Growth studies in commercial broiler birds offered citric acid in formulated feed with low mineral density

Study of 35 days was conducted to evaluate citric acid (CA) as an additive in poultry broiler feed with lower mineral content of calcium (Ca) and total phosphorus (TP) in commercial broiler poultry birds for its effect on growth, nutrient utilization, carcass characteristics, and economics. Vancobb-400 strain day old broiler chicks were divided into four main treatment groups T0, T1, T2, and T3. Treatment groups were further divided into eight replicates with ten chicks in each. T0 served as control, given standard corn-soy flakes-based ration (Pre-starter %: Crude protein (CP)-23, Ca-1.00, TP-0.70; Starter %: CP-22, Ca-1.10, TP-0.72, and Finisher %: CP-20, Ca-0.99, TP-0.70). Treatment T1 served as positive control with added 0.5% CA (Pre-starter %: CP-23, Ca-1.00, TP-0.70; Starter %: CP-22, Ca-1.10, TP-0.72 and Finisher %: CP-20, Ca-0.99, TP-0.70). Treatment T2 was given feed containing 0.5% CA with low Ca and TP content (Pre-starter %: CP-23, Ca-0.90, TP-0.66; Starter %: CP-22, Ca-0.99, TP-0.71 and Finisher %: CP-20, Ca-0.90, TP-0.69), whereas treatment T3 was given feed containing 0.5% CA with moderately low Ca and TP content (Pre-starter %: CP-23, Ca-0.80, TP-0.65; Starter %: CP-22, Ca-0.88, TP-0.70 and Finisher %: CP-20, Ca-0.79, TP-0.68). Birds offered moderately low Ca and TP with 0.5% CA addition, exhibited higher growth rate (P < 0.05), better nutrient utilization with positive influence on dressing percentage and forequarters weight. Economics of broiler feeding revealed that 0.5% CA supplementation fetched highest gross return above feed cost in broiler birds offered feed with moderately low Ca and TP content whereas lowest profit was recorded in feed with low content of Ca and TP. In conclusion, supplementation of 0.5% CA in feed with low and moderately low Ca and TP content positively influenced overall growth, and carcass characteristics. Economics of broiler feeding with moderately low Ca and TP content revealed highest profit with CA (0.5%) supplementation.


Introduction
Gut (or intestinal health) is a key factor that can affect the performance of poultry and thus play key role in profitable poultry production (Samik et al. 2007), and the composition of the intestinal micro flora has a major influence on intestinal health. Antimicrobial drug resistance has alarmed the scientists all over the world especially for the one used as livestock feed additives. Antibiotics added to/used in animal feeding such as tetracycline, bacitracin (Diarra and Malouin, 2014) which are given in preventive doses often lead to survival of pathogenic microbes which in turn develop drug resistance (Diarra et al. 2007;Furtula et al. 2010;Forgetta et al. 2012). Therefore, to avoid drug resistance, there is a search for alternatives to feed grade antibiotics. Moreover, the increasing broiler industry worldwide needs products or supplements which improve quality of produce and at the same time does not compromise yield; thus, there is continual search of such additives which can reduce dependence on antibiotics. Recent reports suggest that organic acids (OA) are being popularly used as non-antibiotic feed additives in poultry nutrition (Windisch et al. 2008). These acids potentially act and exhibit response by reducing pH of intestinal tract and thus favoring beneficial microbes which subsequently suppress the pathogenic microbes (Jin et al.1997;Ghabdan, 2002), reducing the use of antibiotics. Breakdown of protein and fiber in the digestive system is enhanced with citric acid (CA) which is an organic acid (Attapatu and Nelligaswatta 2005) thus reducing the amount of P added to the diets (Boling et al. 2001). Lower intestinal pH less than 7 increases the solubility of calcium (Ca) and thus positively influences the absorption of Ca upon addition of CA (Broz et al. 1994;Boling et al. 1999). Many researchers (Chowdhury et al. 2009;Ghazalah et al. 2011;Islam et al. 2012) have reported that addition of CA @0.5% increases weight gain, feed intake, tibia ash deposition, nonspecific immunity, as well as feed efficiency (Das et al. 2012;Srinivas et al. 2018) and carcass yield (Haq et al. 2014). CA inclusion in diet has been reported to decrease colonization of pathogens, limit production of toxic metabolites, improve availability of protein, Ca, P, Mg, and Zn as well acts as a substrate in intermediary metabolism (Tollba 2010;Adil S. et al. 2011).
The present research experiment was designed to evaluate citric acid (0.5%) addition as feed additive in commercial broiler birds reared on formulated feed with low and moderately low calcium and phosphorus.

Experimental design and management
The experimental plan was approved by the Institutional Animal Ethics Committee of College of Veterinary and Animal Sciences (COVAS) Palampur. The experiment was carried out in an experimental chicken coop on a deep bed in 320-day-old-meat-type chicken (Vencobb-400). The poultry house was kept at 30 ± 1 °C during the first 2 weeks and gradually reduced to 21 ± 1 °C by the end of the experiment. The relative humidity ranged between 40 and 60% throughout the experimental period. Feed was offered ad libitum and water was always accessible for drinking.

Experimental plan
Chicks were reared in electric battery brooders during first week post hatch. Single battery brooder consists of ten compartments placed vertically measuring 36 × 72 in. per compartment. Each compartment was allocated to a specific replicate with ten chicks in complete randomized design during which, chicks were wing tagged, weighed, and randomly distributed according to the experimental plan into four groups T 0 , T 1 , T 2 , and T 3 . Each group was divided into eight replicates with ten chicks in each replicate; thus, each treatment had 80 birds. Birds were shifted from brooder house to deep litter system on the 7th day, and a lighting schedule of 24 h per day was followed till the end of the experiment. The deep litter pen floor was divided using wire mash into cubicles measuring 5 × 4 × 6 ft each, covered with wood shaving up to 6-cm height. T 0 served as control diet and was given standard corn-soy flake-based ration (Prestarter %; CP; Ca; 1.00,0.70,Starter;22,1.10,0.72 and Finisher;20,0.99,0.70). Treatment T 1 served as positive control with added 0.5% CA (Pre-starter %; CP; Ca; TP 23, 1. 00,0.70;Starter 22,1.10,0.72;and Finisher 20,0.99,0.70). Treatment T 2 was given standard corn-soy flake-based ration containing 0.5% CA but with low Ca and TP content (Pre-starter %; CP; Ca; 0.90,0.66;Starter 22,0.99,0.71;and Finisher 20,0.90,0.69). Further treatment T 3 was given standard corn-soy flake-based ration containing 0.5% CA with moderately low Ca and TP content (Pre-starter %; CP; Ca; 0.80,0.65;Starter 22,0.88,0.70;and Finisher 20,0.79,0.68) according to ICAR (2013) standards. The physical and chemical composition of diet for different phases is presented in Table 1.

Digestibility and metabolism studies
The digestibility of the feed was evaluated using the fecal collection method on the 24th day onwards. Two male birds selected randomly from each replicate having similar weight were shifted to electric battery brooders. Birds were offered the same treatment feed for 3 days as in the growth study to provide them adaptation time in the metabolic cages. After adaptation period of 3 days, the weighed quantity of feed for next five consecutive days was offered to each replicate, both during morning at 9:00 a.m. and in evening at 6:00 p.m. Actual consumption of feed was estimated by weighing the residual feed on the 5th day in each replicate. The excreta voided by birds from each replicate were collected in the morning and weighed on daily basis, screened for the presence of feathers and feed particles which were removed manually. Around 10% of the total excreta was taken as final sample. The samples were mixed carefully in plastic tray and further divided into two similar portions for the subsequent determination of nitrogen and dry matter. Twenty mL of 5% sulfuric acid was mixed in the feces collected for nitrogen estimation to avoid nitrogen loss, and the feces collected for dry matter estimation was dried separately at 105 °C in hot air oven till the constant weight was achieved, and then pooled samples were ground and analyzed for various proximate parameters in the laboratory of the Department of Animal Nutrition. The digestibility of nutrients viz dry matter, ether extract, crude fiber, and retention of nitrogen, Ca, and P was calculated by total fecal collection method, which involves measurement of feed intake and excreta voided.
Mortality % was calculated by keeping the record of dead birds during the experiment.

European efficiency factor
Formulae (Bera et al., 2010;Lup et al., 2010) for calculation of economic factors viz. viability and European efficiency factor (EEF) along with cost of 1 kg live weight (cm) production is given below: BW refers to (average body weight in kg), units of age were days, and the calculation of the price for 1 kg live weight production is therefore as follows:

Carcass parameters
At the end of feeding trial, two birds from each replicate of same weight were slaughtered to record carcass weight, eviscerated weight, dressing percentage, abdominal fat, and weight of the heart, liver, and gizzard. The birds were fasted over night to drain the intestinal content and sacrificed to assess the effect of dietary treatments on the carcass weight, dressing percentage, muscle yield, and weight of the heart, liver, and gizzard. At the time of slaughter, pH of the contents of duodenum, jejunum, ileum, caecum, and colon were recorded with the help of pH meter (perfit digital pH meter).

Microbial count
The caecum was removed from birds of respective experimental group at time of slaughter and was collected in glass jars and placed along with ice packs to arrest microbial growth. Caecal content was removed with the help of scalpel blade and forceps into sterilized petri plates. The samples of caecal content were processed by preparing 1:100 dilutions with double distilled water. The dilutions are prepared by thorough mixing of the sample and the diluents. One milliliter of dilution was transferred to the sterile petri dishes from the tube with the highest dilution Viability = {1 − (n of dead birds∕total birds)} × 100 EEF = {BW × viability × 100∕(FCR × age)} 10 −7 . Then to each plate, 15-20 mL molten agar cooled to temperature 50-55 °C was transferred by plate inoculation method. Petri plates were kept inverted for incubation at 37 °C for 24-48 h. After incubation, the colonies were counted with the help of Quebec colony counter at the Department of Veterinary Public Health, COVAS (H.P). Twenty different colonies from each respective treatment group were taken from the petri plates with the help of inoculation loop on the glass slide and were stained with the help of gram staining. Then these slides were observed under the microscope, and the shape of microorganism along with gram stain whether positive or negative was recorded.

Blood profile
Approximately 3 to 5 mL of blood from randomly chosen birds of each replicate was collected from ulnar vein with the help of 24-gauge (24G) needle and collected in 10% heparin-coated centrifuge tubes. Whole blood after collection was centrifuged at 2000 × g for 15 min; thereafter, the separated plasma after collection was transferred into clean polypropylene tube using a Pasteur pipette. Samples were stored at − 20 °C for biochemical analysis in the lab (CHEM 5 χ plasma analyzer, Erba Mannheim-India) with the help of the kits (Agape diagnostics, India). The analysis of whole blood for hematology was performed immediately after collection of the blood in heparin-coated tubes using fully automated hematology analyzer (BC 2800 vet-Agape diagnostics, India). The hematological analysis includes red blood cells (RBC), white blood cells (WBC), hematocrit (Hct), and platelet count.

Tibiae bone analysis
Tibiae bones of two birds from each replica were collected separately in labelled plastic bags at the time of slaughter. The plastic bags containing samples of tibiae bones were stored in freezer till analysis of mineral content. Processing of bone was done as per the method given by Hall et al. (2003) for the estimation of Ca and P by employing methods as discussed above.

Statistical analysis
All the recorded and calculated data were subjected to analysis of variance (ANOVA). Duncan multiple range test (Duncan, 1995) was used for determining the significant difference (at 5% level of significance) using statistical analysis software (version 9.3).

Results
GIW, FI, FCR, GIW/day, FI/day, and mortality % were studied ( Table 2). Results of the experiment revealed no significant (P > 0.05) difference for GIW among control T 0, positive control (PC) T 1; and treatment groups T 2 and T 3 during 1-14 days and 14-21 days. Growth pattern during 21-35 days and 1-35 days revealed significantly (P < 0.05) higher GIW in treatment group T 3 compared to control T 0 . Similarly, PC T 1 also exhibited significantly (P < 0.05) higher GIW for the overall period 1-35 days compared to control T 0 . On an average, feed intake in treatment T 1, T 2 , and T 3 was recorded to be 11.8% higher than control T 0 . FCR value for the period 1-35 days, varied in between 1.65 and 1.81 with lowest in control T 0 followed by treatment T 3 exhibiting significant (P < 0.05) difference as compared to T 2 which recorded highest FCR of 1.81. Highest mortality (Table 2) of 11.25% was exhibited in T 0 which exceeded the normal management norms whereas treatment T 1 , T 2 , and T 3 had mortality % varying between 3.75 and 7.50% acceptable as per management norms. 0.5% CA-supplemented treatments T 3 exhibited higher EEF (Table 5) of 301 compared to 261 and 283 in control T 0 and PC T 1, whereas treatment T 2 exhibited EEF of 260 similar to control T 0. Overall, birds in treatment T 3 exhibited lowest cost (Rs.51.90) of feed per kg live weight gain.
Digestibility studies (Table 3) revealed non-significant (P > 0.05) difference for calcium and phosphorous retention. Positive control T 1 and treatment groups T 2 and T 3 exhibited higher calcium retention, whereas T 2 and T 3 exhibited higher P retention compared to control T 0 . Microbial count ( Table 3) in caeca of broiler chicks exhibited a significant (P < 0.05) reduction in PC T 1 and treatment groups T 2 and T 3 compared to control T 0 . Plasma calcium (mg/dL) ( Table 4) level varied between 9.94 and 11.31 mg/dL and was significantly (P < 0.05) higher in control T 0 compared to PC T 1 and treatment T 2 and T 3 . Similarly, plasma phosphorus level (mg/dL) exhibited significant (P < 0.05) difference and varied between 6.74 and 7.43 mg/dL but were within normal range in control T 0 , PC T 1 , and treatments T 2 and T 3 . Analysis of blood plasma (Table 4) revealed significantly (P < 0.05) low cholesterol in PC T 1 and treatments T 2 and T 3 compared to control group T 0 . Blood hematology revealed no differences for different parameters (Table 4) thus causing no major influence at the cellular level by supplementation of CA. Tibiae ash % (Table 3) exhibited significantly (P < 0.05) low ash content in treatment T 2 compared to control T 0 and T 1 as well as treatment T 3 . Furthermore, tibiae calcium and phosphorus content were significantly (P < 0.05) high in treatment T 2 and T 3 . Results for the carcass characteristics revealed significantly (P < 0.05) higher dressing % (Table 3) in 0.5% CA-supplemented treatment compared to control T 0 with recorded 7.76%; 7.33%; and 4.12% higher dressing yield in T 1 , T 2 , and T 3 respectively. The average value for leg quarter (% live weight), thigh (% live weight), and drum stick (% live weight (Table 3) did not exhibit any difference among T 1 , T 2 , and T 3 but the average value for fore/breast quarter was significantly (P < 0.05) higher compared to control group T 0 .

Discussion
Results of present study are relatable with the experiment of Chowdhury et al. (2009) who reported that supplementation of CA at 0.5% in the corn-soybean-based basal diet of 161 1-day-old broiler Hubbard Classic chicks had positive effects on growth, feed intake, feed efficiency, carcass yield, bone ash, and immune status of broilers; and addition of citric acid also reduced the pH of the formulated diets, whereas Fattah et al. (2008) reported that addition of any level and source of organic acids to the basal diet of 189 1-day-old Hubbard broiler chicks increased feed digestion and absorption as a result of increasing relative pancreas weight and 3.5 b 2.5 b 6.5 a 1.5 b .5 0.001 Bacilli (cfu) × 10 7 16.5 a 17.5 a 13.5 b 18.5 a .5 0.001 Tropical Animal Health and Production (2023) 55:33 small intestine density. CA is a weak acid and is dissociated at a faster rate in low pH in the upper part of the GIT (gastrointestinal tract) activating pepsinogen and other zymogens by adjusting gastric acidity closer to that required for optimal activity resulting in increased enzyme activity, improved digestion of proteins and other nutrients as well (Jongbloed et al. 2000). In our study, no significant (P > 0.05) change in pH of intestinal contents in different segments of intestine was recorded (Table 4). A report by Hume et al. (1993) on metabolism of organic acid in broiler chicks, when given by gavage to determine its chemical fate and distribution among organs and tissues concluded that dietary organic acid is metabolized and absorbed in the foregut and does not reach the intestine or ceca in appreciable amounts, since organic acids are very readily absorbed in the upper part of the GIT, explaining the reason for lack of pH reduction in the lower part of the GIT. Moderately reducing the level of Ca in feed has influenced the growth and FCR. Higher level of Ca in diet causes increase in gastric pH as limestone, as a source of calcium carbonate (Table 1) which is considered to have high acid binding capacity (Lawlor et al. 2005) thus, affect/reduce the protein digestion by reducing the action of pepsin (Walk et al. 2012). Similarly, increasing concentrations of Ca in feed has also been attributed to influence the apparent digestibility of fats by soap formation with fatty acids enhancing its excretion (Ruvini K. et al. 2014). In the present study, digestibility studies revealed non-significant but numerically higher ether extract digestibility in broiler birds offered moderately low Ca (Table 3). Moreover, CA is a weak acid being an essential component of the citric acid cycle, releases energy for physiological functions (Wright, 1976) thus contributing to the net energy availability. Adil S. et al. (2011) also reported that addition of organic acids to the diets of broiler chicken significantly decreased (P < 0.05) the caecal viable coliform counts as compared to the unsupplemented group. Tollba (2010) observed that addition of CA in the feed of 1-week old Hubbard broiler chicks had statistical effects (P < 0.05) regarding the decrease in the counts of pathogenic intestinal bacteria and parasites in ileum, caecum, or fecal matter of birds. Furthermore, it also significantly improved body weight gain, feed consumption, feed conversion, carcass characteristics and mortality rate, thereby leading to an improved gut health parameters and increase in the availability of nutrients. According to Adil S. et al. (2011) modulation of intestinal microbial population beneficially affects the host by positive impact on histology of intestine, thereby enhancing digestion and absorption of nutrients. Katoch et al. (1996) reported higher growth performance and carcass yield in broiler birds with a shift in microbial population by supplementation with direct fed microbial particularly acid producing bacteria. Shambhavi et al. (2020) also reported similar finding with supplementation of direct fed microbial on azolla-based feed to 1-day-old broiler chicks enhancing the positive effect on carcass characteristics of broilers, by enhancing digestion and eventually resulting in extra availability of nutrients for deposition. This microflora has the flexibility to convert the retrograde uric acid (which is being carried to the caecum and coprodeum) to amino acids. Moreover, role of caecum is also vital in amino acids absorption and thus has larger ability to move amino acids towards higher deposition in fore/breast quarter. The results were evident in the present study with significantly (P < 0.05) high fore/ breast weight and dressing % ( The blood Ca level is maintained within very narrow limits by several hormones that control Ca absorption and excretion, as well as bone metabolism. Homeostasis by endocrine regulation (through the action of calcitonin and parathyroid hormone, among other hormones) could be responsible for maintaining the constant Ca and P concentrations in blood, although dietary components influence mineral balance affecting Ca blood levels (Taylor and Dacke. 1984). In the present study, tibial bone Ca and P percent was significantly (P < 0.05) higher in treatment T 2 and T 3 . The results obtained reveal that CA supplementation at 0.5% in low and moderately low Ca and TP ration increase deposition of Ca and P in tibial bone. Earlier report also (Swaminathan et al. 1978) suggests that efficiency of Ca absorption is enhanced in low Ca diets by a positive response of birds to Ca restriction by increased production of 1,25-dihydroxycholecaliferol in the intestine (Edelstein et al. 1975) which activates the expression of Ca binding protein (DeLuca and Schnoes, 1976). Related results have been reported earlier by Brenes et al. (2003) whereas Boling et al. (2001) observed that the CA did not significantly affect the Ca but CA increases P utilization in corn-soybean meal diets and reduces the available P requirement by approximately 0.10% of the diet.
Addition of CA at 0.5% level exerted hypocholesteraemia effect in all the treatment groups. Probable reason for reduced serum cholesterol may be that lactic acid bacteria grow at low pH in intestine and are more resistant to organic acids as compare to E-coli. This lactic acid bacteria produce bile salt hydrolase (BSH) in the intestine which is responsible for bile salt deconjugation. Deconjugated bile acids are less soluble at low pH and less absorbed in the intestine and are more likely to be excreted in the feces, and to maintain bile salt homeostasis, more bile acids need to be synthesized and this in turn will reduce cholesterol in the body pool as cholesterol is the precursor for bile acids. (Haq et al.2014). Tollba (2010) also reported significant reduction in cholesterol when CA was used at the rate of 2% in broilers feed.
CA supplementation in diet with moderately low Ca and TP content exhibited highest EEF of 301 and lowest cost of feed for every 1-kg live weight gain. Economics of broiler feeding revealed that 0.5% CA supplementation fetched highest gross return above feed cost with moderately low Ca and TP content whereas lowest profit was observed in feed with low content of Ca and TP (Table 5).
In conclusion, addition of CA (0.5%) in broiler birds offered corn soya-based feed with low and moderately low Ca and TP content enhanced the overall growth and improved carcass characteristics. Economics of broiler feeding with moderately low Ca and TP content revealed highest profit with CA (0.5%) supplementation. Further validation is needed in the commercial set up to realize the potential gain in increased commercial scale to reap the benefits in terms of economics as well as the unnecessary dependence of antibiotics for growth, prevalent in commercial farming in the region.

Data Availability
The authors confirm that the data supporting the findings of this study are available within the article. Raw data supporting the findings of this study is available from the corresponding author, upon request.

Declarations
Ethics approval All the animal experimentation procedures were approved by the Institutional Animal Ethics Committee (CSKHP Agriculture University), Palampur, HP, India). The study has been performed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki and its later amendments.

Consent for publication
All the authors give their consent for publication of the article.

Conflict of interest
The authors declare no competing interests.