Delving into vitamin A supplementation in poultry nutrition: current knowledge, functional effects, and practical implications

SUMMARY Vitamin A, also known as retinol, is crucial for maintaining various physiological functions, including vision, immunity, epithelial cell growth and differentiation, reproduction, and bone development. Discussions surrounding retinol in commercial poultry farming have recently emerged due to its significant biological importance, as well as concerns regarding its cost and susceptibility to oxidation in both pure form and premixes. Insufficient vitamin A in avian species can lead to reduced performance, increased susceptibility to infectious diseases, and reproductive disorders. Determining the optimal supply of vitamin A poses a challenging task. Poultry’s requirements for retinol depend on factors such as physiological state, age, health, nutritional status, and function. Scientific committees like the National Academies of Sciences, Engineering, and Medicine (NASEM, formerly National Research Council) or Gesellschaft für Ernährungsphysiologie (GfE) provide requirement estimates (NASEM) or allowances (GfE) as foundations for practical considerations. However, when applied in commercial farming scenarios, extrapolating requirement estimates and allowances from controlled research environments may entail intrinsic limitations. Therefore, developing a comprehensive understanding of the effects of supplemental vitamin A on poultry health and metabolism is essential for formulating balanced diets and achieving optimal performance in commercial settings. The objective of this review is to provide an in-depth analysis of this complex and relatively unexplored topic.


Introduction
Vitamin A holds tremendous practical importance and is considered one of the most crucial vitamins (McDowell 2000).The term 'vitamin A' encompasses three chemical compounds: retinol (the alcohol), retinal (the aldehyde), and retinoic acid (all-trans retinoic acid, ATRA) (Carazo et al. 2021).These different oxidation states of vitamin A, collectively known as retinoids, play a vital role in the physiology of the retina (Engelking 2015).Vitamin A fulfils essential roles in vision, immunity, reproduction, CONTACT Yauheni Shastak yauheni.shastak@basf.comBASF SE, Nutrition & Health Division, Business Unit Animal Nutrition, Ludwigshafen am Rhein 67063, Germany growth, and the differentiation of epithelial cells (EFSA 2013).In recent years, retinol has gained attention for its antioxidant function (Abd El-Wahab et al. 2017;Hao et al. 2021).The specific functions of vitamin A are summarised in Table 1.
Vitamin A is naturally found in fish liver oil, whole milk, fish meal, and other animalderived feeds.Additionally, certain plant pigments called carotenoids, such as β-carotene, can metabolically produce retinoids and exhibit vitamin A activity (Combs and McClung 2017).However, the contribution of carotenoids from feed to the vitamin A status of poultry is minimal, leading to the supplementation of synthetic retinyl acetate in commercial diets (Surai et al. 2003).Insufficient vitamin A intake can lead to developmental abnormalities, pathological changes in the skin and mucosa, vision impairments, and an increased susceptibility to diseases, including reproductive disorders (Carazo et al. 2021;Chen et al. 2015).Despite being one of the earliest discovered vitamins, the full range of biological activities and functions mediated by vitamin A is still being explored by researchers, and a better understanding is needed (Carazo et al. 2021;Chen et al. 2015).
There is a great deal of variation in the retinol requirements of poultry based on their physiological state, age, health, nutritional status, and function (McDowell 2006).Therefore, a variety of factors determine how much vitamin A and its metabolites such as 14-hydroxy-4,14-retro-retinol, 3,14-di-OH-retinol, S-4-oxo-9-cis-13,14-dihydro-retinoic acid or 11-cis-retinal are needed to maximise health and performance in domestic fowl (Ortega and Jastrzebska 2019;Schuchardt 2007).Scientific committees, such as GfE 1999 or NASEM 1994, provide nutrient requirement estimates and allowances that are based on the needs of healthy birds from controlled research environments (Taghinejad-Roudbaneh et al. 2013).Nutritionists generally consider the NASEM projections for vitamins as the minimum levels necessary to prevent clinical deficiency (McDowell 1989).However, it is important to note that these vitamin requirement estimates and allowances from scientific committees do not account for potential losses during storage and processing, which can be particularly significant for retinyl acetate (Hirai et al. 2023).According to McDowell (2006), the optimal level of vitamin supplementation is determined by achieving the best growth rate, feed utilisation, and overall health, including immune competency, while also ensuring adequate body reserves.
In this review, we present a comprehensive analysis of vitamin A metabolism in poultry.We also examine the effects of retinol supplementation on different  Fu et al. (2000), Bermudez et al. (1993), Chiba et al. (1996), Hong et al. (2013), Aydelotte (1963), Elvehjem and Neu (1932), Chandra et al. (1984), Bhuiyan et al. (2004) aspects of poultry production, such as reproductive performance, immune function, and antioxidant capacity.Additionally, we highlight important factors to consider in order to ensure adequate vitamin A supply for domestic fowl.Overall, this review aims to provide a well-rounded understanding of the subject matter.

Metabolism of vitamin A in poultry
In poultry diets, vitamin A is primarily present in the form of retinyl esters.The digestion and absorption of retinyl esters in domestic fowl involve several specific steps.Upon ingestion, fats and fat-soluble substances such as retinyl esters are released from the feed matrix through mechanical grinding and the acidic conditions in the proventriculus and gizzard (Shoaib et al. 2022).The subsequent release of retinyl esters from the feed particles allows for their solubilisation and subsequent hydrolysis.In the small intestine, pancreatic esterases produced by the pancreas act on retinyl esters, breaking them down into free retinol and fatty acids (Harrison and Hussain 2001).The process of retinyl ester hydrolysis is essential for the subsequent absorption of retinol into enterocytes.Once hydrolysed, free retinol is then solubilised in mixed micelles, which are formed through the interaction of bile acids, phospholipids, and cholesterol (Cohn et al. 2010;Green and Fascetti 2016).These mixed micelles facilitate the transport of retinol to the apical membrane of enterocytes, where it can be absorbed for further metabolism and utilisation in the body.The absorption of retinol predominantly occurs in the duodenum and upper jejunum of the small intestine in poultry (Hynd 2019;Krogdahl 1985;Noy and Sklan 1995).Specific transporters facilitate the uptake of retinol into enterocytes at the apical membrane.Key transporters involved in retinol absorption in avian species could include STRA6 (stimulated by retinoic acid gene 6) and/or RBPR2 (Retinol Binding Protein 4-receptor 2) (Alapatt et al. 2013;Jackson et al. 2023;Reijntjes et al. 2010).These transporters bind to retinol and mediate its translocation across the membrane.STRA6 and/or RBPR2 function as high-affinity receptors for retinol and facilitate its internalisation into enterocytes (Muenzner et al. 2013).
After being absorbed, retinol undergoes re-esterification within the enterocytes and becomes part of chylomicrons.These chylomicrons are subsequently transported through the lymphatic system and bloodstream, effectively carrying vitamin A to the liver (Carazo et al. 2021).As shown in wild avian species, in the hepatic system, specialised cells known as stellate cells store vitamin A in the form of retinyl esters (Senoo et al. 2012).Whenever necessary, retinyl esters undergo hydrolysis to liberate retinol, which is subsequently conveyed to specific tissues by plasma retinol-binding protein (RBP) through its intricate association with transthyretin (Dhokia and Macip 2021;Monaco 2000).
Once inside an avian cell, retinol is bound to cellular retinol-binding protein (CRBP) within the cytoplasm (Goda and Takase 1989).In chickens, CRBP acts as a carrier protein, ensuring the intracellular transport of retinol to specific cellular compartments where it is utilised (Yin et al. 2014).Within the cell, retinol can undergo various metabolic transformations to meet the cell's specific needs.
One critical pathway involves the oxidation of retinol to retinaldehyde, a crucial precursor for the synthesis of active forms of vitamin A. In mammals, this oxidation reaction is mediated by enzymes known as alcohol dehydrogenases (ADHs), specifically class I ADH and class IV ADH, which convert retinol to retinaldehyde (Kumar et al. 2012).However, research has shown that in avian species, a different enzyme structure is responsible for this conversion.Specifically, an NADP(H)-dependent aldo-keto reductase enzyme has been identified as the key player in avian retinol oxidation (Crosas et al. 2001).This highlights a divergence in the enzymatic mechanisms of retinol metabolism between mammals and avian species, underscoring the need for species-specific investigations in understanding the cellular metabolism of vitamin A in poultry.
Research conducted on chick choroids has demonstrated that retinaldehyde can undergo further metabolism to produce ATRA through the enzymatic activity of retinaldehyde dehydrogenases (RALDHs) (Harper et al. 2016).The primary isoforms responsible for this conversion are RALDH1 and RALDH2 (Harper et al. 2018).ATRA, which is the active form of vitamin A, functions as a ligand for nuclear retinoic acid receptors (RARs) and retinoid X receptors (RXRs).These receptors act as transcription factors that govern the expression of specific target genes in chickens (Seleiro et al. 1994).Upon binding to RARs and RXRs, ATRA forms heterodimers that can attach to specific DNA sequences known as retinoic acid response elements (RAREs).These RAREs are situated in the regulatory regions of target genes (Zhang et al. 2015).In vertebrates, the interaction between ATRA and RAR/RXR heterodimers leads to the activation or suppression of target genes, giving rise to diverse cellular responses (Cunningham and Duester 2015).
The gene regulatory effects of ATRA are extensive and have an impact on various physiological processes in poultry.For example, ATRA plays a vital role in growth by facilitating cellular proliferation and differentiation (Chen and Ross 2004).It stimulates the progression of cells through the cell cycle, particularly the G1 phase, by activating genes that support cell division and suppressing genes associated with cell cycle arrest (Qiu et al. 2020).In quail embryos, Kim et al. (2021) demonstrated that ATRA enhances hypertrophic fat accretion, suggesting significant roles of this vitamin A form in the embryonic development of adipose tissues.Through the activation of specific genes and signalling pathways, ATRA promotes the differentiation of stem cells into specialised cell types, thereby enabling the formation of complex tissues and organs (Gudas and Wagner 2011).Moreover, ATRA serves as a crucial morphogen.By modulating gene expression patterns, ATRA contributes to shaping the overall structure and organisation of developing tissues, ensuring proper growth and morphogenesis (Dubey et al. 2018).
Aside from its involvement in cellular differentiation, ATRA plays a crucial role in the growth and development of bones.Research conducted in avian systems has demonstrated that ATRA influences the production and activation of osteoblasts, which are responsible for the formation of new bone tissue (Chiba et al. 1996).Furthermore, ATRA is involved in the regulation of bone resorption, the natural process of breaking down old bone tissue and replacing it with new tissue (Yee et al. 2021).The available evidence suggests that ATRA promotes periosteal bone resorption by increasing the RANKL/OPG ratio through the activation of RARα receptors.The RANKL/OPG ratio refers to the balance between two proteins involved in bone remodelling: receptor activator of nuclear factor kappa-B ligand (RANKL) and osteoprotegerin (OPG) (Grimaud et al. 2003).Bone health impacts gait by affecting stability, shock absorption, and alignment during movement, potentially leading to compromised mobility and balance.The results of a joint study from Mansoura, Hannover, and Cloppenburg suggest that low vitamin A levels in the diet may be a major cause of abnormal gaits in turkeys (Abd El-Wahab et al. 2017).
In the realm of vision, the significance of vitamin A in form of retinal or retinaldehyde lies in its pivotal role as an integral element of rhodopsin, the visual pigment.As demonstrated in chicken, rhodopsin is primarily located in the retinal rods and holds critical importance for facilitating vision in low-light conditions (Szél et al. 1985).Interestingly, the term 'chicken eyes' is often used to describe night blindness in humans, based on a misconception that chickens lack rods and therefore cannot see well in lowlight situations.However, it is important to note that chickens do possess rods, but their visual system is primarily adapted for daylight vision rather than low-light conditions.When light impinges on the retina, it triggers a transformative alteration in the configuration of rhodopsin, setting in motion a cascade of signalling events that ultimately culminate in the perception of visual stimuli (Palczewski 2014).Rhodopsin itself consists of two essential constituents: opsin, a protein, and 11-cis-retinal, a chromophore derived from retinol (Miyazono et al. 2011).
Moreover, the primary active form of vitamin A, known as ATRA, plays a crucial role in the development and maturation of the reproductive system.It is also essential for modulating the immune response.Furthermore, ATRA is critical for enhancing the antioxidative capacity of the body.In the following sections, we will delve into the specific mechanisms through which supplementary dietary vitamin A affects these functions.

Effect of vitamin A supplementation on immune function
In 1928, Green and Mellanby (1928) made a groundbreaking discovery by identifying the crucial role of vitamin A in modulating the immune system.This significant finding resulted in the recognition of vitamin A as 'the anti-infective vitamin'.Today, retinol stands out as one of the most extensively researched micronutrients in relation to immune function (Gürbüz and Aktaç 2022).
When vitamin A is available in adequate amounts, its active form ATRA affects immune function by promoting the differentiation of regulatory T cells, modulating cytokine production, and enhancing innate immune cell activity, thus helping to regulate immune responses and maintain immune homoeostasis (Bono et al. 2016;Chang and Hou 2015;Oliveira et al. 2018;Shastak and Pelletier 2023;Xiao et al. 2008).In particular, ATRA binds to its specific receptors (RARs) within immune cells (Mora et al. 2008).This binding event triggers the activation of gene transcription, leading to the production of specific proteins such as Interferon Regulatory Factors (IRFs) that are essential for immune cell function (Al Tanoury et al. 2013;Matikainen et al. 1996).These proteins regulate key processes such as cell differentiation, proliferation, and immune response modulation.Ultimately, the influence of vitamin A on gene expression helps maintain a balanced and robust immune system, enhancing the body's ability to fight off infections and maintain overall health (Tourkochristou et al. 2021).
In a study by Lin et al. (2002), dietary supplementation of vitamin A in the form of retinyl acetate at a level of 12,000 IU per kg of feed was found to have a significant impact on the immune function of laying hens.The presence of dietary vitamin A had a notable impact on the immune response, particularly on NDV antibody titre and peripheral T lymphocyte proportion following Newcastle disease virus vaccination, even with high levels of stored vitamin A in the liver.Increased T-lymphocyte counts indicate a positive immune response to vaccination, highlighting the important role of retinol in modulating the immune response in laying hens.Interestingly, the amount of retinol required for optimal immune function under heat and vaccination stress is higher than the NASEM (1994) requirement estimates (Lin et al. 2002).
A recent research study by Zhang et al. (2023) revealed that oral vitamin A supplementation at a dose of 8,000 IU per kg diet improved the immune response of White Leghorn chickens infected with infectious bronchitis virus (IBV).On d 21 posthatch, all chickens were infected with IBV by intraocular and intranasal routes, and blood and tissue samples were collected at days 2 and 5 post-inoculation.Administered until the chickens reached 21 days old, the supplementation of vitamin A resulted in reduced viral replication, increased serum IgG levels, and a decrease in the inflammatory response.Despite no significant effects on disease progression and growth performance, the study highlights the vital role of vitamin A in regulating chicken-IBV interactions and innate immunity.
Investigating various levels of vitamin A in relation to immune response function enables the identification of the most effective range of vitamin A supplementation, fostering the development of well-informed guidelines to enhance the well-being and health of birds in the field of poultry farming.
In a study conducted by Sklan et al. in 1994, the researchers aimed to investigate the influence of dietary vitamin A on antibody production and T cell proliferative response in broiler chickens aged 21 to 39 days.The study centred on observing the chickens' response to two different stimuli introduced at day 21 after hatching: β-casein and Mycobacterium tuberculosis.The experimental diets were designed to include varying levels of vitamin A from the time of hatching.The results of the study indicated that the immune responses of the broiler chickens were most favourable when their dietary intake of vitamin A reached approximately 20,000 IU/kg.Beyond this threshold, the immune responses showed a decline.On the other hand, optimal growth of the chickens required only about 5,000 IU/kg of vitamin A.
In a subsequent trial conducted by Sklan et al. (1995), turkey poults aged 21 -41 days were fed diets with varying levels of vitamin A supplementation, ranging from 0 to 13,200 μg/kg of feed.The researchers measured the antibody production and T-cell proliferative response of the turkeys when they were immunised with NDV and turkey pox vaccines.Similar to the broiler trial, increasing concentrations of dietary vitamin A resulted in an enhanced proliferative response, peaking at a concentration of approximately 18,000 IU/kg.These findings suggest that higher levels of vitamin A supplementation might be necessary for achieving optimal immune function in broiler chickens and growing turkeys compared to the recommendations provided by the NASEM (1994).
Similar positive outcomes of vitamin A supplementation on immune function have also been demonstrated in non-challenged birds.Recent research by Guo et al. (2019) demonstrated that a vitamin A dosage of 6,000 and 15,000 IU/kg feed maximised serum immune factors, such as interleukin-1 (IL-1), interleukin-2 (IL-2), interleukin-6 (IL-6), and tumour necrosis factor-α (TNF-α), in 42-day-old broilers.Additionally, the groups receiving higher vitamin A supplementation demonstrated improved feed conversion ratio (FCR) and body weight gain compared to the group with a lower supplementary level (3,000 IU/kg).These findings suggest that maximising the levels of immune factors had a positive impact on the overall well-being of the birds in the study.Similarly, Sepehri Moghaddam and Emadi (2014) found comparable results in broilers when assessing immunoglobulin titres, cutaneous basophil hypersensitivity, and heterophils and lymphocytes counts.Their study highlighted the importance of higher vitamin A levels (11,000 IU/kg feed) for promoting optimal immune function in growing poultry.
In a 28-day study, goslings were fed diets with varying levels of vitamin A supplementation (0, 3,000, 6,000, 9,000, 12000, and 15,000 IU/kg feed) to assess their impact on intestinal morphology and immune response (Wan et al. 2022).Increasing levels of vitamin A resulted in significant improvements in intestinal structure, including increased villus height, crypt depth, and muscular layer thickness in the duodenum, jejunum, and ileum (P < 0.05).Furthermore, higher levels of vitamin A intake were associated with elevated serum levels of immunoglobulin A and G, indicating enhanced immune response (P < 0.05).
The provision of vitamin A, both during maternal stages and to the offspring, has been shown to have a positive impact on immune function in broiler chickens and geese, as demonstrated by Yang et al. (2020) and Wang et al. (2020).
In a comprehensive investigation, Yang et al. (2020) explored the effects of maternal and offspring dietary vitamin A supplementation on goslings.The researchers administered varying doses of vitamin A to the maternal (0, 4,000, 8,000, 12,000, or 16,000 IU/kg) and offspring (0 or 9,000 IU/kg) diets.The study revealed that supplementing maternal geese with 12000 IU/kg of vitamin A significantly improved immune function and organ development in goslings.Similarly, offspring supplemented with 9,000 IU/kg of vitamin A showed improved indirect immune markers, such as increased bursa weight, as well as enhanced direct immune marker immunoglobulin G content.
In a study conducted by Wang et al. (2020), broiler chickens were provided with a diet that included either 0 or 5000 IU/kg of vitamin A from day 1 to 21 after hatching.The results showed that the broilers in the group supplemented with vitamin A exhibited a higher relative bursa of Fabricius ratio compared to the control group, which received no vitamin A supplementation (0 IU/kg feed), indicating an enhancement in immune function.The relative bursa of Fabricius ratio is a vital indicator of poultry's immune status, with vitamin A playing a crucial role in the development of this lymphoid organ, particularly during the starter phase.To assess the birds' immune health, the immune or lymphoid organs index or ratio is calculated as 100% × immune or lymphoid organ weight/body weight.This index provides valuable insights for guiding nutrition and management practices to ensure proper immune system development in poultry during their early growth stages.
In conclusion, the groundbreaking discovery made by Green and Mellanby in 1928 solidified vitamin A's status as a key player in immune system regulation, earning it the title of 'the anti-infective vitamin'.Extensive research in domestic fowl has demonstrated that ATRA, the active form of vitamin A, exerts profound effects on immune function by influencing T cell activity, cytokine production, and innate immune responses.These findings emphasise the importance of investigating optimal vitamin A supplementation strategies in poultry nutrition to bolster immune responses and ensure the health and productivity of birds in commercial farming settings.

Effect of supplemental vitamin A on antioxidant capacity
In domestic fowl, oxidative stress or redox imbalance denotes a state where the generation of reactive oxygen species (ROS) surpasses the organism's ability to counteract or eliminate them, resulting in harm to cells and disturbance of normal physiological processes (Surai et al. 2019).Cellular metabolism generates ROS as natural byproducts, which play crucial roles in signalling pathways and serve as a defence mechanism against pathogens (Forrester et al. 2018;Lee and Song 2021).Immune cells like macrophages and neutrophils rely on ROS for their proper functioning (Shekhova 2020), but to avoid the harmful consequences of excessive free radical production, they require sufficient levels of antioxidant defences (Victor et al. 2004).In poultry, an excessive build-up of ROS can occur due to a range of factors, including environmental stressors, inadequate elimination of free radicals, diseases, and substances present in their feed (Miazek et al. 2022;Surai et al. 2019).
Exposure to a stressful environment can have detrimental effects on the performance and disease resistance of domestic fowl.Heat, cold, and vaccination stress-related depression in poultry performance has been shown to be alleviated by vitamin A (Abd El-Hack et al. 2019;Demir et al. 1995;Kucuk et al. 2003;Lin et al. 2002;McDowell 1989;Sahin et al. 2001;Shojadoost et al. 2021).A specific study conducted by Kucuk et al. (2003) focused on heat-stressed broiler chickens.They found that birds subjected to heat stress showed improved performance and reduced blood serum levels of malondialdehyde (MDA), a marker for lipid peroxidation, when supplemented with 15,000 IU of vitamin A per kilogram of feed compared to birds without vitamin A supplementation.
Retinol is known to possess direct antioxidant properties, which can be attributed to its hydrophobic polyene tail and the isoprenoid backbone that provides the structural framework for the molecule (Landete 2013).These polyene units possess the capability to effectively counteract thiyl radicals and stabilise peroxyl radicals.On the other hand, when retinol is exposed to elevated oxygen levels, it has the potential to undergo autooxidation.As a result, its role as an antioxidant is most beneficial when functioning within the physiological oxygen tensions naturally present in tissues (Dao et al. 2017).However, as noted by Combs and McClung (2017) and further supported by Blaner et al. (2021), it has been observed that retinol and retinal exhibit only weak capabilities in scavenging ROS in vivo.Modern research indicates that vitamin A's antioxidant defence involves a complex interplay of mechanisms (Shastak et al. 2023).One crucial aspect is the upregulation of genes encoding antioxidant enzymes, which play a pivotal role in neutralising harmful ROS generated during mitochondrial functions (Lee et al. 2020;Surai et al. 2019).This upregulation leads to an elevation of antioxidant enzyme levels, bolstering the cellular antioxidant capacity (Gad et al. 2018;Hao et al. 2021).Specifically, ATRA influences numerous genes that play a role in processes related to antioxidants, such as the metabolism of glutathione (GSH), the activity of superoxide dismutase (SOD), and the regulation of pathways responsible for responding to oxidative stress (Demary et al. 2001;Malivindi et al. 2018).These observations suggest that the mode of action of ATRA is indirect compared to retinol.Research findings have demonstrated that ATRA can increase the expression of genes associated with GSH activity, such as glutathione peroxidase (GSH-PX) and GSH reductase (Brigelius-Flohé and Flohé 2020; Nefedova et al. 2007).Consequently, this reinforces cellular defence mechanisms against oxidative stress in poultry (Surai et al. 2017).
Furthermore, the impact of ATRA on the management of oxidative stress has been observed through its influence on the expression of various genes associated with oxidative stress pathways.For example, a study conducted by Azzam et al. (2022) provided evidence that ATRA has the ability to reduce the expression of NADPH oxidase genes in rat liver cells.These genes play a crucial role in generating ROS, which can lead to oxidative stress.Consequently, the downregulation of these genes led to a decrease in oxidative stress and an enhancement of the antioxidant status of the hepatocytes.
Additionally, multiple research investigations have provided evidence that ATRA has the potential to augment the antioxidative capabilities of mitochondria using diverse mechanisms (Kurekova et al. 2022).As a case in point, ATRA demonstrates the ability to trigger the activation of the manganese superoxide dismutase gene, an antioxidant enzyme situated within the mitochondria (Kiningham et al. 2008).
Finally, ATRA triggers autophagy and promotes cell death via the Bcl-2/Beclin1 pathway (Fang et al. 2020).Autophagy, a cellular response to nutrient deprivation, is a well-preserved mechanism in living cells.It involves the breakdown of organelles and persistent proteins within lysosomes, playing a crucial role in maintaining cellular balance, supporting tissue development, and serving as a defence against aggregated proteins, impaired organelles, and infectious agents (Maier et al. 2013).
A study conducted by Liang et al. (2019) examined the impact of dietary vitamin A supplementation on the early growth performance, antioxidant levels, and tissue vitamin A content of goslings, as well as their mothers.The researchers discovered that administering 9,000 IU/kg of vitamin A significantly enhanced the levels of antioxidant enzymes such as GSH-PX, superoxide dismutase (SOD), total antioxidant capacity (T-AOC), and catalase (CAT), as well as tissue retinol content in the offspring (P < 0.05), when compared to the group not receiving vitamin A (as shown in Table 2).Additionally, the levels of MDA were significantly reduced in the offspring with vitamin A supplementation (P < 0.05).Maternal vitamin A levels also exhibited a significant influence on the levels of GSH, GSH-PX, SOD, MDA, T-AOC, and CAT in the offspring  (P < 0.05).The supplementation of both maternal and offspring geese with vitamin A had interactive effects on weight gain, tissue retinol content, GSH, GSH-PX, SOD, MDA, and CAT in the goslings (P < 0.05).In conclusion, it was observed that maternal supplementation with 12,000 IU/kg of vitamin A and offspring supplementation with 9,000 IU/kg of vitamin A had positive effects on the growth of goslings.These findings underscore the significance of vitamin A supplementation in reducing oxidative stress and maintaining optimal antioxidant levels in both maternal and offspring geese.Similarly, Wang et al. (2020) investigated the impact of maternal and dietary vitamin A on the growth performance, meat quality, and antioxidant status of broiler offspring.The study found that supplementing either maternal or offspring diets with vitamin A alone did not have any effect on the activities of total SOD and GSH-PX in blood serum.However, a statistically significant interaction (P < 0.05) was observed between maternal vitamin A levels and offspring vitamin A levels on the activity of serum GSH-Px in broiler offspring at 63 days post-hatch.Therefore, it may be important to consider supplementing both maternal and offspring diets with vitamin A, as it could potentially help optimise antioxidative function in the offspring.Mahmoud and Hijazi (2007) conducted a study to examine the impact of a very high supplementation of vitamin A (approximately 71,000 IU/kg feed) on the antioxidant defences of broiler chickens against carbon tetrachloride (CCl4)-induced oxidative stress at 4 weeks of age.Following the intraperitoneal injection of CCl4, the birds exhibited a significant decrease (P < 0.05) in plasma GSH-PX activity.While antioxidants, including α-tocopherol, are generally recognised for their ability to counteract harmful free radicals and safeguard cells against oxidative damage, excessive amounts of free radical scavengers can occasionally produce the opposite effect (Bast and Haenen 2013).Specifically, concerning retinol, studies have indicated that at extremely high doses, it can induce the production of free radicals and trigger oxidative stress (Wang et al. 2020).This prooxidant activity of retinol has been observed under specific experimental conditions, such as when high doses are administered alone or in combination with other factors that promote oxidative reactions.
In conclusion, vitamin A supplementation has shown promise in mitigating oxidative stress and maintaining optimal antioxidant levels in domestic fowl.Retinol and ATRA possess antioxidant properties and regulate antioxidant-related genes, while ATRA also enhances mitochondrial performance by promoting mitochondrial biogenesis (Tourniaire et al. 2015), supporting the electron transport chain (Hammerling 2016), and protecting against oxidative damage (Chiu et al. 2008).Adequate vitamin A supplementation in both maternal and offspring diets can improve growth performance, antioxidant enzyme activities, and reduce oxidative stress markers.

Effect on reproductive performance
Vitamin A is an essential micronutrient for reproductive processes in both males and females, as well as for various stages of embryonic development (Clagett-Dame and Knutson 2011).In poultry, the impact of ATRA on reproduction has been observed in Japanese quail and involves interactions with nuclear receptor superfamily members (RARs and RXRs) (Fu et al. 2000).These receptors function as transcription factors, controlling the expression of some specific genes involved in reproductive processes.
One important mechanism through which ATRA influences reproduction is the regulation of gonadal development and sexual differentiation.During early stages of gonadal development, ATRA plays a critical role by promoting the differentiation of bipotential gonads into either testes or ovaries (Endo et al. 2019;Spade et al. 2019).
Moreover, ATRA exerts influence on the synthesis and secretion of reproductive hormones.It can modulate the activity of the hypothalamic-pituitary-gonadal axis, which governs hormone production and release (Abdelhamed et al. 2021).For example, ATRA stimulates the synthesis of gonadotropin-releasing hormone in the hypothalamus, which subsequently triggers the release of luteinising hormone and follicle-stimulating hormone from the pituitary gland (Cho et al. 1998).These hormones play crucial roles in ensuring proper reproductive function, including follicle development, ovulation, and sperm production.However, it is important to note that these results are based on in vitro studies in rats, which does not necessarily mean the same effect in poultry in vivo, and further investigation in avian species is necessary.
Furthermore, ATRA contributes to the maintenance of reproductive tissues and their functional integrity.In chicken, it supports the proliferation and differentiation of germ cells within the testes and ovaries, thereby ensuring the continuous production of viable gametes (Endo et al. 2019;Zhang et al. 2016).
A variety of reproductive performance parameters such as egg weight, egg number, egg mass, egg yield, fertility or albumen quality have been improved by higher levels of vitamin A in breeding and laying hens (Abd El-Hack et al. 2017;Lin et al. 2002;Chen et al. 2015).As Chen et al. (2015) demonstrated, vitamin A supplementation of up to 21,600 IU/kg feed improved broiler breeder fertility and positively affected reproductive organ development by suppressing apoptosis-related gene transcripts and regulating the expression of ovarian hormone receptors (Table 3).
In two experiments conducted by Lin et al. (2002), incremental levels of vitamin A were added to the diets of heat-stressed laying hens at 3000, 6000, 9000, or 12,000 IU per kg of feed.The results showed that in experiment 1, egg yield (%) was significantly higher at the supplementation level of 9000 IU compared to 3000 IU per kg of feed.In experiment 2, it was observed that egg weight significantly improved up to a supplementation level of 12,000 IU/kg of feed.
In their study, Abd El-Hack et al. (2017) examined the effects of dietary vitamin A supplementation on the performance and egg quality of laying hens.They specifically investigated three levels of supplementation: 0 IU/kg, 8,000 IU/kg, and 16,000 IU/kg.The results showed that supplementing vitamin A up to 16,000 IU/kg diet had a significant positive impact (P < 0.05) on various reproductive performance criteria.These included egg number, egg mass, and albumen quality, all of which showed improvement when compared to the control group receiving 0 IU of vitamin A/kg feed.
A study conducted by Yuan et al. (2014) found that 15,000 IU of supplemental vitamin A significantly increased fertile egg percentage compared to the control group (5,000 IU) (Table 4).The same criterion, however, was negatively affected by high vitamin A supplementation levels.As a result, the authors concluded that the maximum amount of vitamin A that broiler breeders could tolerate was 35,000 IU/kg, whereas excessive supplementation could negatively affect liver function, reproduction performance, and immunity.
In summary, vitamin A is crucial for poultry reproduction, impacting gonadal development, hormone synthesis, and tissue maintenance.Higher levels of vitamin A supplementation have been shown to improve various reproductive performance parameters in breeding and laying hens.However, excessive vitamin A supplementation may have negative effects on reproductive performance, emphasising the importance of maintaining an optimal dosage.

Fine-tuning vitamin A supplementation
Considering the effects outlined above, vitamin A supplementation can benefit poultry performance and well-being.Ensuring an adequate level of dietary vitamin A through supplementation is crucial for optimising performance and maintaining the health of commercial poultry flocks.It is worth noting that the last update on the vitamin A requirement estimates for domestic fowl by NASEM (1994) was nearly three decades ago (as shown in Table 5).To arrive at their retinol requirement estimates, NASEM relied on publications dated from 1937 to 1986.However, significant advancements have been made in poultry growth, productivity, body composition, and laying performance over the past 30 years.These advancements have created higher nutritional demands that cannot be met solely by increased feed intake  For instance, in a study by Sklan et al. (1994), optimal immune responses (antibody production and T cell proliferation) in broiler chickens were achieved at a dietary intake of 6,660 µg/kg (approx.20000 IU/kg) of vitamin A. Similarly, in a subsequent trial with turkey poults (Sklan et al. 1995), the optimal proliferative response was observed at 6.0 μg/g (approx.18000 IU/kg) of dietary vitamin A. Recent research by Guo et al. (2019) demonstrated that a vitamin A dosage of 6,000 and 15,000 IU/kg feed maximised serum immune factors, such as interleukin-1 (IL-1), interleukin-2 (IL-2), interleukin-6 (IL-6), and tumour necrosis factor-α (TNF-α), in 42-day-old broilers.Similarly, Sepehri Moghaddam and Emadi (2014) found that the current recommended vitamin A requirement of 1,500 IU/kg feed for broilers, as suggested by NASEM (1994), may not adequately meet the needs of modern high-performing broiler strains.Their findings, based on immunoglobulin titres, cutaneous basophil hypersensitivity, and heterophils and lymphocytes counts, emphasise the significance of higher vitamin A levels for optimal immune function in growing poultry.These studies highlight the importance of higher vitamin A levels for immune function in both broiler chickens and turkey poults.
Feeding guidelines set by poultry breeding companies can provide insights into actual vitamin A requirements.These recommendations are based on scientific publications and data, and they likely consider the inclusion of an appropriate safety margin to account for potential storage and processing losses of vitamins.According to Leeson and Summers (2001), when practical requirements for vitamin A are being set, the following points must be considered: (1) Requirements may vary depending on genetics.
(2) There may be variations in carry-over from laying hen to chick.
(3) Differences in bioavailability between different vitamin A sources. (4) Vitamin A loss through oxidation, e.g.due to catalytic effects of trace minerals, peroxidised polyunsaturated fats and thermal treatment of the feed.(5) Destruction of vitamin A in the gastro-intestinal tract by a variety of factors.(6) Poor absorption of fat-soluble vitamins in the small intestine as a result of parasites, mycotoxins, inadequate dietary fat, or other factors.(7) Insufficient protein or lipid status in the animal which impairs formation of ßlipoproteins and/or retinol binding proteins for vitamin A transport.(8) A higher requirement for vitamin A due to illness or any other type of stress.
An overview of vitamin A recommendations for broiler, turkey, and laying hen breeds is presented in Tables 6-8.
Finally, when utilising the commercial form of vitamin A, such as retinyl acetate, in poultry nutrition, it is crucial to prioritise adherence to the local regulatory requirements and conditions of use.The safe and appropriate application of vitamin A in poultry farming necessitates adherence to specific directives.These directives limit vitamin A supplementation to the recommended intake levels set by authoritative bodies, such as the European Commission in the European Union, relying on scientific opinions provided by the European Food Safety Authority (EFSA).Consequently, any application of vitamin A in nutrition of domestic fowl must strictly adhere to regulatory framework and directives to prevent excessive supplementation and potential adverse effects on bird health and welfare.By following these guidelines, we can ensure that the use of vitamin A as a supplement in poultry farming is both safe and effective in promoting optimal health and productivity.It is worth noting that the occurrence of hypervitaminosis A in poultry is   T. heavy lines 11,000-12,000 8,000-10,000 7,000-8,000 6,000-7,000 5,000-6,000 Nicholas and B.U. T. heavy lines Feeding Guidelines (2015) Nicholas and B.U.
highly improbable and has not been reported in practical farming or husbandry scenarios, owing to the stringent regulation of vitamin A levels in poultry feed (EFSA 2008).

Conclusions
Vitamin A is essential in avian nutrition, yet the majority of feedstuffs used in compounding poultry feeds lack sufficient retinol.As a result, supplementation with synthetic retinyl acetate is necessary to meet the dietary requirements and must be added in the appropriate quantity.Studies have shown that vitamin A supplementation can enhance reproductive performance, antioxidant capacity, growth, and immunity in poultry.
Referring to breeder recommendations for vitamin A can be beneficial in practical applications.The ability to design optimal poultry diets that maximise bird health and performance under specific conditions relies on a deep understanding of the correct timing and dosage of vitamin A. In this context, it is important to consider the complexity of feed compositions and interactions among fat-soluble vitamins, particularly vitamins A, E, and D. Further research is warranted to explore the role of vitamin A in poultry physiology, optimise feed supplementation, and investigate potential interactions with other feed components.Moreover, an update of the vitamin A requirement estimates and allowances by scientific committees, considering the current state of scientific knowledge on vitamin A function, genetics, and poultry farming methods, is called for.

Table 1 .
Functions and signs of vitamin A insufficiency in poultry.

Table 2 .
Liang et al. 2019)vitamin A levels in maternal and offspring diets on liver antioxidant index of goslings at one day old and seven days old* (adapted fromLiang et al. 2019).

Table 3 .
Chen et al. 2015) vitamin A on broiler breeder production and reproductive organs from 46 to 54 weeks of age* (adapted fromChen et al. 2015).46-week-old birds were fed experimental diets for a duration of 9 weeks; a,b,c Mean values within a row with no common superscript differ significantly (P < 0.05). *

Table 4 .
Yuan et al. 2014)tile eggs in broiler breeders fed varying levels of vitamin A at weeks 43 and 55 of their life* (adapted fromYuan et al. 2014).

Table 5 .
(Applegate and Angel 2014)equirement estimates for poultry according to GfE and NASEM guidelines.(Applegate and Angel 2014).Thus, in contemporary times, it is possible that the vitamin A requirements for commercial poultry production could exceed the levels previously established for healthy birds in controlled research settings.Stress, infection, and illness, all cause additional vitamin A requirements.Such needs must still be accounted for in practical situations.It is therefore imperative to conduct extensive research into the actual retinol requirements in poultry during all stages of production.

Table 6 .
Vitamin A recommendations for selected commercial broiler breeds*.The amounts of supplemental vitamin A in feed shall not exceed the local regulatory maximum. *

Table 7 .
Vitamin A recommendations for selected commercial layers breeds*.The amounts of supplemental vitamin A in feed shall not exceed the local regulatory maximum. *

Table 8 .
Vitamin A recommendations for selected commercial turkey breeds*.