommended dose (100 g/ton); T4: Lincomix half the recommended dose (50 g/ton) + Butirex C4 0.5 kg/ton at 0-15 d and then 0.25 kg/ton; T5: Lincomix half the recommended dose (50 g/ton) + Butirex C4 1 kg/ton at 0-15 d and then 0.5 kg/ton.Number of sampled birds (N) = 5 birds/group

Fig. 5
Fig. 5 Influence of dietary sodium butyrate and lincomycin on intestinal histomorphometry of broiler chickens (day 35) Photomicrograph of jejunum.H&E.V: intestinal villi, Arrow: intestinal crypt and Lines: intestinal length.A: The control group showing the intestinal villi (V) and intestinal crypts (arrow).B: The Butirex (1 kg) fed group showing significant increase in the intestinal villi length (red lines) compared to control group.C: The Lincomix (100 g) fed group revealing significant increase in the length of villi (red lines) compared to control group.D: The Lincomix (50 g) plus Butirex (0.5 kg then 0.250 kg) fed group showing increase in the length of intestinal villi but not significant compared to groups 1, 2, and 3. E: The Lincomix (50 g) plus Butirex (1 kg then 0.5 kg) fed group exhibiting increase in the length of intestinal villi but not significant compared to groups 1, 2, 3, and 4. Number of sampled birds (N) = 5 birds/group

Table 1
Physical and chemical compositions of basal diets for each growing period

Table 3
Influence of dietary sodium butyrate and lincomycin on weekly growth performance parameters of broiler chickens

Table 4
Influence of dietary sodium butyrate and lincomycin on cumulative growth performance parameters of broiler chickens (days

Table 5
35)luence of dietary sodium butyrate and lincomycin on carcass characteristics and immune organs of broiler chickens (day35) a,b,c Mean values with different superscripts in the same column indicate significant difference (Tukey's test; P ≤ 0.05)

Table 7
Influence of dietary sodium butyrate and lincomycin on histomorphometry of the small intestine of broiler chickens (mean µm ± SE)