Application of dietary organic acids in laying hens production: a comprehensive review

Formic acid and salts of butyric, propionic, and lactic acids supplemented in 53-week-old White Lohmann LSL hens for 16 weeks increased body weight and HDEP, alongside increases in EM and better FCR, while FI remained unchanged [5]. Similarly, formic acid supplementation in drinking water for 68-week-old Hy-Line W-98 White Leghorns under heat stress increased HDEP and EW and improved FCR [23]. The positive effects were attributed to enhanced digestive enzyme activity, microbial phytase activity, and pancreatic secretions [19,24]. A multi-acid mixture containing formic, lactic, malic, citric, and tartaric acids in 32-week-old W-36 layers improved EW [25]. Another mixture (15% formic acid, 14% acetic acid, 7% propionic acid, etc.) in 73-week-old Shaver 579 hens improved HDEP and FCR when provided via drinking water [26]. Acidifier blends containing formic, propionic, and acetic acids with cinnamaldehyde improved BW and weekly HDEP in long-term trials [27,28]. Overall, formic acid and multi-acid blends consistently improve HDEP, EW, and FCR, especially under heat stress, by enhancing enzyme activity, nutrient absorption, and gut health.

Acetic acid supplementation in drinking water (200, 400, and 600 ppm) in 30-40-week-old Brown Leghorn hens under heat stress (35°C) increased egg production and significantly improved EW at higher inclusion levels [29]. Since heat stress reduces serum and tissue mineral concentrations, thereby impairing egg production [30], acetic acid likely enhanced nutrient digestibility and helped maintain microbial balance. Additionally, silicic acid powder containing bamboo vinegar (rich in acetic acid), supplemented at 2-4 g/kg for 16 weeks in 25-week-old ISA Brown hens, significantly improved HDEP during the later weeks of lay [31]. Bamboo vinegar-derived acetic acid inhibits pathogenic bacteria and promotes beneficial microbiota [32], while silicic acid supports skeletal development [33] and collagen biosynthesis [34]. Collectively, acetic acid is particularly effective under heat stress conditions, improving HDEP and EW through enhanced mineral absorption and modulation of gut microbiota.

Dietary supplementation of 0.1% propionic acid, 0.2% butyric acid, and 0.3% acetic acid in 47-week-old Bowens hens increased FI [35], although no detailed mechanism was provided. Youssef et al. [36] evaluated the impact of an OAs mixture (0.06% fumaric acid, calcium formate, calcium propionate, potassium sorbate, and hydrogenated vegetable oil) on 27-week-old Hy-Line Brown hens over 12 weeks and reported improved HDEP and EM. A mixture of calcium-formate, calcium-propionate, calcium-lactate, and citric acid added to the diet of 75-week-old Hy-Line Browns reduced soft-shell and broken eggs while improving FCR [4]. These findings suggest propionic acid and its calcium salts support better nutrient solubility and gut balance, sustaining HDEP and reducing egg defects under stress conditions.

Sodium butyrate supplementation (0.05%, 0.10%, 0.20%) in 65-week-old Hy-Line Brown layers for 8 weeks significantly reduced broken egg percentage, with the highest inclusion showing the lowest breakage rates [37]. Butyrate supports mineral utilization, improving eggshell integrity, especially in older hens [38,39]. Calcium propionate and calcium butyrate supplementation (0.5%) in 24-week-old White Leghorns for 16 weeks significantly reduced FCR and increased EM [40]. However, in 70-week-old ISA Browns, FI decreased with the addition of calcium propionate or butyrate, while HDEP remained unaffected [41]. A microencapsulated OAs containing MCFAs (capric and caprylic acid) and butyric acid in 25-week-old Hy-Line Browns improved HDEP [42], likely due to antimicrobial effects that selectively alter gut microbiota. In summary, butyric acid enhances nutrient utilization and eggshell quality, while microencapsulated blends with MCFAs further improve laying performance via antimicrobial effects.

A mixture of citric, butyric, and fumaric acids under NPP-deficient conditions in 34-week-old Hy-Line W-36 hens improved HDEP and maintained EM, showing significant OAs×NPP interactions [43]. Citric acid can chelate calcium and prevent the formation of insoluble calcium-phytate complexes, thereby enhancing mineral bioavailability [13,27,44]. Similarly, fumaric acid inclusion with calcium formate/propionate in 27-week-old Hy-Line Browns increased HDEP and EM [36]. Citric acid-dextrose supplementation also increased HDEP and reduced FCR in APEC-infected ISA Browns [45], mitigating pathogen-induced performance decline. Together, citric and fumaric acids enhance mineral utilization, maintain laying performance under phosphorus-deficient diets, and mitigate the effects of pathogenic stress.

Kucukersan et al. [46] reported that supplementing humic acid (85% polymeric polyhydroxy acid at 30 and 60 g/ton) to 36-week-old Hybrid Hysex Brown hens for 16 weeks significantly enhanced HDEP, EW, and feed efficiency by stabilizing intestinal microbiota. Similarly, humic acid supplementation (100 or 200 mg/kg) for 24 weeks in 20-week-old Gimmizah hens increased HDEP and EW, while reducing the age at first egg, suggesting improved sexual maturity [47]. Benzoic acid (1,000–2,000 mg/kg) in 45-week-old Lohmann Pink-Shell hens for 16 weeks decreased EW at high doses [48], illustrating variability in acidifier responses due to dietary buffering and gut microbiota differences [49,50]. In contrast, caprylic acid (500–2,000 mg/kg) in 24-week-old Lohmann Browns improved BW, HDEP, EM, and feed efficiency by reducing bacterial competition for nutrients and minimizing toxic metabolites [6,51]. Overall, humic and caprylic acids enhance nutrient utilization and microbial stability, thereby improving overall laying performance. In contrast, benzoic acid exhibits dose-dependent effects on laying performance.

Formic acid and salts of butyric, propionic, and lactic acids were examined in 53-week-old White Lohmann LSL hens over 16 weeks, improving shell thickness and reducing the proportion of thin and broken shells at later stages of lay [5]. These improvements were attributed to enhanced calcium and protein deposition facilitated by better nutrient absorption. A mixture containing formic, lactic, malic, citric, and tartaric acids supplemented to 32-week-old W-36 hens improved albumen quality and eggshell parameters [25]. Another OAs blend (15% formic acid, 14% acetic acid, 7% propionic acid, and others) in 73-week-old Shaver 579 hens improved eggshell thickness and albumen percentage [26]. Formic acid supplementation in drinking water for 68-week-old Hy-Line W-98 Leghorns under heat stress improved shell thickness and HU, supporting shell quality and albumen freshness even under high temperatures [23]. Gul et al. [52] also reported improved yolk color in Lohmann Brown hens with formic acid mixtures. These studies suggest formic acid and blends enhance mineral utilization, albumen quality, and overall egg freshness.

Acetic acid supplementation in drinking water for 30-week-old Brown Leghorn hens under heat stress conditions improved albumen height, albumen percentage, and HU [29]. Since heat stress negatively affects protein metabolism [30], acetic acid likely helps maintain protein quality through better nutrient digestibility. Similarly, bamboo vinegar, rich in acetic acid, improved shell thickness and yolk color in 25-week-old ISA Brown hens [31]. Dietary bamboo vinegar inhibits pathogenic bacteria [32], while enhanced mineral utilization supports skeletal metabolism [33,34] and protein digestion [53], thereby contributing to better albumen and shell quality. Collectively, acetic acid helps maintain albumen and shell quality, especially under heat stress, by stabilizing gut microbiota and supporting protein metabolism.

A mixture of calcium-formate, calcium-propionate, calcium-lactate, and citric acid in the diet of 75-week-old Hy-Line Browns reduced the incidence of soft and broken shells [4]. Another combination of fumaric acid, calcium formate, calcium propionate, and potassium sorbate improved shell thickness and yolk color in 27-week-old Hy-Line Browns [36]. Propionic acid in such blends enhances nutrient solubility, resulting in improved shell integrity and yolk pigmentation.

Sodium butyrate supplementation in 65-week-old Hy-Line Brown hens improved eggshell strength and reduced the percentage of broken eggs [37]. This improvement was associated with improved mineral utilization and gut health [38,39]. A protected sodium butyrate diet improved shell thickness, eggshell percentage, and shell strength in Dekalb White hens, while also reducing yolk index values, indicating better protein incorporation into the eggshell membrane [54]. Encapsulated butyric acid and protected sodium butyrate in Lohmann Brown hens enhanced shell thickness and eggshell weight as a proportion of EW [55]. Calcium propionate and calcium butyrate supplementation in ISA Brown hens improved eggshell percentage, thickness, and strength [41]. These findings suggest that butyrate-based acidifiers enhance intestinal function and mineral absorption, resulting in stronger shells and improved shell quality.

Citric acid, by lowering gastrointestinal pH, enhances mineral solubility and phytase activity [27,56]. Co-supplementation of phytase with citric acid improved shell quality in Brown Nick layers on a low-phosphorus diet [57]. Fumaric acid inclusion in Hy-Line Browns improved shell thickness and yolk color [36]. Citric, butyric, and fumaric acids tested in Hy-Line W-36 hens improved shell thickness and yolk index under different phosphorus levels, suggesting better mineral utilization even under nutritional challenge [43]. Microencapsulated blends containing fumaric and citric acids in Hy-Line Browns improved HU and shell strength [42]. Together, citric and fumaric acids enhance calcium utilization and improve shell quality and albumen quality.

Humic acid supplementation in Gimmizah hens improved eggshell percentage and shell thickness, which was supported by higher plasma calcium levels [47]. Similarly, humic acid increased albumen index and HU in Lohmann Brown Lite hens [58]. Benzoic acid supplementation in Lohmann Pink-Shell hens improved albumen height and HU, suggesting better protein quality in the egg white [48]. However, responses to benzoic acid vary due to age, breed, and dietary factors, and the exact mechanisms underlying these variations remain unclear. MCFAs blends containing caprylic acid improved shell quality in Bovans Brown hens, enhancing shell-breaking strength, shell percentage, and shell density at different laying phases [59]. These effects were linked to better gut health and mineral availability. Overall, humic, benzoic, and caprylic acids improve both shell quality and albumen freshness by supporting calcium metabolism and protein utilization.

Supplementation with formic acid and its salts, including butyric, propionic, and lactic acids, has been shown to enhance blood protein profiles and mineral metabolism in laying hens [5]. In 53-week-old White Lohmann LSL hens, Soltan [5] observed that dietary inclusion of these acids for 16 weeks significantly increased serum total protein and albumin concentrations, likely due to improved gut health that facilitated the absorption of nitrogen and calcium. Similarly, sodium formate administered to hens exposed to heat stress increased plasma calcium and phosphorus concentrations while simultaneously reducing total lipid and cholesterol levels, suggesting improved mineral availability and modulation of microbial activity in the gastrointestinal tract [60,61]. These findings align with the observation that OAs blends, particularly those containing fumaric and citric acids, improved serum calcium concentrations in Hy-Line Browns by lowering intestinal pH and enhancing nutrient solubility [42]. Moreover, citric and fumaric acids have been linked to enhanced phytase activity, thereby improving the release and utilization of bound minerals [27,56,57].

OAs also play an essential role in regulating immune function and mitigating stress in laying hens. Formic acid supplementation in drinking water for 68-week-old Hy-Line W-98 hens under heat stress increased hemagglutination inhibition titers against Newcastle Disease, indicating enhanced systemic immune responses through the suppression of pathogenic gut microbiota [23]. Propionic acid-based blends have demonstrated similar immunomodulatory effects, increasing plasma anti-Salmonella IgA levels following vaccination while modulating IgG titers depending on the infection challenge [27]. Humic acid, when fed to Gimmizah hens, improved hematological parameters, including red and white blood cell counts, hemoglobin levels, plasma calcium levels, and total protein levels [47]. Furthermore, humic acid supplementation in high-density housing conditions significantly reduced heterophil counts and heterophil-to-lymphocyte ratios, while increasing lymphocyte counts, indicating reduced stress responses that may be associated with enhanced interleukin-2 signaling and reduced stress hormone secretion [62–64]. These results collectively suggest that OAs modulate both innate and adaptive immunity while mitigating environmental and social stressors.

Beyond their effects on minerals and immunity, OAs influence lipid and energy metabolism. Sodium formate and citric acid supplementation reduced total cholesterol and low-density lipoprotein (LDL) levels while increasing high-density lipoprotein (HDL) concentrations in Hy-Line W-36 hens, suggesting improved lipid profiles [43,60]. Benzoic acid exhibited similar lipid-lowering effects in Hy-Line White layers without altering yolk cholesterol levels, indicating systemic lipid regulation without affecting egg composition [48,65]. Moreover, acidification of the diet with fumaric, citric, and propionic acids was suggested to stimulate glycogenesis by increasing glucose-6-phosphate influx into glycogen synthesis pathways, possibly through the inhibition of glycolysis via elevated citrate levels [66,67]. Medium-chain fatty acids, such as caprylic acid, when combined with OAs blends, further enhance gut health, indirectly supporting serum mineral balance and overall metabolic stability [42,59]. Together, these findings highlight the role of OAs in optimizing lipid metabolism, reducing energy losses, and improving the overall metabolic resilience of laying hens.

Dietary OAs significantly influenced villus and crypt development in laying hens, primarily by lowering intestinal pH and modulating microbial colonization. Supplementation with mixtures containing formic, propionic, and citric acids in Lohmann Brown hens increased VH over a 10-week period [52], while increasing inclusion levels linearly enhanced VW and CD in Lohman layers [66,70]. These morphological improvements are associated with reduced intestinal colonization by both pathogenic and non-pathogenic bacteria, thereby lowering local inflammation and promoting nutrient absorption. Longer villi increase the absorptive surface area, improving digestive efficiency and nutrient uptake [71,72]. Similarly, supplementation of day-old layer chicks with an OAs blend of formic, propionic, and acetic acids plus cinnamaldehyde extended jejunal VH following Salmonella Enteritidis vaccination [27]. However, benzoic acid supplementation exhibited segment-specific effects, with duodenal VH and CD increasing at higher doses, while altering VH:CD ratios differently in the jejunum and ileum [48]. This variability may result from differences in benzoic acid formulation, dosage, or experimental conditions, and excessive inclusion levels could induce mild toxicity, impairing gut architecture.

Beyond villus morphology, OAs support epithelial cell proliferation and the development of the mucosal layer. Encapsulated butyric acid and protected sodium butyrate improved glandular layer thickness, VH, and CD in the ileum of Lohmann Brown hens over a 24-week period, with encapsulated butyric acid showing superior effects [55]. Butyrate serves as a direct energy source for epithelial cells, promoting mucosal repair and proliferation [73]. Supplementation with silicic acid powder containing bamboo vinegar in ISA Brown hens enhanced jejunum VH and increased villus areas in the duodenum and jejunum at optimal inclusion levels, likely by suppressing pathogenic bacteria and reducing villus atrophy [31]. These findings collectively indicate that OAs and their derivatives modulate gut morphology by maintaining a balanced microbial ecosystem, reducing intestinal inflammation, and providing metabolic substrates that enhance epithelial growth, ultimately improving nutrient absorption and intestinal health.

OAs supplementation significantly alters the composition and diversity of intestinal microbiota in laying hens by lowering gut pH and creating an environment favorable for beneficial bacteria. Sodium butyrate supplementation in Hy-Line Brown hens increased total anaerobic bacteria and Lactobacillus spp., which are essential for maintaining gut homeostasis through epithelial stimulation, immune enhancement, and nutrient metabolism [37,76]. Similarly, microencapsulated blends containing fumaric, citric, and malic acids along with MCFAs promoted Lactobacillus populations and reduced Escherichia coli counts in Hy-Line Browns [42]. In vitro analyses also showed that sodium butyrate shifted microbial populations toward carbohydrate fermenters such as Bacteroides and Faecalibacterium, while reducing populations of nitrogenous fermentation bacteria, including Desulfovibrio, Helicobacter, and Campylobacter, ultimately mitigating ammonia production [77]. Additionally, butyric, citric, and fumaric acids enhanced the abundance of beneficial Lactobacillus while simultaneously suppressing Salmonella in the ileum of Hy-Line W-36 hens [43]. These effects are attributed to the ability of OAs to penetrate bacterial cell membranes and disrupt intracellular metabolism, favoring probiotic populations over pathogenic species.

Beyond promoting beneficial microbiota, OAs directly inhibit pathogens and restore microbial balance. Acidifier supplementation containing formic, propionic, and acetic acids, combined with cinnamaldehyde, significantly reduced cecal Salmonella Enteritidis in vaccinated layer chicks and simultaneously increased Bifidobacterium populations at multiple post-challenge time points, supporting the competitive exclusion of pathogens [27,78–80]. Fumaric acid is particularly effective in suppressing coliforms and Salmonella while sparing beneficial Lactobacillus due to its slow dissociation rate and the inherent acid tolerance of Lactobacillus strains [81–84]. On the other hand, benzoic acid supplementation in Lohmann Pink-Shell hens exhibited dose-dependent effects; moderate inclusion enhanced the Firmicutes/Bacteroidetes ratio and enriched beneficial bacterial families such as Clostridiales and Lachnospiraceae, whereas higher doses reduced microbial richness and diversity and selectively enriched Bacteroides caecicola [48,85,86]. These findings underscore the importance of achieving optimal inclusion rates to maintain eubiosis and prevent potential dysbiosis. Overall, OAs act as selective antimicrobial agents, reducing pathogenic loads while supporting the growth of probiotic communities, thereby improving gut health and nutrient utilization in laying hens.

Supplementation with specific OAs improves calcium absorption and supports bone development in laying hens. Encapsulated butyric acid at 500 g/ton in Lohmann Brown hens increased tibia length compared with both the basal diet and higher inclusion of protected sodium butyrate, indicating reduced mobilization of bone calcium and enhanced mineral retention in the skeleton [55]. Similarly, boric acid supplementation, either alone or in combination with ascorbic acid, elevated tibia calcium content in Hy-Line White hens, highlighting boron’s role in stimulating the formation and maturation of the bone matrix required for optimal mineralization [65,87,88]. These results suggest that specific OAs and mineral-associated compounds can positively modulate bone composition by improving gastrointestinal calcium uptake and overall mineral utilization.

Citric, fumaric, and butyric acids also enhance phosphorus utilization by promoting phytate hydrolysis. In Hy-Line W-36 hens fed non-NPP-deficient diets, fumaric acid supplementation increased tibia ash content. In contrast, citric acid increased tibia phosphorus levels, demonstrating a significant interaction between OAs and NPP levels [43]. These effects are attributed to the acidification of the gastrointestinal tract, which creates a favorable environment for endogenous phytase activity and improves the release of phytate-bound phosphorus. Such mechanisms highlight the indirect benefits of OAs on bone mineralization, which is achieved through improved nutrient availability.

While some OAs enhance bone health, others may influence bone turnover differently depending on diet composition and age. Calcium butyrate supplementation in older Isa Brown hens decreased tibia calcium and phosphorus content, although tibia bone index and bending strength remained unaffected [41]. This reduction may be linked to increased medullary bone resorption during eggshell formation, a process that can be exacerbated by metabolic acids stimulating osteoclast activity through extracellular acidification, which lowers intracellular pH and destabilizes calcium [89–91]. These findings suggest that while OAs can enhance bone mineral metabolism under optimal conditions, specific organic acids and physiological demands, such as prolonged egg production, may alter their effects to promote increased bone turnover.