Changes in the Histological Structure of Adrenal Glands and Corticosterone Level after Whey Protein or Bee Pollen Supplementation in Running and Non-Running Rats

**Background:** Stress activates the hypothalamic–pituitary–adrenal (HPA) axis, leading to adrenal gland secretion of stress hormones. Bee pollen and whey protein are widely used dietary supplements with reported health-promoting properties, but their effects on adrenal structure and function had not been studied. This study aimed to assess whether these supplements can influence adrenal gland histology and corticosterone levels in rats under running and non-running conditions.

**Methods:** Thirty 8-week-old male Wistar rats were divided into six equal groups (n=5 per group): non-running control, running control, non-running + whey protein, non-running + bee pollen, running + whey protein, and running + bee pollen. The experimental phase lasted 8 weeks. Running rats exercised on a treadmill five times per week (5 min per session, average velocity 6 km/h). Bee pollen (collected near Lublin, Poland) contained approximately 31 g carbohydrates, 23 g protein, 5 g lipids, and 0.8 g vitamins per 100 g. Whey protein concentrate contained 77 g protein, 6 g carbohydrates, and 7 g lipids per 100 g. At the end of the experiment, rats were decapitated, adrenal glands were collected and weighed, and paraffin slides were prepared for H&E and Masson's trichrome staining. Corticosterone levels were measured in urine and feces using ELISA kits. Statistical analysis used the Kruskal–Wallis test with significance set at p < 0.05.

**Key Results:** Daily whey protein intake was similar between running (5.19 g/rat) and non-running (5.18 g/rat) groups, while bee pollen consumption was significantly higher in non-running rats (13.45 g/rat) compared to running rats (11.96 g/rat) (p < 0.05). No statistically significant differences were found in adrenal gland mass (p = 0.65) or adrenal cortex layer thickness between groups (p > 0.05). Statistically significant differences were observed in cell nuclei diameter across all cortex layers and medulla between groups. Sinusoid width differences were also statistically significant. Capsule thickness increased in all four experimental groups compared to controls. No significant fibrosis was observed except in the bee-pollen-supplemented running group, which showed mild fibrosis in all adrenal cortex layers. Urine corticosterone concentrations significantly differed across all groups (p < 0.05): both non-running supplemented groups showed lower values than the non-running control, while both running supplemented groups showed higher values than the running control. Whey-protein-supplemented groups had lower urinary corticosterone than bee-pollen-supplemented groups. The non-running control showed higher urinary corticosterone than the running control. No statistically significant changes were found in feces corticosterone concentration. In non-running experimental groups, both bee pollen and whey protein supplementation resulted in a lower mean percentage of pyknotic nuclei in all adrenal cortex zones compared to the non-running control.

**Clinical Implications:** This is the first study to evaluate the effects of bee pollen and whey protein on adrenal structure and function. The results suggest that both supplements have limited but promising stress-reducing potential, as evidenced by reduced urinary corticosterone levels and decreased pyknotic nuclei in supplemented non-running rats. Whey protein appeared more effective than bee pollen in reducing corticosterone levels. The higher consumption of bee pollen (a carbohydrate-rich food) by non-running rats aligns with the known tendency of stressed animals to seek comfort foods. However, the small sample size (n=5 per group) and potential stress from metabolic cage housing are limitations. Further studies in humans are needed to verify these effects in clinical populations.