Effects of organic acids on growth performance and gut microbial composition in Japanese quail

D M Ridoy¹, P K Sarkar¹, M I Moon², B Bala², S Das³, F I Rume³, M S Alam⁴ and S K Fouzder¹

¹ Department of Poultry Science, Patuakhali Science and Technology University (PSTU), Babuganj, Barishal-8210, Bangladesh
prodip_bau@pstu.ac.bd
² Faculty of Animal Science and Veterinary Medicine, PSTU, Babuganj, Barishal-8210, Bangladesh
³ Department of Microbiology and Public Health, PSTU, Babuganj, Barishal-8210, Bangladesh
⁴ Department of General Animal Science and Animal Nutrition, PSTU, Babuganj, Barishal-8210, Bangladesh

Abstract

The increasing demand for safe meat and eggs has led the use of organic acids as alternatives to antibiotics in animal feed to ensure human health. The present study aimed to investigate the impacts of organic acids on the growth and gut microbial composition of Japanese quail in a dose-dependent manner. One hundred fifty (150) day-old Japanese quail chicks were randomly distributed to five treatments, with 10 birds per replication. The experimental treatment comprised with organic acid solutions at concentration of 0, 1, 2, 3, and 4 ml/L of water, respectively. The birds were provided handmade feed on an ad libitum basis. After 42 days of rearing, the final body weights of quails were 135.86, 145.01, 156.61, 141.42, and 151.96 g/bird for the organic acid solutions of 0, 1, 2, 3, and 4 ml/L of water, respectively. Organic acids significantly improved the final body weight and body weight grain in quails at 2 and 4 ml/L organic acid solution. A microbiological analysis was carried out to check the status of gut microflora in various organic acid treated groups. The total viable count (TVC) (CFU/ml) was 5.8×10⁷, 5.2×10⁶, 4.2×10⁶, 4.8×10⁶, and 4.0×10⁶ at organic acid concentrations of 0, 1, 2, 3, and 4 ml/L, respectively. The high growth performance of quails may be due to low pathogenic colonization and multiple health benefits associated with the addition of organic acids to the drinking water. No mortality was recorded in organic acid-treated groups, suggesting that organic acids could have a significant impact on the poultry industry.

Keywords: antibiotic alternatives, growth performance, gut health, organic acids, microbial load

Introduction

Poultry meat is of one of the vital sources of animal protein worldwide. Its demand has been increasing over times due to its diverse uses, affordability, balanced amino acid profile, and ease of digestibility (Kleyn and Ciacciariello 2021). The demand for lean meat such as poultry breast meat is also steadily rising as well. This growing demand needs boosting of poultry productivity. Use of antibiotic growth promoters (AGP) in diet is still an important strategy to accelerate poultry productivity and profitability. It is well established that AGP in poultry diets improves growth performance compared to antibiotic-free feed (Fernández Miyakawa et al 2024) by reducing organism load and the risk of illness, as well as promoting growth. But, antibiotic use in poultry diets contributed to develop antibiotic resistance (Kousar et al 2021). Therefore, concerning public health issues, the poultry industry has started to reduce its usages and is exploring alternative options (Azizi et al 2024).

It is difficult to raise poultry without antibiotics, particularly in hot-humid countries like Bangladesh, where antibiotic usages is widespread and antibiotic-free poultry production is becoming more challenging (Hasan et al 2021). Commercial poultry producers in Bangladesh indiscriminately use antibiotics showing the thumbs against the legislations on withdrawal period, either due to ignorance or getting maximum profit (Tasmim et al 2023). Sattar et al (2014) found residual effects of antibiotic in the thigh meat, liver, kidney, and breast meat of broilers.

Recently in Bangladesh, government and poultry industry are trying to focus the issues of maximum poultry productivity and profitability maintaining food safely, public health, animal and human welfare, environment, etc. for sustainable poultry production. As consumers become increasingly concerned about the effects of antibiotics and antimicrobial resistance, producing poultry meat without the use of antibiotics is essential to ensure safety of public health (Haque et al 2020). To address this issue, poultry nutritional biotechnology may play an immense role. It may enhance the efficiency of poultry production (Rafiq et al 2022). Scientists have started exploring the application of non-therapeutic substitutes as feed additives including organic acids, essential oils, prebiotics, probiotics, enzymes, herbs, and immune stimulants (Abd El-Hack et al 2022). Organic acid therapies, either single or a mixture, exhibit antibacterial functions, and they may be an alternative to antibiotics. Organic acids can influence growth performance, feed conversion ratio (FCR), body weight gain, egg production, gut health, and microbial load in the intestines of poultry (Khan et al 2022). While several studies have explored the effectiveness of organic acids in chicken production, their use in quail production has given less attention and remains unexplored. Therefore, the study aims to investigate the efficiency of organic acids and their impact on growth performance and gut microbiota in Japanese quail. The research emphasizes the potential of organic acid supplementation to promote safe poultry production.

Materials and methods

Ethical approval

We obtained ethical approval from our institutional ethical committee to handle and operate the experimental birds. The approval number was PSTU/IEC/2018/33(4), dated May 16, 2022.

Experimental birds and diets

A total of 150 Japanese quail chicks were reared at the departmental poultry farm of Patuakhali Science and Technology University, Babuganj, Barishal. Before chick arrival, the poultry shed and its premises were cleaned, washed, and disinfected properly to control insects, rodents and germs. Poultry farm equipment and instruments were also cleaned and disinfected properly. After drying, the farm equipment and materials were placed in the shed. The chicks were brooded for two weeks in an open sided house and provided feed and water on ad libitum basis. Thereafter, all birds were weighed individually and distributed randomly to five dietary treatment groups in cages having 10 quails in each group with 3 replications each for a period of 4-weeks. Hand-mixed mash ration was formulated using the locally available feed ingredients, following the ration used in our previous experiment (Sarkar et al 2023). The formulated ration was analyzed in the laboratory of the Department of Livestock Services, Dhaka (Table 1 and Table 2). Rations were supplied twice a day. Feed was provided ad libitum to the birds in each replication per day. We also did microbiological analysis in the laboratory of the Department of Microbiology and Public Health, PSTU.

Preparation of organic acid solutions

Organic acid liquid was mixed with water. The chemical composition of organic acids is citric acid 2.5%, sodium formate 8.67%, formic acid 17.55%, acetic acid 7.6%, lactic acid 3.75%, propionic acid 2.31%, ammonium propionate 8.32%, and moisture up to 100%. Organic acids were provided to birds at the doses of 0, 1, 2, 3 and 4ml/L of water.

Housing management

Quails were reared in cage and the birds, with the cage space of 30 cm × 30 cm for 10 birds. The droppings were collected on trays. Dirt, droppings, waste feed and cage were cleaned twice in a week. The feeders and drinkers were cleaned regularly in accordance with the previous research by Sarkar et al 2008. The ambient temperature and humidity varied based on environmental conditions and ranged from 26.1 to 35.4 ℃ and 61 to 89%, respectively during the experimental period. The farm fences were prepared with nets to facilitate smooth ventilation. Proper biosecurity was maintained throughout the entire experimental period.

Feeder and drinker management

The drinkers and feeders were cleaned every morning. The feces and feed that felt into the drinker were removed and cleaned the drinker using fresh water. Feeders were cleaned and the old feeds and droppings were removed by using a brush.

Body weight measurement

The birds were weighed on a weekly basis at 10 AM every Thursday for 6 weeks. The initial body weight and weekly body weight of each replicate were recorded to calculate the body weight gain and final body weight of quails.

Measurement of water quality

The water quality was measured after mixing experimental doses of organic acids into water with a water quality tester (model: EZ-9909SP, China). Different concentrations of organic acids (0, 1, 2, 3, and 4 ml/L of water) in water were prepared and the water quality was assessed. Initially, 6.86, 4.00, and 9.18 pH solutions were used to calibrate the water quality tester. After that, the electrode was submerged in different concentrations of organic acids (0, 1, 2, 3, and 4 ml/L of water) to measure the pH, salinity (%),total dissolved solids (TDS) (ppm), temperature (°C), and electrical conductivity (EC) (μS/cm).

Microbiological analysis

Birds from each replication were sacrificed for microbiological analysis. After slaughtering, the blood was allowed to drain out, and the guts with their contents were immediately kept in ice box and were sent to the Microbiology and Public Health Laboratory, PSTU. The gut fluids were collected by squeezing the guts and the samples were stored at 4°C overnight. Then, the frozen samples were thawed at room temperature for 3-5 hours. For isolation, identification and characterization of bacteria, the bacteriological media such as Nutrient agar, Eosin-Methylene-Blue agar, Blood agar, Plate count agar, Salmonella-Shigella (SS) agar base, Brilliant Green agar powder, and MacConkey (MC) agar, Nutrient Broth and Methyl-Red Voges-Proskauer (MR-VP) broth etc. were used in this experiment. The total viable bacterial count of the gut microbiota was determined, and Escherichia coli and Staphylococcus spp. were isolated and identified based on their morphological, cultural, staining, hemolytic, and biochemical properties, following the methods described by Chessbrough (1985).

Data analysis

Data were analyzed with the help of IBM SPSS version 20. Differences between treatments were examined with the help of Tukey's honestly significant difference test as described by our previous research (Khan et al 2023). The significance level was set at p<0.05.

Results

Growth performance in Japanese quail

At the end of 6^th week of rearing, the final body weight of quails differed significantly among the treatment groups. The final body weights of Japanese quail were 143, 152, 163, 148 and 159 g/bird at organic acids concentrations of 0, 1, 2, 3 and 4 ml/L of water, respectively (table 3). The weight gain of birds followed similar trend. Final body weight gain was 135, 145, 156, 141, and 151 g/bird at organic acid concentrations of 0, 1, 2, 3 and 4 ml/L water, respectively.

Figure 1. Body weight gain (BWG) of quails at different concentrations of organic acids	Figure 2. Final body weight of quails at different concentrations of organic acids

Figure 3. Feed conversion ratio of quails at different concentrations of organic acids

Figure 1 and figure 2 illustrates the gradual increase in the body weight and final body weight of quails with the increasing doses of organic acids up to 2 ml/L, respectively. However, no significant improvement in body weight was observed when an organic acid concentration of 3ml/L was added. Notably, the body weight of the birds increased significantly (p <0.05) at concentration of 2 ml/L and 4 ml/L.

Feed intake and FCR

The findings of feed intake and feed conversion ratio in quails are presented in Table 3. After 6 weeks rearing period, the feed intake was 585.75, 578.25, 564.75, 549.15, and 552.15 g/bird, corresponding to feed conversion ratios of 4.1, 3.8, 3.45, 3.70, and 3.47 for the dietary groups of 0, 1, 2, 3 and 4 ml/L, respectively (Figure 3).

Mortality

No mortality was observed during the experimental period.

Microbial load

Microbial load in the gastrointestinal tract is presented in Table 4. The total viable counts were 5.8×10⁷, 5.2×10⁶, 4.2×10⁶, 4.8×10⁶, and 4.0×10⁶ CFU/ml of intestinal fluid content when organic acids were applied at concentration of 0, 1, 2, 3, and 4 ml/L, respectively. E. coli was not found in any of the groups, and Staphylococcus was present only in the control group.

Water quality

The water quality at different concentrations of organic acids is presented in table 5. The pH of water decreased with the increased concentrations of organic acid solutions. The pH values of water were 7.11, 5.8, 4.59, 4.47 and 4.25 at organic acid concentrations of 0, 1, 2, 3, and 4 ml/L, respectively. Both TDS and EC increased with higher doses of organic acids solutions. TDS values were 993.33, 1113.33, 1303.33, 1426.67 and 1650 while the EC values were 2016.67, 2233.33, 2600, 2866.67 and 3316.67, respectively at organic acid concentrations of 0, 1, 2, 3, and 4 ml/L. The salinity of water ranged from 0.1-0.16 at various concentrations of organic acids.

Discussion

In the current study, the growth of Japanese quail was significantly improved in the organic acids treatment groups compared to the control group. Similarly, R. U. Khan et al (2016) observed significantly higher body weights in quails fed organic acids after 42-day trial. Fikry et al (2021) also reported a significant impact of organic acids on growth performance, feed intake and feed conversion in Japanese quail. Several studies have reported that feed intake is higher in the control group compared to the organic acids treatment group, while the organic acids treatment groups showed better FCR than the control group. In another study, Reda et al (2021)found higher feed intake in quail in the control group, leading to low feed conversion efficiency compared to organic acids treatment groups. There are several factors responsible for influencing the growth performance. Among the factors, gut health is one of the most important considerations for achieving feed efficiency and growth in birds. Research findings have shown that supplementation of organic acids in feed or water improves the height and width of villus, and surface area for nutrient absorption. According to Aliverdi-Nasab et al (2023) supplementation of formic acid resulted in longer villi in quails. Another reason for improved growth is the impact of organic acids on nutrient digestibility. Ghazalah et al (2011) observed that the inclusion of fumaric or formic acid at a concentration of 0.5%, as well as acetic or citric acid at concentrations of 0.75% and 2% in the diet, led to enhancements in both metabolizable energy (ME) and the digestibility of nutrients in broilers. Castillo et al (2014) stated that the organic acids enhance the breakdown of proteins in younger animals by promoting the release of enzymes from the pancreas. Organic acids also increase gastric proteolysis; thereby enhance protein and amino acids digestibility in birds. Additionally, protein digestibility increases when the pH of the chyme decreases due to supplementation of organic acids. Furthermore, the organic acids supplements enhances digestibility of crude protein and ME by decreasing microbial competition of microbes with the host, reducing the loss of nitrogen from the body, and lowering ammonia production (Khan and Iqbal 2016).

In present study, we observed that the microbial load is lower in the organic acids treated groups than control. It was also found that Staphylococcus spp. was only present in the control group. Many researchers also reported the effects of organic acid in reducing bacterial populations in the gut of birds. Açikgöz et al (2011) observed the total counts and E. coli populations were higher in control group compared to acidified water groups. Short-chain acids, among all the organic acids, show the strongest antibacterial effects, which can enhance productivity in poultry. Antimicrobial action of organic acids is closely related to their dissociation properties. Upon penetrating the cell, the acid dissociates, and releases H+ due to the internal pH being greater than the pKa. The cell utilizes a substantial quantity of energy to eliminate the H+ ions, leading to cell death. Additionally, the RCOO- ions generated from the acids interact with bacterial DNA, inhibiting microbial growth (Abd El-Ghany 2024).

Adding organic acids to drinking water also changes the composition of water such as pH, TDS, salinity, electrical conductivity etc. In our study, the pH of untreated water was 7.11 while the pH of organic acids treated water ranged from 5.8 to 4.25. Ali et al (2020) also showed that the pH of untreated water was 7.6 while a lower pH of water treated with organic acids ranged from 2.5 to 3.7. The reduction of pH of water after adding acids is dependent on the dissociation capacity of acid. In essence, acid dissociation is the release of H+, which lowers pH and produces the acidifier effect. We observed TDS value increased linearly as the amount of organic acid increased. There is a positive relationship between conductivity and TDS. As TDS influences pH, conductivity also increases and pH decreases (Islam et al 2017).

Conclusion

The supplementation of organic acids in water improves growth performance in Japanese quail. Specifically, it improves growth rate, final body weight, and feed conversion ratio. The microbial load is also lower in organic acids treated groups compared to control. The growth benefit is likely due to low microbial load in the gut and multiple health benefits associated with the using of organic acids in poultry. Therefore, organic acids could play an immense role in the poultry industry to produce safe poultry.

Acknowledgments

The authors express gratitude to the Research and Training Centre, Patuakhali Science and Technology University, who financed the research.

Conflict of interest

The authors declare no conflict of interest.

Authors’ contribution

Dip Majumder Ridoy conducted the experiment, collected the data, and wrote the manuscript. Prodip Kumar Sarkar planned the experimental design, analyzed the data, prepared graph, tables, and contributed to writing the manuscript. Mehedi Islam Moon, Bishnu Bala, Smita Das and Farzana Islam Rume conducted the experiment, and collected the data. Muhammad Shahbubul Alam and Swapon Kumar Fouzder reviewed the manuscript. All the authors confirmed the data and approved the final manuscript.

References

Abd El-Ghany W A 2024 Applications of organic acids in poultry production: an updated and comprehensive review. Agriculture (Switzerland), 14(10). https://doi.org/10.3390/agriculture14101756

Abd El-Hack M E, El-Saadony M T, Salem H M, El-Tahan A M, Soliman M M, Youssef G B A, Taha A E, Soliman S M, Ahmed A E, El-kott A F, Al Syaad K M and Swelum A A 2022 Alternatives to antibiotics for organic poultry production: types, modes of action and impacts on bird’s health and production. Poultry Science, 101: 4. https://doi.org/10.1016/j.psj.2022.101696

A çikgöz Z, Bayraktar H and Altan Ö 2011 Effects of formic acid administration in the drinking water on performance, intestinal microflora and carcass contamination in male broilers under high ambient temperature. Asian-Australasian Journal of Animal Sciences, 24(1). https://doi.org/10.5713/ajas.2011.10195

Ali A M, El Agrab H M, Hamoud M M, Gama A M, Mousa M R, Nasr S A E, El Shater M A H, Laban S E, Zahran O K and Ali M M 2020 Effect of acidified drinking water by organic acids on broiler performance and gut health. Advances in Animal and Veterinary Sciences, 8(12). https://doi.org/10.17582/journal.aavs/2020/8.12.1301.1309

Aliverdi-Nasab K, Zhandi M, Yousefi A R, Zahedi V and Rafieian-Naeini H R 2023 The effect of acidifier supplementation on egg production performance and intestinal histology of Japanese quail (Coturnix japonica). Veterinary Medicine and Science, 9(1). https://doi.org/10.1002/vms3.1043

Azizi M N, Zahir A, Mahaq O and Aminullah N 2024 The alternatives of antibiotics in poultry production for reducing antimicrobial resistance. World’s Veterinary Journal, 14(2), 270–283. https://doi.org/10.54203/scil.2024.wvj34

Castillo S, Rosales M, Pohlenz C and Gatlin D M 2014 Effects of organic acids on growth performance and digestive enzyme activities of juvenile red drum Sciaenops ocellatus. Aquaculture, 433. https://doi.org/10.1016/j.aquaculture.2014.05.038

Cheesbrough M 1985 Medical laboratory manual for tropical countries. Vol. II. Microbiology. pp. 400-480.

Fernández Miyakawa M E, Casanova N A and Kogut M H 2024 How did antibiotic growth promoters increase growth and feed efficiency in poultry? Poultry Science, 103(2). https://doi.org/10.1016/j.psj.2023.103278

Fikry A M, Attia A I, Ismail I E, Alagawany M and Reda F M 2021 Dietary citric acid enhances growth performance, nutrient digestibility, intestinal microbiota, antioxidant status, and immunity of Japanese quails. Poultry Science, 100(9). https://doi.org/10.1016/j.psj.2021.101326

Ghazalah A A, Atta A M, Elkloub K, Moustafa M E L and Shata, R F H 2011 Effect of dietary supplementation of organic acids on performance, nutrients digestibility and health of broiler chicks. International Journal of Poultry Science, 10(3). https://doi.org/10.3923/ijps.2011.176.184

Haque M H, Sarker S, Islam M S, Islam M A, Karim M R, Kayesh M E H, Shiddiky M J A, Anwer M S 2020 Sustainable antibiotic-free broiler meat production: Current trends, challenges, and possibilities in a developing country perspective. Biology (Basel) (Vol. 9, Issue 11). https://doi.org/10.3390/biology9110411

Hasan S M M, Sherer P P, Sakcamduang W, Das S, Islam M S and Sringernyuang L 2021 Antibiotic use in commercial poultry production in bangladesh: Stakeholders roles and dependencies for antibiotic transaction. Journal of Public Health and Development, 19(3). https://he01.tci-thaijo.org/index.php/AIHD-MU/article/view/251545

Islam R, Faysal M, Matin Juliana F, Johirul Islam M, Jahangir Alam M, Islam R, Md Faysal S, Ruhul Amin M, Nazir Hossain M and Asaduzzaman M 2017 Assessment of pH and total dissolved substances (TDS) in the commercially available bottled drinking water. IOSR Journal of Nursing and Health Science, 6(5).

Khan R U, Chand N and Ali A 2016 Effect of organic acids on the performance of Japanese quails. Pakistan Journal of Zoology, 48(6).

Khan R U, Naz S, Raziq F, Qudratullah Q, Khan N A, Laudadio V Tufarelli V and Ragni M 2022 Prospects of organic acids as safe alternative to antibiotics in broiler chickens diet. In Environmental Science and Pollution Research (Vol. 29, Issue 22). https://doi.org/10.1007/s11356-022-19241-8

Khan S H and Iqbal J 2016 Recent advances in the role of organic acids in poultry nutrition. Journal of Applied Animal Research, 44(1). https://doi.org/10.1080/09712119.2015.1079527

Khan S U, Fouzder S K and Sarkar P K 2023 Productivity and profitability of commercial broiler chickens under various farming conditions. Journal of Experimental Biology and Agricultural Sciences, 11(1), 209–215. https://doi.org/10.18006/2023.11(1).209.215

Kleyn F J and Ciacciariello M 2021 Future demands of the poultry industry: will we meet our commitments sustainably in developed and developing economies? In World’s Poultry Science Journal (Vol. 77, Issue 2). https://doi.org/10.1080/00439339.2021.1904314

Kousar S, Rehman N, Javed A, Hussain A, Naeem M, Masood S, Ali H A, Manzoor A, Khan A A, Akrem A, Iqbal F, Zulfiqar A, Jamshaid M B, Waqas M, Waseem A and Saeed M Q 2021 Intensive poultry farming practices influence antibiotic resistance profiles in Pseudomonas aeruginosa inhabiting nearby soils. Infection and Drug Resistance, 14. https://doi.org/10.2147/IDR.S324055

Rafiq K, Tofazzal Hossain M, Ahmed R, Hasan M M, Islam R, Hossen M I, Shaha S N and Islam M R 2022 Role of different growth enhancers as alternative to in-feed antibiotics in poultry industry. In Frontiers in Veterinary Science (Vol. 8). https://doi.org/10.3389/fvets.2021.794588

Reda F M, Ismail I E, Attia A I, Fikry A M, Khalifa E and Alagawany M 2021 Use of fumaric acid as a feed additive in quail’s nutrition: its effect on growth rate, carcass, nutrient digestibility, digestive enzymes, blood metabolites, and intestinal microbiota. Poultry Science, 100(12). https://doi.org/10.1016/j.psj.2021.101493

Sarkar P K, Chowdhury S D, Kabir M H and Sarker P K 2008 Comparative study on the productivity and profitability of commercial broiler, cockerel of a layer strain and cross-bred (RIR × Fayoumi) chicks. Bangladesh Journal of Animal Science. https://doi.org/10.3329/bjas.v37i2.9886

Sarkar P K, Ridoy D M, Moon M I and Fouzder S K 2023 Use of probiotics for safe quail meat production. Turkish Journal of Agriculture - Food Science and Technology, 11(12). https://doi.org/10.24925/turjaf.v11i12.2402-2406.6473

Sattar S, Hassan M M, Islam S K M A, Alam M, Faruk M S Al, Chowdhury S and Saifuddin A K M 2014 Antibiotic residues in broiler and layer meat in Chittagong district of Bangladesh. Veterinary World, 7(9). https://doi.org/10.14202/vetworld.2014.738-743

Tasmim S T, Hasan M M, Talukder S, Mandal A K, Parvin M S, Ali M Y, Ehsan M A and Islam M T 2023 Sociodemographic determinants of use and misuse of antibiotics in commercial poultry farms in Bangladesh. IJID Regions, 7. https://doi.org/10.1016/j.ijregi.2023.01.001