Kosali cattle are recognized as the first registered cattle breed of Chhattisgarh and the thirty-sixth indigenous cattle breed of India. This breed represents an important native bovine genetic resource and is predominantly reared by rural farming communities under low-input production systems, where animals are sustained mainly on agricultural by-products such as paddy straw. Kosali cattle are particularly valued for their adaptability to harsh climatic conditions, disease resistance and ability to maintain productivity under minimal management inputs (Jain et al., 2017). The spleen is the largest secondary lymphoid organ and serves as a major site for blood filtration, fetal hematopoiesis and immunological surveillance against blood-borne antigens. It harbors nearly one-fourth of the body’s lymphocyte population and plays a central role in the orchestration of innate and adaptive immune responses (Gadre et al., 1985; Kraal and Mebius, 2006). The splenic capsule is covered by mesothelium on its peritoneal surface and consists of an outer layer of slightly dense connective tissue and an inner fibromuscular layer. Histologically, the splenic parenchyma is organized into red pulp and white pulp. The red pulp is involved in erythrocyte sequestration, blood storage and phagocytosis, whereas the white pulp functions as the principal immunological compartment responsible for lymphocyte maturation and antigen-dependent immune activation (Gupta et al., 2017). The white pulp is further differentiated into periarteriolar lymphoid sheath (PALS), lymphoid follicles and marginal zone. Despite extensive reports on postnatal splenic histology in domestic animals, information concerning prenatal splenic histogenesis remains fragmentary, particularly in indigenous bovine breeds. Recent developmental studies have emphasized that prenatal splenic differentiation is crucial for establishment of fetal immune competence and hematopoietic microenvironment. However, systematic data on prenatal splenic development in Kosali cattle are lacking. Therefore, the present study was undertaken to characterize the sequential histomorphological changes during prenatal development of the spleen in Kosali cattle and to establish baseline developmental data for future comparative and functional investigations.

Place of research
 
The present investigation was conducted in the Department of Veterinary Anatomy, College of Veterinary Science and Animal Husbandry, Anjora, Durg, Chhattisgarh, India. A total of 24 prenatal Kosali fetuses were taken for the study. The fetuses were collected immediately after the accidental death or slaughter of pregnant Kosali cows from nearby districts of Rajnandgaon, Balod and Durg. All procedures involving animal materials were reviewed and approved by the Institutional Animal Ethics Committee (IAEC), in accordance with the guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Government of India.
 
Study design
 
Immediately after collection, fetal body weight, volume and crown-rump length (CRL) were recorded following the method described by Joubert (1956). Fetuses were collected immediately after the death of Kosali cows from gravid uterus at local abattoirs. Only apparently normal fetuses without visible congenital anomalies were included in the study to avoid bias related to developmental abnormalities. A total of 24 fetuses were selected and distributed evenly across developmental stages to ensure representative sampling. 
 
Grouping criteria
 
Based on CRL, fetuses were categorized into four developmental groups, each comprising six fetuses: Group I (<20 cm CRL), Group II (20-40 cm CRL), Group III (40-60 cm CRL) and Group IV (>60 cm CRL). This classification was adopted to represent distinct phases of fetal development (early, mid, advanced and near-term stages). CRL-based grouping is preferred because it reflects continuous somatic growth and minimizes variability associated with unknown conception dates. Dividing fetuses into fixed CRL intervals ensures balanced group sizes and facilitates comparative developmental analysis across stages.
 
Histomorphological and histochemical analysis
 
Spleen samples were carefully collected following dissection by making a mid-ventral skin incision extending from the body of the mandible to the inguinal region. The spleen was then located, carefully excised and rinsed thoroughly in normal saline. The collected spleen samples were subsequently fixed in 10% neutral buffered formalin as per Luna (1968). Following fixation, tissue samples were processed routinely for paraffin embedding using paraffin wax with a melting point of 58-60°C. Paraffin sections of 3-5 µm thickness were prepared using a rotary microtome. The sections were stained with Hematoxylin and Eosin for general histological evaluation. Special histochemical stains included Van Gieson’s stain for collagen fibers, Verhoeff’s stain for elastic fibers and Gomori’s silver impregnation method for reticular fibers, following the procedures described by Singh and Sulochana (1996).
 
Statistical analysis
 
Data obtained from histomorphometric parameters were subjected to one-way analysis of variance (ANOVA) followed by Duncan’s Multiple Range Test (DMRT) for comparison of mean values among groups using SPSS software version 20.0. Results were expressed as mean ± standard error (Mean±SE) and differences were considered statistically significant at p<0.05.

Histomorphology of spleen
 
Stroma
 
The splenic capsule and trabecular framework showed progressive structural differentiation with advancing gestation. In Group I, the capsule was thin and composed of loose to moderately dense connective tissue, lined externally by mesothelial cells and underlain by a thin smooth muscle layer (Fig 1). Trabeculae extended into the parenchyma, indicating early establishment of the stromal framework. Similar early organization has been reported in fetal goat and sheep spleens (Nishant et al., 2020; Bhagyalakshmi et al., 2025). In Group II, the capsule exhibited a bilaminar arrangement with an outer connective tissue layer and an inner fibromuscular layer, along with increased vascularity (Fig 2). This stratification reflects advancing stromal differentiation and increased metabolic demand associated with hematopoietic and immunological development, consistent with earlier reports in domestic ruminants (Waghaye et al., 2017; Haldar et al., 2021). Group III showed a significant (p<0.05) increase in capsular and trabecular thickness, with enhanced vascularization and the appearance of subcapsular sinuses (Fig 3). These changes likely support the expanding splenic parenchyma and indicate maturation of the microcirculatory framework. Similar findings have been described in mid-gestation fetal spleens of goats and sheep (Nishant et al., 2020; Bhagyalakshmi et al., 2025). In Group IV, these features were most pronounced, with a thick, well-organized capsule, prominent trabeculae and an extensive vascular network including trabecular sinuses (Fig 4). This advanced structural organization suggests near-complete maturation of the splenic stroma and readiness for postnatal immunohematopoietic function. Comparable observations have been reported in late gestational bovine and ovine fetuses (Haldar et al., 2021; Holkunde et al., 2023). Overall, the progressive increase in capsular thickness, trabecular development and vascularization highlights dynamic prenatal remodeling of the spleen, essential for its functional maturation.








 
Parenchyma development
 
The splenic parenchyma showed progressive differentiation with advancing gestation, transitioning from a primitive, poorly organized structure to a well-defined architecture. In Groups I-III, the parenchyma consisted mainly of diffusely distributed lymphocytes and erythrocytes within an immature stromal framework, indicating early developmental and hematopoietic activity. Similar observations have been reported in fetal goat and sheep spleens during early and mid-gestation (Nishant et al., 2020; Bhagyalakshmi et al., 2025). In Group IV, clear demarcation into red pulp and white pulp was evident, along with well-developed splenic nodules and an extensive vascular network (Fig 5). This structural differentiation indicates advanced maturation and functional compartmentalization of the spleen, consistent with findings in bovine and ovine fetuses (Haldar et al., 2021; Holkunde et al., 2023). Megakaryocytes were sparsely observed across all groups, suggesting limited thrombopoietic activity during the studied gestational period. The white pulp showed progressive organization from loose lymphoid aggregates in Group I to well-defined periarteriolar lymphoid sheaths (PALS) and distinct nodules in Group IV. This gradual lymphoid maturation reflects the establishment of splenic immunocompetence, in agreement with previous reports in domestic mammals (Kraal and Mebius, 2006; Haldar et al., 2021). Overall, the findings demonstrate a sequential and coordinated prenatal development of splenic architecture in Kosali cattle, culminating in structural and functional maturity during late gestation.


 
Histomorphometric parameters
 
Prenatal splenic development in Kosali cattle showed a significant (p<0.05) increase in lymphoid organization with advancing gestation (Table 1). The mean number of splenic nodules per 10X field increased from 1.00±0.00 in Group I to 4.67±0.33 in Group IV, indicating progressive lymphoid proliferation. Similar increases in lymphoid follicle density during fetal development have been reported in goats, sheep and cattle (Chaurasia et al., 2020; Haldar et al., 2021; Bhagyalakshmi et al., 2025). Morphometric observations revealed significant enlargement of white pulp components. The maximum and minimum diameters of white pulp increased from 363.33±9.45 µ to 497.50±15.90 µ and from 183.83±5.06 µ to 269.67±7.54 µ, respectively, while nodular arteriole diameter increased from 12.33±1.35 µ to 23.83±2.41 µ. These findings indicate enhanced vascularization and maturation of the white pulp, consistent with reports in ovine and caprine fetuses (Rahmoun et al., 2020; Holkunde et al., 2023). A significant increase in white pulp area (150000±16124.51 µ² to 278333.33±13519.53 µ²) with a concomitant decrease in red pulp area (750000±16124.50 µ² to 635000.33±16881.94 µ²) resulted in more than a two-fold increase in pulp index (20.27±2.56 to 44.26±3.32). This shift reflects transition from hematopoietic to immunological predominance, in agreement with earlier findings in fetal goats and lambs (Waghaye et al., 2017; Nishant et al., 2020; Rahmoun et al., 2020). Histologically, the red pulp consisted of splenic cords containing erythrocytes, lymphocytes, macrophages and occasional megakaryocytes arranged around developing sinusoids. The presence of megakaryocytes in Groups II and III supports the transient hematopoietic role of the spleen during mid-gestation (Haldar et al., 2021; Bhagyalakshmi et al., 2025). In Group IV, the white pulp was well organized into distinct splenic nodules with periarteriolar lymphoid sheaths (PALS) and a clearly defined marginal zone composed of reticular cells, fibers and macrophages. Similar structural differentiation of the marginal zone has been described in advanced fetal spleens of domestic animals (Bacha and Bacha, 2000; Holkunde et al., 2023). Overall, the findings demonstrate progressive prenatal maturation of the spleen in Kosali cattle, characterized by coordinated development of hematopoietic and immunological components essential for postnatal immune function.


 
Histochemical assessment
 
Connective tissue fibers analysis
 
Progressive maturation of the fetal spleen was accompanied by a marked increase in the deposition, organization and complexity of connective tissue fibers, including collagen, elastic and reticular fibers, within the capsule, trabeculae and splenic parenchyma across successive developmental groups. These changes reflect gradual strengthening of the stromal framework essential for structural support and functional compartmentalization of the developing spleen. Collagen fibers were absent in Group I, indicating the primitive and immature nature of the early splenic stroma. From Group II onward, collagen fibers became distinctly evident within the capsule and trabeculae, where their density progressively increased and paralleled that of smooth muscle fibers. This continued increase through Groups III and IV suggests progressive reinforcement of the splenic supporting framework in response to rapid expansion of vascular and parenchymal elements (Fig 6). Similar gestational increases in collagen deposition have been reported in prenatal goat and sheep spleens, where collagen accumulation was associated with stromal maturation and capsular strengthening (Gupta et al., 2017; Haldar et al., 2021; Bhagyalakshmi et al., 2025).


 
Elastic fibers analysis
 
Elastic fibers were sparsely discernible even in Group I; however, their distribution became progressively more prominent in deeper regions of the splenic capsule and trabeculae with advancing gestation, attaining maximal density in Group IV (Fig 7). The increasing abundance of elastic fibers likely corresponds to the growing requirement for tissue elasticity and resilience, particularly within vascularized stromal components, as splenic blood flow and mechanical stress increase during fetal growth.


 
Reticular fibers analysis
 
Reticular fibers first appeared as fine, delicate and loosely arranged fibrils in Group I. In Group II, they remained sparse and short, with minimal increase in density through Group III. By Group IV, however, reticular fibers became markedly more prominent, particularly within the fibromuscular subcapsular layer and trabeculae, where they appeared thicker, coarser and more undulating in arrangement. Within the red pulp, reticular fibers formed a characteristic wavy meshwork around splenic cords, likely contributing to the establishment of the microreticular framework necessary for hematopoietic and immunological cell support (Fig 8, 9). Similar progressive reticulin network formation has been documented during prenatal splenic maturation in domestic ruminants and is considered a hallmark of stromal differentiation and lymphoid organ maturation (Djalal et al., 2020; Holkunde et al., 2023). The presence of transversely oriented reticular fibers in the hilar region further suggests regional variation in trabecular organization and stromal specialization within the developing spleen. Collectively, the increasing density and complexity of connective tissue fibers observed with advancing gestational age indicate progressive microarchitectural maturation of the fetal spleen, facilitating enhanced mechanical integrity, vascular support and establishment of the hematopoietic and immunological microenvironment necessary for postnatal function.

The present investigation provides comprehensive baseline information on the prenatal histomorphological development of the spleen in Kosali cattle and demonstrates that splenic maturation occurs through a sequential and progressive process during fetal life, preparing the organ for its postnatal hematological and immunological functions. Hence, the present study establishes fundamental developmental data on prenatal splenic histogenesis in Kosali cattle and provides a valuable anatomical reference for future investigations in developmental biology, comparative anatomy, fetal immunology and indigenous bovine reproductive research.

The present study was supported by Dau Shri Vasudev Chandrakar Kamdhenu Vishwavidyalya (DSVCKV), Chhattisgarh.
 
Animal ethical approval
 
All procedures involving animal materials were reviewed and approved by the Institutional Animal Ethics Committee (IAEC) of College of Veterinary Science and A.H., Anjora Durg (C.G.), in accordance with the guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Government of India.
 
Disclaimers
 
The views and conclusions expressed in this article are solely those of the authors and do not necessarily represent the views of their affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.

The authors declare that there are no conflicts of interest regarding the publication of this article. No funding or sponsorship influenced the design of the study, data collection, analysis, decision to publish, or preparation of the manuscript.