Gross topography and morphology
The thymus consists of a paired cervical part and an unpaired thoracic part, as observed from groups I to IV
Chaurasia et al. (2019). The cervical thymus has right and left limbs: the right limb extends cranially to the middle part of the thyroid cartilage, while the left limb reaches the level of the cricoid cartilage across all groups (Fig 1 and 2). These findings are consistent with previous research by
Nickel et al. (1977) in calves,
Dyce et al. (1987),
Machado et al. (2001) in buffalo,
Cunningham et al. (2001) in unborn lambs and
Baishya et al. (2001) in pig fetuses, as well as
Konig and Leibich (2004). The significant increase in crown-rump length, fetal weight, thymic weight and volume from group I to group IV indicate symmetrical exponential growth of the thymus relative to crown-rump length and fetal size. Similar reports have emerged, such as the one by
Baishya et al. (2001), which documented that in crossbred pig fetuses, thymic weight displayed ascending ontogeny tendencies with advancing fetal age.
Press and Landswork (2006) found that the thymus progressively increases in size up to the age of puberty.
Igbowke and Ezenwaka (2017) reported that in indigenous Large White pigs, the absolute weight of the thymus shows significant (p≤0.05) increases in size with advancing fetal age, though the increase is not significant in the postnatal period.
The weight and volume of the left limb of the cervical thymus increased, showing a maximum ten- and eleven-fold rise respectively from group I to group II, although this change was not statistically significant (Fig 2). In contrast, the minimum increases in weight and volume were recorded from group III to group IV, which were significant. The thymic index of the left limb exhibited highly significant differences from group II to group III, peaking at that stage (Table 1). The presence of paired cervical and junctional parts alongside the unpaired thoracic part in group I (corresponding to the apparent first trimester of pregnancy) contradicts previous reports by
Baishya et al. (2001), who noted that all fetuses had cranial and cervical lobes by days 45 to 46, while the thoracic lobe became evident between days 50 and 52 (second trimester of pregnancy). Similarly,
Hyttel et al. (2010) found that in horses at 60 days and dogs at 30 days of gestation, the thoracic part forms only after these time frames. According to
Press and Landswork (2006), the thymus originates as a solid epithelial outgrowth from the epithelium of the third pharyngeal pouch, developing into an irregular bi-lobed structure located in the cranial mediastinum during early life, with further development occurring until puberty.
Ramayya et al. (2008) reported that buffalo fetuses possess paired cervical and cranial parts, as well as unpaired thoracic parts within their thymus.
Hyttel et al. (2010) documented that developed thymus structures consist of a paired cervical part and an unpaired thoracic part in ruminants by 40 days of gestation. The significant reduction in the number of lobes from groups I to IV in this study may be attributed to the fusion of lobes with advancing age and the resultant increase in lobe size. The recorded weight, volume, length and width of the right limb of the thymus were greater than those of the left limb across groups I to IV, which contrasts with findings by
Ramayya et al. (2008) in buffalo fetuses, where they noted that the left limb was marginally longer than the right.
Histomorphological analysis
In the samples from all four regions of the thymus, specifically the thoracic area, cervical-thoracic junction and right and left cervical limbs, the capsule was primarily composed of loose to slightly dense areolar connective tissue (groups I to IV). Some samples from group I contained sections of various blood vessels. The vascularity of the capsule increased from group I to group IV, with notable aggregations of blood vessels present in multiple locations. This increase in vascularity was particularly evident in tissue samples from the cervicothoracic junction and thoracic regions of the thymus from groups II to IV. The average thickness of the capsule increased significantly from 38.12 ± 5.95 µm in group I to 138.8±3.5 µm in group IV (Table 2). The increase was significant between groups I and IV, II and IV, as well as between groups I, II and III. However, the difference between groups III and IV was not significant. Furthermore, the stroma of the thymus consisted of interlobar and interlobular septa made of loose to slightly dense irregular connective tissue (groups I to IV). The interlobar septa extended from the capsule, while the interlobular septa originated either from the capsule or the interlobar septa. The initiation of interlobular septa formation was observed in the thoracic and cervicothoracic junction samples of group I, but from groups II to IV, these septa distinctly divided the lobes into incomplete lobules across all thymic regions corroborating the findings of
Kadam et al. (2023). They reported that the thin connective tissue capsule of thymic lobes gives rise to septa to divide lobes in to lobules. In the samples of group I, the lobes were primarily oval in shape. In contrast, from groups II to IV, the lobules were typically oval, elongated oval, or polygonal. The average maximum diameter of the lobule increased from 498.83±27.16 µm in group I to 755.31±3.41 µm in group IV. Similarly, the average minimum diameter rose from 291.67±27.16 µm in group I to 426.67±6.16 µm in group IV. The increase in maximum diameter was significant between groups II and III, but not between groups III and IV. Conversely, the increase in minimum diameter was significant across groups II, III and IV. Additionally, the average number of thymic lobules per field (10X) decreased from 13.00±6.16 in group II to 5.33±0.21 in group IV. The decrease in the number of lobules per field was significant between groups II to IV. Lobules were not clearly distinguishable in group I. The septa contained sections of blood vessels, with their vascularity increasing from group I to group IV, particularly in the cervicothoracic junction and thoracic tissue samples from groups II to IV. The thickness of the septa gradually decreased from group I to group IV, while their vascularity increased during the same progression.
The stroma of the thymus consisted of interlobar and interlobular septa made of loose to slightly dense irregular connective tissue (across groups I to IV). The interlobar septa penetrated the parenchyma from the capsule, while the interlobular septa descended from either the capsule or interlobar septa (Fig 3). These septa contained segments of blood vessels, with both the density and size of these vessels increasing from groups I to IV. The loose connective tissue progressively transformed into dense connective tissue with fetal age. The thickness of the septa decreased from groups II to IV, corroborating the findings of
Lieshchova and Gavrilin (2020),
Ahmed et al. (2025), who recorded an increase in connective tissue in septa up to two months, followed by a decrease in
Bos taurus fetuses. Conversely,
Mainde et al. (2017) reported that the thickness of septa increased consistently until 142 days of gestation in goat fetuses, nearly reaching the end of the gestation period. The parenchyma of the lobes and lobules comprised different stages of lymphocytes, reticular cells, macrophages, mast cells, mesenchymal cells and other free connective tissue cells, along with fine connective tissue fibers (Fig 4). The initiation of differentiation of thymic lobular parenchyma into peripheral cortex and central medulla began in group I, though the boundary between these two areas was not distinct. A clear division between the cortex and medulla was observed starting from group II. The medulla served as a common entity for all lobules within a lobe, with interlobular septa dividing the lobes into incomplete lobules. Similar differentiation into cortex and medulla was noted at mid-gestation by
Chandra and Parmar (2004) and at 50 days of gestation by
Mainde et al. (2017) in goat fetuses and at 41 to 55 days by
Baishya et al. (2001) and at 60 to 75 days by
Igbowke and Ezenwaka (2017) in pig fetuses.
Histochemical analysis
Distinct cortex and medullary areas have been reported during fetal development. Well-developed collagen fiber bundles with a wavy course were observed in the capsule and interlobar septa of the cervical limb of the thymus in samples from group IV (Fig 5). This supports the findings of
Sharma et al. (2004), who noted that collagen fibers increase in number in piglets as they age. In groups I to III, collagen fibers appeared during the maturation phase, indicated by a gradual increase in staining intensity. The density of collagen fibers decreased from the capsule to the deeper parts of the interlobular septa. Additionally,
Baishya et al. (2001) reported the presence of collagen fibers in the capsule and trabecula of the thymus in pig foeti, noting that smooth muscle and collagen fibers together outnumber reticular fibers. Sparse, very thin, unbranched elastic fibers were recorded in the septa of group I. These fibers developed in both structure and density from group II to IV, with the most developed elastic fibers observed in the interlobular septa of group IV, as well as in the capsule and walls of blood vessels. This observation aligns with findings by
Uppal et al. (2007), who reported a limited number of elastic fibers in the thymic capsule of neonatal buffalo calves. The density of reticular fibers was greatest in group II, found in the capsule (Fig 6), septa and parenchyma of the lobule. However, their density decreased from groups II to IV, indicating that as lymphoid cells increased in number, connective tissue decreased with the advancing age of the foetus. This finding supports the observations of
Lieshchova and Gavrilin (2020) in
Bos taurus foeti. Similar distributions of reticular fibers in the thymus have also been documented by
Baishya et al. (2001) in pig foeti,
Chandra and Parmar (2004) and
Mainde et al. (2017) in goat foeti,
Uppal et al. (2007) in neonatal buffalo calves and
Ramayya et al. (2008) in buffalo foeti.
