Use of condensed fermented corn extractives (liquid steep liquor) as a potential alternative for organic acids and probiotics in broiler ration

Abstract Condensed fermented corn extractives (liquid steep liquor) was assessed as an alternative to organic acids in broiler chicken. Day-old Ross 308 chicken (n = 180) were allocated into six treatments (basal diet with no supplement, basal diet supplemented with 1 and 2 cc/kg liquid corn steep liquor (CSL) or 1 and 2 cc/kg formic acid along with basal diet supplemented with probiotic (1 × 108 CFU/g, Lactobacillus acidophilus, Lactobacillus casei) in a completely randomised design with five replicate pens per treatment and six chicks in each pen. Broilers were assessed for performance measures and intestinal and serum characteristics. The results indicated that inclusion of CSL increased (p < .05) feed intake (FI) on days 1–21. Relative weight and length of different organs showed great differences among the treatments (p < .01). The pH content of different parts of the gastrointestinal tract reduced by inclusion of 1 cc/kg of CSL and formic acid (p < .05). Formic acid inclusion reduced villi length in comparison to the control group (p < .05). The lowest villi length to crypt depth ratio was determined in chicks fed diets supplemented with 1 cc/kg of CSL. Ileal and caecal microbial population did not alter by CSL or formic acid supplementation. CSL supplementation reduced the concentration of blood triglyceride, total and LDL cholesterol compared to the control group (p < .05). Results obtained from current trial revealed that CSL is an organic substance that have an acidic pH and its inclusion (1 cc/kg of diet) improved body weight on starter period and reduced digesta pH in gastrointestinal tract comparable to liquid formic acid, however, supplementation of CSL on higher dose (2 cc/kg od diet) did not change digesta pH considerably. HIGHLIGHTS Condensed fermented corn extractives (liquid Steep Liquor) was produced from Wet-milling of corn. Inclusion of 1 cc/kg of condensed fermented corn extractives improved growth performance of broiler chicken on starter period. Inclusion of 1 cc/kg of condensed fermented corn extractives reduced digesta pH.


Introduction
Improving the intestinal health by inclusion of antibiotic growth promoters (AGP) since 1940s has led to control and eradicate many infectious and bacterial diseases, enhanced broilers' growth rate, and overall productiveness during the last 50 years.However, an increase in demand for antibiotic free broilers and regulatory pressure to remove AGP have been resulted in a greater frequency of clinical and subclinical gastrointestinal health problems and challenges for the broiler industry.Therefore, improving both productivity and health condition of avian species using alternate safe products (e.g.plant extracts, essential oils, organic acids, prebiotics, probiotics, synbiotics) is necessary for the poultry industry.Acidification of poultry diets using various organic compounds such as formic, acetic, propionic, and butyric acid (Dibner and Buttin 2002;Taylor et al. 2012, Sobotik et al. 2021) has been proposed and tested in poultry ration as an alternative feed additives instead of growth promoter antibiotics to control and eliminate the pathogen bacteria (Al-Tarazi and Alshawabkeh, 2003;Moharrery and Mahzonieh 2005;Koyuncu et al. 2013) from feed ingredients in order to improve bird health through reduction of the feed and gastro intestinal pH (Dibner and Buttin 2002), reduction of the pathogens colonisation and the buffering capacity of diets (Langhout 2000;Nguyen et al. 2018), and increase in gut health and nutrient digestion (Langhout 2000).Previous studies have reported that inclusion of a blend of organic acids significantly improved nutrient utilisation and immune response due to the increase in relative weight and length of intestinal section and weight of immune-related organs such as bursa of Fabricius in broilers (Mohamed et al. 2014;Sultan et al. 2014;Nguyen et al. 2018).Elnaggar and Abo El-Maaty (2017) reported that supplementing organic acid at 0.5 and 1.0% in the diet of broiler chickens increased the relative weights of total edible pasts and dressing when compared to the control group.However, despite such benefits, application of the organic acids in liquid and powder forms remains controversial due to the concerns about the metabolism, conversion and deactivation of these products in the poultry foregut (Khan and Iqbal 2016).Several approaches such as coating has been proposed to overcome these limitations (Upadhaya et al. 2014a(Upadhaya et al. , 2014b)).Nonetheless, using the coated organic acids may lead to poor acidification in the poultry foregut.Thus, acidification of diets using certain grain refining by-products, which are high in acidity, may be one of the alternate choice to overcome the mentioned limitation in this concept.
Corn and corn by-products are abundant materials and most of which are utilised as ingredients in poultry diets.Wet-milling, dry-milling and corn-distilling process are the three major processes which have been used to manufacture food products from corn (Loy and Lundy 2019).In the wet milling process of corn, almost 25% of corn is converted to by-or coproduct materials (Loy and Lundy 2019).Corn steep liquor (condensed fermented corn extractives) is one of the important by-products of the corn-steeping process, which is rich in small peptides, free amino acids (Christianson et al. 1965), several vitamins, trace elements, and lactic acid (Liggett and Koffler 1948).Lactic acid (10-30%, dry basis) is the main acidic component which has been produced via fermentation by desirable lactic acid-producing microorganisms that are able to use different carbohydrates during the steeps process (Liggett and Koffler 1948).Lactic acid is present in the form of potassium lactate salts in the steep liquor.The rare research report have indicated some kind of chick-growth-stimulating component in steep liquor (Russo et al. 1960).Russo and Heman (1959) replaced 5% corn, corn-soybean meal mixture and menhaden fish meal with 5% corn fermented condense soluble in broiler diet and showed considerable increase in growth rate.It is reported that the feeding high levels of dry steep liquor to laying hens improved Hugh units and egg white integrity (Hazen and Waldrup 1972;Lilburn and Jensen 1984).Most studies with corn steep liquor in poultry have been carried out using the solid steep liquor as substitute for corn and protein supplements and there is no study considering its effect as a fermentable or acidic liquid agent to acidification of poultry diets.Hence, evaluating the potency of corn by-product as an acidic solution with low pH to improve the intestinal pH and health may introduce new and safe acidic compound for broiler chickens.
Therefore, the current study explored the possibility of using the corn steep liquor as a fermented and acidic by-product to impact intestinal health and pH in comparison to the formic acid or lactic acid bacteria (LAB) in broiler ration.

Diets and experimental design
The corn steep liquor (CSL) was obtained from commercial corn processing company Glucosan (Qazvin Province, Alborz Industrial City, North Ebn Sina St., Iran).The lactic acid content was determined using high-performance liquid chromatography (HPLC) (Waters 600, Tokyo, Japan) according to the procedure described by Muck and Dickerson (1987).Chicks were fed different phased diets in mash form during starter (days 0 À 11), grower (days 12 À 24) and finisher (days 25 À 42) phases to meet the Ross 308 strain recommendations, respectively.Six dietary treatments were used, including a control diet, control diet supplemented with 1 and 2 cc/kg liquid CSL, control diet supplemented with 1 and 2 cc/kg formic acid and control diet supplemented with 0.2 g/kg LAB (1 Â 10 8 CFU/g, Lactobacillus acidophilus, Lactobacillus casei).Control and experimental diets were mixed using a stainless steel twin-shaft paddle mixer with a capacity of 200 kg for 4 min.Calculated and analysed nutrient content of the basal diet is presented in Table 1.

Birds rearing managements
All animals were reared according to the protocols approved by Animal Care Council (1995).In total, 180 one-day-old male Ross-308 broilers (39 ± 1 g) were individually weighed and divided at random into 30 pens (1 Â 1 m 2 ) (five pen with six chicks each).Rearing house was cleaned and disinfected prior to chick arrival.Heaters were turned on 24 h before chick arrival and room temperature was adjusted to 30 C at chick's height and 28-30 C at floor using two gas heaters at the first three days of life and then reduced gradually from 31 to 24 C at 18 days of age.Each pen was lined with wood shavings as bedding material and spread at depth of 5 cm.Pens equipped with one tube feeder and one cup drinker until approximately 7 to 10 days of age and one bell drinker from 7 days onward.Chicks received a light intensity of 30 lux from 0-7 days of age and 10 lux thereafter with constant day length of 23 h from 0-7 days and 16 h light and 8 h of dark period thereafter.Chicks had unobstructed access to both feed and water throughout the experimental period.Feed intake (FI) and weight gain (BW gain) were measured weekly and mortality were recorded daily.Average FI and body weight gain were corrected for mortality when calculating feed conversion ratio (FCR) for each pen.

pH measurement
The pH of CSL was determined by inserting the sterile glass pH electrode probe of a portable ADWA pH metre (model AD800; Adwa Instruments, Romania) into liquid from different angles.pH metre was calibrated before testing the CSL and digesta pH using two standard buffer solutions with pH values of 7 and 4, respectively.On day 14, 10 birds per treatment (two birds from each pen) were randomly picked and were euthanised by cervical dislocation and immediately after opening up the abdominal cavity, the pH of the digesta contents in the proventriculus, gizzard, duodenum, jejunum and ileum were determined using the same pH metre (model AD800; Adwa Instruments, Romania) in three independent location as described by Adejumo et al. (2021).

Ileal morphology
On day 42, ileum tissue samples were obtained from two chicks and washed three times in physiological saline solution to preserve cells from autolysis and stabilised the cellular and tissue constituents so that they withstand the subsequent stages of tissue processing and thereafter tissue specimens were transferred immediately into the formalin and fixed in 10% buffered formalin.A single 0.5-cm sample was cut from each ileal section, dehydrated using increasing ethanol concentrations, cleared with xylene and placed into polyfin embedding wax.Paraffin sections are cut into 8.4 a Vitamin premix supplied the following per kg of diet: transretinol: 13 mg; cholecalciferol: 0.5 mg; a tocopherol acetate: 80 mg; menadione: 3 mg; thiamine: 3 mg, riboflavin: 8 mg; pyridoxine: 5 mg; cyanocobalamin: 0.017 mg; nicotinic acid: 60 mg; folic acid: 2 mg; Ca pantothenate: 20 mg; choline: 1000 mg b The mineral premix provided the following per kg of diet: Mn: 120 mg; Zn: 110 mg; Cu: 16 mg; I: 1.25 mg; Se: 0.3 mg and Fe: 20 mg 2-4 mm thickness using microtome (Easy cut 202, Italy), floated onto slides and stained with haematoxylin (Gill number 2; Sigma, St. Louis, MO) and eosin (Sigma Aldrich, Darmstadt, Germany).For each sample, a comparative photographic analysis was carried out to analyse the changes in villus height, crypt depth, villi height to crypt depth ratio, lamina propria thickness and epithelial cell layer thickness by taking images using a digital camera that had light microscopy (Motic-SMZ-140, Germany).Twenty-five images from 10 tissue sections of each ileal section were taken and the changes were measured by imaging software.

Microflora population
Ileum and caecal microbiota were assayed according to the gut microflora cultures methodology described by Khalaji et al. (2011).Briefly, on day 14, two birds from each pen were euthanised by cervical dislocation.Ileum and caeca digesta contents were collected into the 15 mL sterile tube.Coliform, salmonella and E. coli counts were obtained by serial dilution (10 À5 to 10 À6 ) in anaerobic diluents (saline solution 9%) after inoculation in culture media plates in petri dishes of sterile agar as described by Bryant and Burkey (1953).Salmonella shigella agar (Liofilchem, Zona Ind.le-Roseto d.Abruzzi-Lot: 111816503, Italy) is used for salmonella culture and McConkey agar (Liofilchem, Zona Ind.le-Roseto d.Abruzzi-Lot: 120316502, Italy) for coliforms and E. coli plates were incubated at 37 C and were counted between 24 and 48 h after inoculation.Colony-forming units were counted immediately after removal from the incubator (GmbH, D-91126, Germany).

Blood characteristics
On days 14 and 42, blood samples were taken from two birds in each pen and kept at room temperature for 2 h and then centrifuged at 1300 Â g for 10 min.Plasma total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C), and triglyceride concentration were analysed using commercial kits (Pars Azmoon Inc., Tehran, Iran) and an automatic biochemical analyser (Hitachi 717, Boehringer Mannheim, Ingelheim am Rhein, Germany) by the assessment of an enzymatic method with colorimetric detection.The two other blood samples were taken from two birds in each pen and were drawn into heparinised microcapillary tubes for cell count.Differential cell counts were conducted by staining the blood specimen with May-Gr€ unwald-Giemsa stain.One hundred cells were counted, including heterophils, lymphocytes, monocytes, eosinophils and basophils.

Humoral immune response to the vaccine
For assessment of antibody titre against the Newcastle disease and Avian influenza (AI) on days 31 and 42, 2 mL blood specimen was taken by wing vein puncturing of two birds per pen into the non-heparinised tubes and tubes were kept for 2 h at an angle of 45 and serum was harvested for analysing antibody titres by haemagglutination inhibition test.

Relative organ weights and intestinal lengths
On days 14 and 42, two chicks from each pen were chosen randomly, weighed, feed removed from feeders for 6 h to permit intestinal emptying of birds and then the chicks were slaughtered by cervical dislocation.The digestive tract from the proventriculus to caeca was carefully excised, and adherent fat was removed.The length of duodenum, jejunum and ileum were recorded according to the procedure reported by Amerah et al. (2008b) and reported as cm/kg of BW.The empty weights of carcase, liver, spleen and bursa of Fabricius were determined and reported as g/kg of BW.

Statistical analysis
A one-way analysis of variance (ANOVA) was used to investigate the significance of the dietary treatment using the MIXED procedure of SAS Version 9.1 (SAS Institute 2003) with dietary treatment (n ¼ 6) as fixed effect.Each treatment had five replicate pens with six chicks per pen.Homogeneity of variances (Levene's test) and normality (Shapiro-Wilk's test) of the residual was tested using the UNIVARIATE procedure of SAS.When the effect was significant, differences among treatment means were tested using Tukey's multiple comparison tests.Statistical significance was declared at p .05.

Chemical composition of corn steep liquor
The DM content of CSL which was used in the current trial was 43% and contained 31.7% crude protein, 11.3% lactic acid (dry basis) with pH of 4.2.

Performance
The results of adding CSL on performance of broilers are presented in Tables 2 and 3. Throughout the duration of trial, no differences in overall BW, feed consumption or FCR (p !0.05) were observed among control, formic acid, CSL and LAB supplemented group, however, BW and FI were consistently greater (p .05) in chicks fed diets supplemented with 1 cc/kg of CSL compared with chicks fed 1 cc/kg formic acid on day 7, 14 and 21.Meanwhile, no significant differences (p !.05) in FI and body weight were observed when birds fed with 2 cc/kg of CSL or formic acid during the trial.

pH measurement
The pH content of CSL was 4.2.The effect of dietary treatment on proventriculus, gizzard, duodenum, jejunum and ileum digesta content are presented in Table 4. Digesta pH value throughout the gastrointestinal tract was lowest (p .01) in birds fed diets supplemented with 1 cc/kg of CSL and highest in birds fed diets supplemented with LAB (p .01)among the treatments.Except for gizzard, inclusion of CSL reduced digesta pH significantly (p .01)compared to the formic acid throughout the gastrointestinal tract.

Ileal morphology and microflora population
Villi length and crypt depth of birds fed CSL supplemented diets were similar to that of the control group, whereas villi length was significantly lower (p .01) in birds fed 2 cc/kg formic acid compared to the other treatment groups (Table 4).Villi to crypt ratio was lowest (p 0.01) in birds fed diets supplemented with 1 cc/kg of CSL and highest in birds fed diets supplemented with LAB (p .01)among the treatments.Epithelial cell layer thickness was reduced significantly (p .01) in birds fed formic acid or LAB supplemented diets compared to the birds fed control diet or diet supplemented with CSL.Neither ileal nor caeca Salmonella, Coliforms and E. Coli population were altered by supplementation of CSL, formic acid or LAB into the control diet (Table 4).

Blood characteristics
Effect of different treatments on blood lipid profile and white blood cell counts are given in Table 5.No significant differences were observed among dietary treatments for any of the assayed plasma lipid content during the 14 days of age, however, triglyceride and total cholesterol content of plasma were reduced by inclusion of CSL significantly (p .05) on day 42.Birds fed 1 cc/kg of formic acid had lower (p .05)total and LDL-cholesterol and triglyceride compared with birds .748a-d Means within a column without a common superscript significantly differ (p < .05)Body weight gain (g/period); FI: feed intake (g/period); FCR: feed conversion ratio (g of feed/g of body weight gain); CSL: corn steep liquor; LAB: lactic acid bacteria There were six broilers per pen and five pens per diet.Pens means were used to calculate each treatment means..650a-d Means within a column without a common superscript significantly differ (p < .05).Body weight gain (g/period); FI: feed intake (g/period); FCR: feed conversion ratio (g of feed/g of body weight gain); CSL: corn steep liquor; LAB: lactic acid bacteria.There were six broilers per pen and five pens per diet.Pens means were used to calculate each treatment means.Humoral immune response to the vaccine No significant differences of Newcastle disease and Avian influenza (AI) vaccine titre were detected among the treatments in broilers on day 31 (Table 6).However, supplementation of CSL, formic acid and LAB significantly (p .01)increased AI vaccine titre measured at 42 days of age.

Relative organ weights and intestinal lengths
There was no significant (p !.05)differences in the carcase, thigh muscle, liver, duodenum and jejunum weight in birds among all treatments on day 14 (Table 6).However, breast muscle and ileum relative weight increased in birds supplemented with CSL, formic acid and LAB.Birds fed 1 cc/kg of formic acid showed lowest (p .05)gizzard and spleen relative weight among all the groups.The relative lengths of duodenum, jejunum, and ileum were similar (p !.05)among all treatments.

Discussion
Several alternatives such as plant extracts, essential oils, organic acids, prebiotics, probiotics and synbiotics with encouraging outcomes have been used to increase the gastrointestinal health status of broiler chickens and to reduce or eliminate the use of growth-promoting antibiotics in poultry production (Abd El-Ghany et al. 2022).It is reported that organic acids could enhance the broiler performance through the reduction of the gastrointestinal pH, suppressing the pathogenic intestinal bacteria, lowering the levels of harmful bacterial metabolites and improving the protein and energy digestibility (Baurhoo et al. 2007;Hajati 2018;Jadhao et al. 2019).However, reviewing the research finding for inclusion of these compounds into the poultry rations have been shown that the protective efficacies and cost-effectiveness of these therapeutics agents were questionable, inconsistent and variable.Interestingly, CSL (condensed fermented corn extractives) is low cost (0.5-1$per kg vs 3-4$per kg of common acidifiers in poultry feed), acidic byproduct rich in LAB for poultry production.Accordingly, the current study was designed to compare the efficacy of CSL with organic acid and dietary probiotic lactobacilli in commercial broiler chickens.
No differences were found in body weight, FI, and/or FCR during the trial, when formic acid, LAB or CSL were fed to the broiler chickens in current trail.There was only an inconsistent improvement in BW and FI of broiler chickens by inclusion of CSL specially when added in 1 cc/kg dose to diets at early stage of birds life (until 21 days of age).It is reported (Menconi et al. 2014) that acidification of feed and drinking water for poultry for the first seven days of life, when the birds are first placed into the house, is very critical, because the crop and intestinal microbial morphology would still be under development.According to Liggett and Koffler (1948) the CSL is a rich source of lactic acid (11.3%).Consequently, the reduction of crop pH by LAB in newly hatched chicks by inclusion of CSL helps to enhance the survival and colonisation of LAB and eventually results in improving the live production performance.Similarly, previous studies related to the organic acids research area in poultry has been stated inconsistent results with respect to the growth benefit or reduction of pH and pathogen bacteria (Hernandez et al. 2006;Ragaa and Korany, 2016;Adhikari et al. 2020).Many studies that have been done from 1980 till to date for exploring the effects of supplementing organic acid in chickens, and their results were highly variable.Previous studies have been postulated that these differences could be due to the feed form, feed contamination, organic acids sources and dosage, routes of administration (by feed or water) and bird health status (challenged or healthy birds in controlled environment) (Koyuncu et al. 2013;Adhikari et al. 2020).Papatsiros et al. (2013) and Adhikari et al. (2020) also reported that the positive response on growth performance by organic acid in early life could be because of decrease in pH of digesta in upper part of the gastrointestinal tract and increasing the proteolytic enzyme activity and nutrient digestibility along with bacteriostatic and bactericidal action to the pathogenic bacteria.In addition, CSL contains relatively high levels of several important vitamins, trace elements, and lactic acid.The lactic acid is the major acidic part (10%-30%, dry basis) of CSL, which is synthesised by desirable lactic-acid-producing organisms (Liggett and Koffler 1948).As mentioned earlier, the CSL contained 11.3% lactic acid in current trial.Adil et al. (2010) and Sultan et al. (2014) reported considerable improvement of broiler growth performance by addition of lactic acid to the diet.Abdel-Fattah et al. (2008) reported that organic acids can boost performance by reduction of digesta pH and hampering the growth of pathogenic bacteria throughout the gastrointestinal tract leading to increase in the digestion and absorption capacity of chicken intestine to uptake nutrient for growth by increasing villi length and shifting the nutrient usage from pathogenic bacteria to the host (Abdel-Fattah et al. 2008;Pirgozliev et al. 2008;Upadhaya et al. 2016).
Bacterial enumeration in the present study revealed no differences in Salmonella, E. coli and coliform population of ileal and caeca digesta among dietary treatments.Although no significant differences in the intestinal microbiota population were detected, CSL inclusion reduced digesta pH throughout the gastrointestinal tract and reduced the villi to crypt ratio.Intestinal pH reduction by inclusion of 1 cc/kg of CSL was comparable to that of control, formic acid-and LAB-fed birds.No response of bacterial population to CSL or formic acid in this study may partly due to the liquid form of these products, their concentration and sampling location in the intestinal lumen.It is reported that LAB and liquid formic acid were highly effective to reduce pH and Salmonella contamination in upper part of gastrointestinal tract especially at birds crop (Byrd et al. 2001).Compared with the control, CSL, formic acid and LAB did not increase ileal villi length considerably.However, crypt depth was narrow in birds fed 1 and 2 cc/kg of CSL or 1 cc/kg of formic acid and thereby villi to crypt ratio was reduced in these groups compared to the control or LAB fed birds.Markovi c et al. (2009) reported that longer villi length correlates to a larger surface area for absorbing nutrients.Crypt sites are the location of enterocyte proliferation, and shallow crypts indicate less need for epithelial cell turnover, taking energy that would go towards cell turnover and placing it towards animal growth.Although, increase in villus length is associated with an increase in digestion and absorption of nutrients (Awad et al. 2017).Similarly, Fatholahi et al. (2021) noted that crypts constantly renew the epithelial cells lining the intestinal lumen by migration of new cells from the crypts to the villus tip.During this cell migration, the villous epithelial cells growth in longitude and come directly in contact with the lumen content and are therefore prone to damage, which often results in an increased loss of villous epithelial cells in cases of intestinal health problems such as challenged conditions (Awad et al. 2017;De Grande et al. 2020).The constant villus length without accompanying increase in crypt depth or decrease in crypt depth as observed in current trail, may indicate that there is less villous epithelial cell loss at the villus tip by inclusion of CSL or formic acids in diets.Therefore, lower villus length in CSL or formic acid-supplemented group compared to non-supplemented control group can be associated with low pathogen bacteria load in feed and no further need for cell proliferation and cell renew in host intestinal lumen.
Cholesterol and triglyceride concentration of plasma was reduced by inclusion of 1 cc/kg CSL.Several animal and human studies have proven in vitro or in vivo the cholesterol-lowering effect of lactic acids and LAB, especially strains of Lactobacillus and Bifidobacterium (Ooi and Liong 2010;Homayouni et al. 2012;€ Oner et al. 2014).According to the Ooi and Liong (2010), several mechanisms have been proposed for cholesterol and triglyceride-lowering effect of organic acids and Lactobacillus, which include enzymatic deconjugation of bile acids, assimilation of cholesterol by probiotics, co-precipitation of cholesterol with deconjugated bile, cholesterol binding to cell walls of probiotics, incorporation of cholesterol into the cellular membranes of probiotics during growth, conversion of cholesterol into coprostanol, and production of short-chain fatty acids upon fermentation by probiotics in the presence of prebiotics (Pereira and Gibson 2002;Liong and Shah 2005;Lambert et al. 2008;Lye et al. 2010).
Findings from the present study revealed that the heterophils and heterophils to lymphocyte ratio and humoral immune response to AI were enhanced by inclusion of CSL, formic acid and LAB.It is reported that increases in circulating chorticosterone concentrations indicate the activation of the acute stress response, whereas increases in H:L ratios indicate chronic stress and basal inflammation in the intestine which could influence the development of the cellular and humoral immune systems in chickens (Gross and Siegel 1983;Shini et al. 2008;Weimer et al. 2018).Furthermore, inclusion of CSL and formic acid increased relative weight of spleen.Similarly, previous studies have reported that addition of a blend of organic acids significantly increased relative weight of the bursa of Fabricius in broilers (Mohamed et al. 2014;Sultan et al. 2014;Nguyen et al. 2018).Measurement of immune organ weight is a traditional way for evaluation of immune status in chickens (Heckert et al. 2002).Immune organs constitute the immune system of the body together with lymphoid tissue and immune cells (Li and Verma 2002).
Generation, proliferation, differentiation and maturation of immune cells usually take place in the thymus, spleen and bursa (Brekelmans and Van Ewijk 1990).Therefore, the immune organ index is an important indicator reflecting immune competence.Consequently, increase in H:L ratios and spleen weight by inclusion of CSL, formic acid and LAB may be due to the response to chronic stress on the intestine of broiler chicken due promote basal inflammatory cytokines and resulting in improved immune response to AI vaccine and improved immune organs growth.
Carcase yield was not affected by addition of CSL, formic acid and LAB.There is no report considering the effect of CSL on organs relative weight or length and also, there are a limited number of studies evaluating the effects of organic acids on relative organ weights.Wang et al. (2010) also did not observe any significant effects on relative weights of gizzard, liver, spleen, bursa of Fabricius and breast muscle by dietary organic acid supplementation.Our results agree with the study that have been done by De Souza et al. (2018) who found no statistical differences in carcase weight by addition of Lactobacillus acidophilus into the broiler diet.In contrast, breast muscle, gizzard, spleen and ileum relative weight increased substantially in birds fed CSL, formic acid and LAB-supplemented diets.Elnaggar and Abo El-Maaty (2017) reported that supplementing organic acid at 0.5 and 1.0% in the diet of broiler chicks increased the relative weights of total edible pasts and dressing when compared to the control group.However, Islam et al. (1970) and Reda et al. (2021) found no significant differences in percentages of liver, heart and spleen of broiler chicks by addition of different levels of organic acids.

Conclusions
In conclusion, results obtained from the current research revealed that CSL is an organic substance that have an acidic pH with almost 11% lactic acid, and its inclusion (1 cc/kg of diet) could be effective to promote body weight on starter period.Inclusion of 1 cc/kg of CSL reduced digesta pH throughout the gastrointestinal tract comparable to liquid and powder formic acid, however, the ileum villi length was reduced and supplementation of CSL on higher dose (2 cc/kg od diet) did not affect growth performance and digesta pH considerably.

Table 1 .
Ingredient and calculated and analysed composition of the diets (g/kg) on as fed basis.

Table 2 .
Production parameters such as body weight, feed intake and feed conversion ratio from one to 21 days of age.

Table 3 .
Production parameters such as body weight, feed intake and feed conversion ratio from 21 to 42 d of age.

Table 4 .
pH content of gastrointestinal tract, ileal morphology and ileal and caeca microbial population at Days 14 in broilers fed diets supplemented with different additives.

Table 5 .
Blood characteristics at Days 14 and 42 in broilers fed diets supplemented with different additives.

Table 6 .
Humoral immune responses to inactivated Newcastle disease and avian influenza vaccines and carcase analysis of broilers fed diets supplemented with different additives.