Maintaining optimal growth during the early stages of life is a crucial issue in broiler chicken production, as this phase significantly impacts subsequent performance. Post-hatch handling practices, particularly delays in feeding and watering, can induce metabolic stress, reduce environmental adaptability(Wijnen et al., 2022), and accelerate oxidative damage due to exposure to free radicals. This phenomenon is more common in tropical hatcheries, where day-old chick (DOC) distribution is often hampered by long shipping distances and relatively high ambient temperatures. Under these conditions, post-hatch feeding delay becomes a major risk factor for oxidative stress. Oxidative stress occurs when the production of reactive oxygen species (ROS) exceeds the capacity of the endogenous antioxidant system, negatively impacting cell integrity and physiological function(Khelfi, 2024).

Vitamin E and selenium are commonly added to broiler chicken feed as part of a standard nutritional strategy to support antioxidant balance.

Vitamin E plays a role in protecting cell membrane structure, while selenium is an essential component of the enzyme glutathione peroxidase. These two nutrients function to support the regulation of oxidative processes, including by suppressing lipid peroxidation and facilitating enzymatic detoxification of free radicals(Çalik et al., 2022). In this study, vitamin E and selenium were administered uniformly to all treatment groups, so they were positioned as having the same nutritional background and not as the main treatment factor.

Oxidative stress associated with delayed post-hatching feeding has the potential to disrupt intestinal tissue integrity and immune response. Increased malondialdehyde (MDA) levels reflect lipid peroxidation, while changes in the proportion of CD4⁺ and CD8⁺ T cells indicate changes in adaptive immune status(Yang et al., 2023). Although feed with antioxidant support has been reported to help maintain physiological stability under environmental stress conditions, the extent to which delayed post-hatch feeding itself affects oxidative status, intestinal morphology, and immune response under uniform nutritional conditions has not been widely studied, especially in commercial broiler production systems in tropical regions(Özlü et al., 2022).

Therefore, this study aims to examine the relationship between post-hatch holding time (24–60 hours) and physiological responses related to oxidative status, intestinal morphology, and immune function in broiler chickens reared on uniformly supplemented vitamin E-selenium feed. This approach allows for the evaluation of the effect of holding time duration under supported nutritional conditions, rather than assessing the direct effects of antioxidant supplementation. The working hypothesis of this study is that shorter holding times (≤36 hours) tend to support better physiological adaptation, whereas longer delays may exceed adaptive capacity, even under supported nutritional conditions.

Post-hatch (HT) time is a crucial factor affecting the initial performance, immune status, and physiological balance of broiler chickens. During HT up to 36 hours, the embryo’s energy reserves are sufficient to support the initial adaptation process. However, as the duration is extended, glycogen and lipid reserves are depleted, triggering metabolic stress and increasing the accumulation of reactive oxygen species (ROS), which can potentially damage cell membranes and functional proteins(Gregrova et al., 2024). Under these conditions, the observed physiological responses reflect the interaction between post-hatch holding time and the birds’ antioxidant-supported adaptive capacity, rather than a direct protective effect of vitamin E and selenium alone. Vitamin E protects membrane phospholipids from lipid peroxidation, while selenium supports the activity of the glutathione peroxidase enzyme, thereby contributing to the modulation of oxidative balance when nutritional access occurs within a tolerable holding time window(Khelfi, 2024).

In terms of production performance, post-hatch (HT) removal for ≥48 hours causes decreased feed consumption, low body weight gain (BWG), and a worsening feed conversion ratio (FCR), as shown in Table 2. This condition is related to slow enterocyte maturation and impaired nutrient absorption due toincreased oxidative stress in the digestive tract(Desai et al., 2025). Within this context, antioxidant supplementation was associated with numerical improvements in several intestinal histological parameters when the holding time was 24–36 hours, including villus height, crypt depth balance, and absorptive surface area (Table 3)(Boyner et al., 2025);(Marcato et al., 2024). However, these differences were not statistically significant for some parameters and should therefore be interpreted as indicative trends rather than definitive biological effects. These tendencies were not maintained at prolonged holding times (≥48–60 hours), suggesting limited physiological compensation despite antioxidant-supported nutrition.

The numerical decrease in intestinal surface area and crypt depth with increasing HT (Table 3) suggests that delayed feed access may constrain intestinal structural development. Early access to feed and water at 24 hours HT was associated with a tendency toward improved villus maturation and nutrient absorption efficiency, as reflected by a higher villus to crypt ratio. These findings suggest that early post-hatch feeding facilitates intestinal adaptation, while antioxidant supplementation functions as a supportive factor rather than a sole determinant of intestinal integrity. This pattern is consistent with previous reports confirming that early post-hatch feeding improves broiler intestinal morphology, function, and overall health(Proszkowiec-Weglarz et al., 2022);(Syamsuryadi et al., 2025);(Zhang et al., 2025);(Boyner et al., 2025).

An increase in the H/L ratio in HT ≥48 hours indicates activation of the systemic stress response associated with stimulation of the hypothalamic pituitary adrenal (HPA) axis, as illustrated in Figure 1. Prolonged feed deprivation increases heterophil numbers and decreases lymphocytes through increased corticosterone secretion, further prolonging the immune status and inflammatory response(Madej et al., 2024);(Wlaźlak et al., 2023). Conversely, earlier access to food and air (≤36 hours) helps suppress the physiological stress response by maintaining energy and hydration balance. This stress-mitigating effect appears to be conditional on the duration of holding time, even under antioxidant supplementation, as longer delays still amplified oxidative stress and stress hormone production (Figure 1) (Figure 2).

From an immunological perspective, shorter post-hatch feeding times (≤36 hours) increased CD4⁺ and CD8⁺ T lymphocyte populations, reflecting more effective activation of adaptive immunity (Figure 3) (Figure 4). It should be emphasized that the CD4⁺ and CD8⁺ values reported in this study represent relative proportions of lymphocyte subpopulations within peripheral blood, rather than absolute cell counts. Consequently, the relatively low percentages (<2%) observed are consistent with flow cytometry–based immunophenotyping studies in poultry, in which T-cell subsets are typically reported as fractions of total leukocytes or lymphocytes. In contrast, at HT ≥48 hours, both subpopulations significantly decreased (Figure 5) (Figure 6), suggesting impaired T cell differentiation associated with excessive oxidative stress that could not be fully compensated by antioxidant-supported nutrition(Madej et al., 2024);(Wlaźlak et al., 2023). Prolonged feed deprivation is known to inhibit cytokine signaling and lymphocyte proliferation, thereby suppressing the adaptive immune response(Syamsuryadi et al., 2025). Overall, these findings indicate that the benefits of vitamin E and selenium are expressed optimally when early nutritional access is ensured, highlighting a time-dependent interaction rather than a direct causal immunoprotective effect.

The results of this study indicate that post-hatch holding time is a critical determinant of broiler performance, physiological balance, and immune development. When nutritional access occurs within 36 hours after hatching, the birds can still adapt physiologically to antioxidant-supported nutrition, resulting in lower oxidative stress and better overall responses. In contrast, prolonged holding time (≥48 hours) exceeds the birds’ adaptive capacity, leading to impaired intestinal condition, performance, hematological stability, and immune responses, even with antioxidant supplementation. These findings highlight that early nutritional access, rather than antioxidant supplementation alone, is essential for achieving optimal broiler growth under tropical production conditions.

The results of this study indicated that the duration of post-hatch shedding significantly determines the production performance, physiological homeostasis, and immune system development of broiler chickens. Vitamin E and Selenium supplementation effectively suppressed oxidative stress and maintained physiological integrity, but were only optimal at HT ≤36 hours. Delays of up to ≥48 hours reduced protection effectiveness, worsening intestinal conditions, performance, hematology, and immune responses. The study also reported that early nutritional access was crucial to optimizing the benefits of antioxidants and supporting optimal broiler chicken growth. These findings imply that minimizing post-hatch holding time to 36 hours or less should be prioritized in commercial broiler management, particularly under tropical conditions, to enhance production efficiency and maintain physiological and immune stability.