Comparative histoarchitecture of the adrenal gland in buffalo and cattle during different reproductive stages

Introduction

In the view of most comparative endocrinologists, the adrenal gland occupies a unique position among endocrine organs because it combines two embryologically distinct tissues the mesodermal cortex and the ectodermal medulla into a single capsule whose output governs the organism's response to virtually every form of physiological stress [1] . The cortex produces mineralocorticoids (zona glomerulosa), glucocorticoids (zona fasciculata) and androgens (zona reticularis), while the medulla secretes catecholamines [2] . During pregnancy, the adrenal cortex undergoes hyperplasia to meet the increased demand for cortisol, which plays roles in foetal organ maturation, mammary differentiation and parturition timing [3] . Water buffalo and cattle, despite belonging to the same family Bovidae, diverge in several endocrine parameters. Buffalo cortisol concentrations are typically 15-25 percent higher than those of dairy cattle under comparable management conditions [4] . Buffalo exhibit a more exaggerated cortisol surge at parturition and a slower post-partum decline [5] . Whether these physiological differences are reflected in adrenal gland structure specifically, in the relative thickness of the steroidogenic cortical zones and the size of medullary chromaffin cells has not been systematically examined. Existing histological descriptions of ruminant adrenal glands are largely confined to single-species, single-stage studies, often performed decades ago with limited morphometric tools [6] . Modern digital image analysis allows precise measurement of zonal thickness and cellular dimensions across multiple specimens and conditions, enabling the kind of quantitative comparison that earlier work could only approximate [7] Japan's regulated abattoir system and its co-location of buffalo (imported as draft and dairy animals for subtropical esearch stations) and cattle processing facilities make it an ideal setting for obtaining paired tissue samples under standardised post-mortem protocols [8] . The present research aimed to (a) compare adrenal gland weight, cortical zonal thickness and medullary cell size between buffalo and cattle, (b) assess the effect of reproductive stage on adrenal morphometry within each species, and (c) identify histochemical differences using Masson's trichrome and PAS staining.

Material and Methods

Study area description

Adrenal glands were collected from a licensed abattoir in Ibaraki Prefecture (36.34°N, 140.44°E), Japan, which processes both cattle and water buffalo under the supervision of a government veterinary inspector. Ambient temperature at the collection facility was maintained at 4 °C during postmortem processing. Ibaraki Prefecture was selected because it houses both a Holstein-dominant dairy cattle population and a small research herd of Murrah-cross buffalo maintained by the National Agriculture and Food Research Organization (NARO).

Material

Forty-eight adrenal glands (right gland only, to standardise laterality) were collected from 24 water buffalo and 24 cattle females, distributed across three reproductive stages: nonpregnant (n = 8 per species), pregnant in the third trimester (n = 8 per species) and post-partum within 7 days of calving (n = 8 per species). Reproductive status was confirmed by ovarian and uterine examination at slaughter. Animals ranged in age from 4 to 9 years and in body weight from 380 to 620 kg. All animals were clinically healthy at ante-mortem inspection. Ethical approval was obtained from the Tokyo Institute of Comparative Veterinary Anatomy Animal Ethics Committee (Protocol TICVA-AEC-2023-011, approved May 2023).

Reagents and Chemicals: Tissues were fixed in 10 percent

Neutral buffered formalin (pH 7.2) for 48 hours, dehydrated through graded ethanol (70-100%), cleared in xylene and embedded in paraffin. Sections (5 µm) were stained with Harris's haematoxylin and eosin (H&E) for general morphology, Masson's trichrome (Sigma-Aldrich HT15) for collagen visualisation and periodic acid-Schiff (PAS, Sigma-Aldrich 395B) for glycogen and glycoprotein localisation.

Quality Control

To ensure reproducibility, all morphometric measurements were performed by a single trained observer using ImageJ v1.54 (NIH) on digitised micrographs captured with an Olympus BX53 microscope and DP74 camera at 40× and 100× magnification. Ten random fields per section per zone were measured for thickness, and 50 calls per section per zone were measured for diameter. Intra-observer repeatability was assessed by re-measuring 20 randomly selected sections after a two-week interval (ICC = 0.94). Staining batch controls (rat adrenal tissue with known morphometry) were included in every processing run.

Methods

Zonal thickness was measured as the perpendicular distance from the outer to inner boundary of each zone at 10 equidistant points around the gland circumference, averaged per animal. Medullary chromaffin cell diameter was measured on the longest axis. Data were analysed by two-way ANOVA (species × reproductive stage) with Bonferroni post-hoc tests; significance was set at p<0.05. All analyses were performed in R v4.3.

Results

All 48 adrenal glands were suitable for histological analysis. Mean gland weight was significantly higher in buffalo than in cattle across all reproductive stages (overall mean 12.8±1.4 g versus 10.6±1.2 g, p<0.001). Pregnancy increased gland weight in both species (p<0.01), with a proportionally greater increase in buffalo (15.8% versus 11.2% in cattle). 1.00 1.12 1.00 1.12 *p<0.05 versus non-pregnant within species. Buffalo values significantly higher than cattle at all stages (p<0.01). fasciculata, zona reticularis and medulla) with annotated species-specific differences between buffalo and cattle.

Discussion

The 12.4 percent greater fasciculata thickness in pregnant buffalo compared with pregnant cattle is the most functionally significant finding of this research, because the zona fasciculata is the primary site of cortisol synthesis. This structural difference parallels the 15-25 percent higher circulating cortisol concentrations consistently reported in buffalo under both basal and stressed conditions [4,5] . A thicker fasciculata implies a larger mass of steroidogenic cells, which in turn provides greater secretory capacity a straightforward structure-function correlation.

The larger medullary chromaffin cells in buffalo (18.7 versus 15.4 µm) suggest a greater capacity for catecholamine synthesis and storage. Buffalo are known to exhibit more pronounced "fight-or-flight" responses than cattle, with higher peak adrenaline concentrations during handling stress [5] .

Whether the cell-size difference reflects a genuinely higher catecholamine content per cell or simply a larger cytoplasmic volume without proportional granule density cannot be resolved by light microscopy alone; electron microscopy would be needed. The denser PAS staining in the buffalo zona reticularis points to greater glycogen storage, which may serve as a rapidly mobilisable energy substrate for adrenal steroidogenesis during acute stress episodes. This observation is novel and has not been reported in prior comparative histology of ruminant adrenal glands [6] . Its functional significance awaits confirmation through biochemical quantification of glycogen content per unit tissue mass. The pregnancy effect was significant for all three cortical zones in both species but proportionally greater in buffalo, consistent with the greater adrenocortical demand during buffalo gestation. Post-partum regression was incomplete at the 7-day time point for the fasciculata in both species, suggesting that full involution requires more than one week an important consideration for interpreting early post-partum cortisol data in clinical settings.

Conclusion

The adrenal gland, despite its small size, is a critical regulator of the endocrine stress response in domestic ruminants, and its architecture carries species-specific features that have functional implications for both veterinary medicine and comparative physiology. This research, based on 48 glands from buffalo and cattle across three reproductive stages, provides the first systematic quantitative comparison of adrenal histoarchitecture in these two bovid species under standardised conditions. Three principal findings emerge. First, buffalo adrenal glands are heavier and possess a thicker zona fasciculata than cattle, providing a structural basis for the higher cortisol output documented in physiological studies. Second, medullary chromaffin cells are significantly larger in buffalo, suggesting a greater catecholamine storage capacity. Third, pregnancy amplifies cortical zonal thickness in both species, but the effect is proportionally greater in buffalo, consistent with the more demanding endocrine milieu of buffalo gestation. These morphometric reference data should serve as a baseline for future studies investigating adrenal pathology (hyperplasia, neoplasia, exhaustion) in either species. The histochemical differences detected by PAS and trichrome staining open additional avenues for functional investigation at the ultrastructural and biochemical levels. Future research should incorporate immunohistochemistry for steroidogenic enzymes (CYP11A1, CYP17A1, 3β-HSD) to map the enzymatic correlates of the zonal thickness differences, and should extend the post-partum sampling window to 30 days to characterise the full involution trajectory.

Acknowledgement

Acknowledgements Contributions not qualifying for authorshipThe authors thank the staff of the Ibaraki Prefecture abattoir for facilitating tissue collection under government veterinary inspection, and Ms. Ayumi Nakamura for technical assistance with histological processing and digital imaging.

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