Ultrastructural Organization of Epididymis in the Cattle Egret (Bubulcus ibis) During Breeding Season

ABSTRACT

This study presents an ultrastructural analysis of the epididymal epithelium in the cattle egret ( Bubulcus ibis ), aimed at elucidating its cellular diversity and regional specialization. Using transmission electron microscopy, we examined the proximal and caudal regions of the epididymis to identify and compare constituent cell types based on established morphological criteria. Principal cells were distinguished by large euchromatic nuclei and abundant rough endoplasmic reticulum, narrow cells by elongated nuclei and slender cytoplasmic profiles, and clear cells by pale cytoplasm with endocytic vesicles. Myeloid cells exhibited irregular nuclear morphology and cytoplasmic vacuolation, with two morphologically distinct populations observed. The distribution of these cell types across proximal and distal regions underscores the compartmentalization necessary for sperm maturation, storage and protection. Specialized organelles, including large mitochondria, secretory granules and rough endoplasmic reticulum, highlight the metabolic and secretory demands placed on the epithelium. Observations of apoptotic bodies, immune cell heterogeneity and clearance mechanisms suggest ongoing cellular turnover and possible immune involvement. However, given the descriptive nature of this study and the absence of complementary molecular or immunohistochemical data, interpretations regarding immune surveillance must remain tentative. Collectively, these findings provide the first detailed ultrastructural description of the cattle egret epididymis, contributing to a broader understanding of avian reproductive physiology and comparative anatomy. Future studies integrating advanced imaging, immunohistochemistry and molecular profiling will be essential to validate the hypothesized roles of these cell populations and to clarify their contributions to male fertility and reproductive health.

1. Introduction

The epididymis is a critical component of the male reproductive system in vertebrates, functioning as the site of sperm maturation, storage and transport (Jones 1999; Sullivan et al. 2005). In avian species, as in mammals, the proper structural and functional organization of the epididymis is indispensable for reproductive success, directly influencing fertility potential and the quality of spermatozoa (Dacheux et al. 2016; Bashiri et al. 2021). The ultrastructural integrity of this organ ensures that spermatozoa acquire motility and fertilizing capacity, processes that are fundamental to species propagation (James et al. 2020; Rowlison et al. 2018). Despite its biological importance, detailed investigations into the fine architecture of the avian epididymis remain relatively scarce. Much of the available literature has concentrated on domestic or commercially significant birds such as chickens, turkeys and ducks, leaving a considerable gap in knowledge regarding wild or non‐domesticated species (Izquierdo et al. 2025).

The cattle egret ( Bubulcus ibis ), a member of the Ardeidae family, is a widely distributed wading bird inhabiting diverse ecosystems across Africa, Asia, Europe and the Americas (Massa et al. 2014; Abdel‐Gaber et al. 2023). Its remarkable ecological adaptability and reproductive strategies have enabled it to thrive in both pristine natural habitats and environments heavily influenced by human activity (Talbi et al. 2023). While the cattle egret has been the subject of ecological and behavioural studies (Katzir et al. 1999; Kuang et al. 2025), there is a notable paucity of information concerning the ultrastructure of its reproductive organs. A comprehensive understanding of the epididymal ultrastructure in this species could provide valuable insights into the mechanisms of sperm maturation and storage, as well as contribute to broader discussions on avian reproductive physiology, evolutionary adaptation and species resilience in changing environments.

Transmission electron microscopy (TEM) represents a powerful tool for exploring tissue organization at the cellular and subcellular levels (Miranda et al. 2015; Radulović et al. 2022; Zhao et al. 2024). Unlike light microscopy, TEM allows for the visualization of intricate ultrastructural details, including the organization of the epididymal epithelium, the identification of specialized cell types, and the characterization of organelles and intercellular junctions that underpin functional processes (Long et al. 2024; Li et al. 2025). Such ultrastructural characterization is particularly relevant for understanding how the epididymis supports sperm maturation, survival and transport under varying ecological conditions (Akbarsha et al. 2015). Moreover, ultrastructural studies can reveal subtle adaptations that may reflect evolutionary pressures unique to wild bird species (Pap et al. 2015; Terrill and Shultz 2023; Madkour 2024).

To date, no detailed ultrastructural analyses of the epididymis have been reported in the cattle egret. Addressing this gap, the present study aims to provide a comprehensive description of the epididymal ultrastructure in B. ibis using transmission electron microscopy. By documenting the fine architecture of this organ, the study seeks to establish a baseline reference for future comparative investigations in avian reproductive biology. The findings are expected to enhance our understanding of sperm maturation processes in birds, inform comparative anatomy across taxa, and shed light on the potential influence of environmental factors on reproductive health. Additionally, this work may contribute to conservation and management strategies for wild bird populations by deepening our knowledge of their reproductive physiology and adaptive mechanisms. The ultrastructural characterization of the epididymis in B. ibis establishes a critical baseline for evaluating reproductive health in wild populations. Because sperm maturation is tightly coupled to male fertility, deviations from the documented fine architecture may serve as sensitive biomarkers of environmental stress and early indicators of declining population viability. By integrating detailed morphological insights with conservation biology, our study advances comparative avian reproductive science while providing practical tools for monitoring ecosystem integrity. Ultimately, this work bridges fundamental reproductive biology with applied conservation, offering a framework for assessing fertility and guiding management strategies in free‐living bird populations.

2. Methodology

2.1. Animals Used for the Study

A total of ten (10) male cattle egrets were used for this study. The cattle egrets were sourced from Zuru town, Kebbi State, in North‐western Nigeria.

2.2. The Source of the Animal and Ethical Approval

The birds used for this study were obtained during the rainy season of Nigeria (May to September), with approvals from the Federal Ministry of Agriculture of Nigeria and the Institutional Animal Care and Use Committee of Ahmadu Bello University, Zaria, Nigeria, with approval no. ABUCAUC/2025/004. Only birds displaying the breeding plumage of red feet and red beaks were selected. The birds were captured using a trap with fishing nets placed around the top branches of the tree where the birds nest at night, as previously described (Oliveira et al. 2015). The birds were held in plastic cages and transported to the Gross Anatomy Laboratory of the Faculty of Veterinary Medicine at Ahmadu Bello University, Zaria, Kaduna State.

2.3. Consent for Publication

Not applicable.

2.4. Necropsy and Organ Harvesting

The birds were weighed, and thereafter euthanasia was performed using ketamine hydrochloride administered intramuscularly into the pectoral muscles at a dosage of 50 mg/kg body weight. This procedure was conducted in accordance with the approved protocol of the Institutional Animal Care and Use Committee of Ahmadu Bello University, Zaria (approval no. ABUCAUC/2025/004). The birds were placed in lateral recumbency, and an incision was made, extending from the commissure of the beak to the pubic symphysis (vent). The peritoneum was reflected, and the intestines were displaced to gain access to the reproductive system. The organs were examined in situ for any gross abnormality, and thereafter exteriorized by gentle dissection and trimmed.

2.5. Transmission Electron Microscopy

Small blocks (~1 mm³) of the different parts of the epididymis were trimmed by chopping immediately after isolation of the reproductive segment (testis) and fixed by immersion in small vials containing 4% glutaraldehyde in 0.13 M Milliong's phosphate buffer (pH 7.4), for 24 h. The samples were post‐fixed in 1% osmium tetroxide for 2 h. Then the tissue samples were rinsed in 0.1 M Millonig's buffer, dehydrated in graded concentrations of alcohol, and embedded in epoxy resin at a ratio of 1:2 for 1 h, 1:1 for 2 h and 100% resin overnight.

Semi‐thin sections of 1 μm thickness were cut using a glass knife and stained with toluidine blue. Ultra‐thin (50–90 nm thick) sections of selected areas were cut on a Reichert‐Jung Ultracut (C. Reichart AG, Vienna, Austria), using a diamond knife, collected onto copper grids, and stained with Reynold's lead citrate. The sections were counterstained with an aqueous saturated solution of uranyl acetate. The sections were examined in a Phillips CM10 transmission electron microscope (Phillips Electron Optical Division, Eindhoven, The Netherlands), operated at 80 kV. A megaview III side‐mounted digital camera (Olympus Soft Imaging Solutions GmbH, Munster, Germany) was used to capture the images, and iTEM software (Olympus Soft Imaging Solutions GmbH, Munster, Germany) was used to adjust the brightness and contrast.

3. Results

The ultrastructural examination of the epididymis in the cattle egret ( B. ibis ) revealed distinct regional variations in epithelial organization and cellular composition. In the caudal region, numerous principal cells were identified, each exhibiting diverse nuclear morphologies. Some nuclei contained centrally located dark nucleoli, while others displayed nucleoli positioned at the periphery. The general architecture of the principal cell nuclei appeared opaque and irregular, underscoring their structural complexity. In contrast, the proximal region was dominated by narrow epithelial cells with elongated nuclei, although occasional clear cells were also observed (Figure 1a,b).

FIGURE 1.

Electron micrographs of epididymis of cattle egret. First, the distal part of the tubule contains principal cells (PC) that are surrounded by secretory vesicles (SV) and numerous vacuoles (V) (a). Next, the proximal part of the tubule (b) shows narrow cells (NC) and clear cells (CC).

Unique ultrastructural features of myeloid cells were also noted. One type exhibited a large, elongated nucleus with chromatin material concentrated at the nuclear periphery, while another type appeared smaller and oval, with dark chromatin diffusely scattered throughout the nucleus. The latter cell type contained numerous cytoplasmic vacuoles (Figure 2a,b).

FIGURE 2.

Electron micrographs of epididymis of cattle egret showing myeloid cells with a large elongated nucleus and vacuoles (a). A small myeloid cell with dark chromatin material, epididymal duct (ED) and secretory vesicles (SV) (b).

In certain epididymal segments, a mixture of clear and narrow cells was evident, accompanied by large oval mitochondria, secretory granules and dark apoptotic bodies. The epididymal lumen contained spermatozoa interspersed with cellular debris (Figure 3a,b). Numerous electron‐dense secretory granules and clusters of rough endoplasmic reticulum were observed within principal cells (Figure 4a,b).

FIGURE 3.

Electron micrographs of epididymis of cattle egret (a, b) showing clear cells (CC), large oval mitochondria (M), connecting duct (CD), apoptotic body (AB), dark secretory granules (SG) and sperm cells and cellular debris within the epididymal lumen (EL).

FIGURE 4.

Electron micrographs of epididymis of cattle egret (a, b) showing large oval mitochondria (M), epididymal duct (ED), apoptotic body (AB), clusters of rough endoplasmic reticulum (rER), dark secretory granules (SG) and narrow cells (NC).

Overall, the coexistence of principal, narrow, clear and myeloid cells across both proximal and distal regions of the epididymis represents a unique and intriguing finding.

4. Discussion

The ultrastructural findings of the epididymal epithelium in the cattle egret ( B. ibis ) provide descriptive insights into the cellular diversity and regional specialization that may underpin its roles in sperm maturation, storage and transport. The distinct epididymal cell populations—including principal, narrow, clear and myeloid cells—were identified based on established ultrastructural criteria. Principal cells were characterized by their relatively large size, euchromatic nuclei with prominent nucleoli, and abundant rough endoplasmic reticulum. Narrow cells were distinguished by elongated, basally located nuclei and slender cytoplasmic profiles. Clear cells were recognized by their pale cytoplasm, sparse organelles and the presence of endocytic vesicles. These morphological features are consistent with descriptions in both mammalian and avian epididymis (Jain et al. 1991; Shum et al. 2011; Breton et al. 2019; Hermo et al. 2025; Zini et al. 2026), and formed the basis for classification in this study. Myeloid cells, identified by irregular nuclear morphology and cytoplasmic vacuolation, were further subdivided into two morphologically distinct populations, as previously described (Krause 2000).

Such cellular heterogeneity likely contributes to the establishment of microenvironments necessary for the sequential maturation of spermatozoa, although functional confirmation of these roles in birds remains to be established (Miyaso et al. 2022; Zhuang et al. 2023).

Principal cells, predominant in the caudal region, exhibited variable nuclear morphology, including differences in nucleolar positioning and nuclear opacity. These variations may reflect differential transcriptional or metabolic activity, potentially correlating with region‐specific demands such as ion transport, protein secretion and absorptive processes (Skinner and Johnson 2017; Bhiwgade and Menon 2023). The presence of both centrally and peripherally located nucleoli within principal cell nuclei suggests either dynamic stages of transcriptional activity or the existence of subpopulations with specialized functions (Lacroix and Audas 2022). While these interpretations are consistent with mammalian data, further molecular or functional studies are required to confirm whether similar mechanisms operate in avian epididymis.

In the proximal epididymis, the abundance of narrow cells with elongated nuclei and the occasional appearance of clear cells highlight the diversity of epithelial composition along the duct. Narrow cells are thought to contribute to luminal acidification, a process essential for maintaining sperm quiescence until ejaculation, while clear cells are implicated in endocytosis and the removal of luminal debris (Shum et al. 2011; Breton et al. 2019; Ribeiro et al. 2024). The identification of these cell types in the cattle egret supports the concept of regionally specialized functions along the epididymal duct. However, without complementary functional assays, their precise physiological contributions in birds remain hypothetical.

The observation of two morphologically distinct populations of myeloid cells within the epididymal epithelium suggests a structural heterogeneity that may be relevant to immune and phagocytic functions. Larger, elongated myeloid cells with peripheral chromatin could be more actively engaged in nuclear regulation or migratory activity (Battistone et al. 2024; Maxwell et al. 2024), whereas smaller, vacuolated cells may participate in phagocytosis of apoptotic bodies and luminal debris (Torroella‐Kouri et al. 2013; Jenkins and Gregory 2025). While such heterogeneity aligns with evidence of immune activity in mammalian epididymis (Barrachina et al. 2023), the absence of molecular or immunohistochemical data in the present study precludes definitive conclusions.

The frequent observation of large oval mitochondria, abundant secretory granules and clusters of rough endoplasmic reticulum, particularly in segments with mixed cell populations, underscores the high metabolic and secretory demands imposed on the epididymal epithelium (Lorenzana et al. 2007; Bhiwgade and Menon 2023). These organelles are essential for the synthesis and secretion of proteins, glycoproteins and other factors that may modulate sperm maturation and protection (Sharma and Agarwal 2011; Zhang et al. 2025). The presence of apoptotic bodies and cellular debris within the lumen further points to ongoing cellular turnover and clearance mechanisms, although functional confirmation of these processes in avian epididymis remains necessary (Nakidkina and Kuzmina 2019; Xue et al. 2025).

Collectively, these ultrastructural features highlight the functional complexity of the cattle egret epididymis. The coexistence of multiple specialized cell types, coupled with the presence of distinct organelles and secretory structures, points to a system that is likely highly regulated to coordinate sperm maturation, storage and possibly immune protection. However, given the descriptive nature of this study, interpretations regarding functional roles must remain tentative. Future investigations incorporating molecular, immunohistochemical and functional analyses will be essential to validate these hypotheses and to clarify the evolutionary adaptations underlying avian reproductive physiology.

5. Limitations of the Study

A limitation of this study is the exclusion of the vas deferens from the investigation. While the primary focus was on the epididymal regions, the vas deferens represents an important component of the male reproductive tract, and its omission restricts the comprehensiveness of the findings. Future studies incorporating the vas deferens would provide a more complete understanding of the structural and functional continuum of the excurrent ducts. Additionally, the present work emphasized ultrastructural identification of cell types rather than quantitative morphometric analysis. Parameters such as epithelial thickness, epithelial height and luminal diameter are commonly employed to distinguish proximal and distal regions of the epididymis. The absence of these measurements limits the ability to provide precise regional comparisons. Subsequent investigations that integrate morphometric data with ultrastructural observations will strengthen the classification of epididymal segments and enhance the reproducibility of findings across species.

6. Conclusion

The ultrastructural analysis of the epididymal epithelium in the cattle egret ( B. ibis ) reveals a complex and regionally specialized tissue architecture, characterized by the coexistence of principal, narrow, clear and diverse myeloid cell populations. The classification of these epithelial cell types was based on established morphological criteria, ensuring consistency with prior mammalian and avian studies. The distinct distribution and features of these cells across proximal and distal regions underscore the compartmentalization necessary for the unique functions of the epididymis. The presence of specialized organelles, including large mitochondria, secretory granules and rough endoplasmic reticulum, highlights the metabolic and secretory demands placed on the epithelium to support sperm maturation, storage and protection. Observations of apoptotic bodies, immune cell heterogeneity and clearance mechanisms suggest ongoing cellular turnover and possible immune involvement. However, given the descriptive nature of this study and the absence of complementary molecular or immunohistochemical data, interpretations regarding immune surveillance and functional specialization must remain tentative. This work provides the first detailed ultrastructural description of the cattle egret epididymis, contributing to a broader understanding of avian reproductive physiology and comparative anatomy. Future studies integrating advanced imaging, immunohistochemistry and molecular profiling will be essential to validate the hypothesized roles of these cell populations and to clarify their contributions to male fertility. Such investigations may also illuminate how environmental pressures shape reproductive strategies in wild bird populations, thereby informing conservation and management efforts.

Author Contributions

S. O. Ajeigbe: methodology, formal investigation, writing and editing. M. Zakariah: conceptualisation, methodology, formal analysis, writing of original draft. J. Imam: supervision, writing and editing. T. A. Muazu: writing and editing. M. Zubair: writing and editing. H. Owolabi: writing and editing. M. H. Suleiman: conceptualisation, supervision, project administration, writing and editing.

Conflicts of Interest

The authors declare no conflicts of interest.

Data Availability Statement

Data will be available on request.