Histomorphological studies
The present investigation comprehensively studied the histological, histochemical and ultrastructural features of the thyroid gland of Murrah buffalo (
Bubalis bubalis). The thyroid gland was enclosed in a tri-layered connective tissue capsule (Fig 1) comparable to cattle (
Igbokwe and Ezeasor, 2015a) and goat (
Joshi, 2016) but contrarily,
Baishya et al., (1998) and
Hussain and Al-Taay (2009) reported bi-layered capsule in yak and buffalo, respectively.
The outer layer contained collagen fibers parallel to the parenchyma (Fig 2) with few reticular fibers, fibroblasts and sparse elastic fibers similar to the cattle (
Igbokwe and Ezeasor, 2015a). The middle layer was thickest and most loosely packed, comprised adipose tissue, blood vessels, nerves and collagen fibers. The inner layer adhered closely to glandular parenchyma similar to goat (
Shehan, 2017). The mean capsular thickness of the thyroid gland in buffalo was 344.37±9.46 µ.
The glandular parenchyma was separated into incomplete lobules by the connective tissue trabeculae emerging from the capsule (Fig 3) and large-sized adipocytes were observed within prominent trabeculae consistent with previous findings in goat (
Joshi, 2016). The parenchyma was composed of clumps of follicles within the lobules, surrounded by a small amount of interfollicular stroma similar to goat (
Shehan, 2017). Interfollicular spaces comprised of connective tissue (Fig 3), blood vessels, nerves, fibroblasts, fibrocytes, mast cells and parafollicular cells. Follicles varied greatly in shape and size (Fig 1 and 4) due to various irregularly arranged follicles cut at different planes in a particular section and according to glandular activity. The shape of follicles was predominantly round to oval, but triangular, quadrilateral, polygonal, pyriform, bilobed and tubular-shaped follicles were also observed (Fig 4 and 5). The follicles were classified as small (0 µ to 50 µ), medium (50 µ to 100 µ) and large (> 100 µ) follicles based on their diameter which aligned with the results in sheep (
Khaleel and Salih, 2017). Small, medium and large thyroid follicles had mean diameters of 36.18±1.04 µ, 72.75±1.38 and 185.98±6.65 µ, respectively. However, according to
Baishya et al., (1998), the average diameter of thyroid follicles in mithun and yak was 89±7.06 µ and 92.80±6.60 µ, respectively.
The follicular lumen was filled with a viscous, homogeneous fluid called colloid, which was eosinophilic and stained differently across follicles, suggesting varying stages of metabolic activity (
Igbokwe and Ezeasor 2015b). The majority of the small and medium-sized follicles were found closer to the margin of the gland (
Hussain and Al-Taay, 2009) and were primarily round to oval in shape. Medium-sized follicles were the most numerous, followed by large and small follicles. The majority of small and medium-sized follicles contained light-colored, less viscous colloids with a variable number of peripherally located vacuoles (Fig 4). It could be considered in active metabolic stages, as they were lined mainly by low-columnar or high-cuboidal epithelium with a central spheroid nucleus and eosinophilic cytoplasm consistent to the previous findings in camel (
Abdel-Magied et al., 2000) and sheep
(Nabi et al., 2018).
The large follicles were mostly asymmetrical and found in the deeper portion of the gland, in consonance to the observations in sheep
(Nabi et al., 2018) while
Peksa et al., (2011) reported the uniform follicular distribution in cattle. Presence of squamous epithelium and dark-colored, semi-solid colloid in majority of the large follicles consistent to the previous reports in sheep
(Ali et al., 2020). A few bilobed, half-filled, empty and ruptured follicles were also observed. A few ruptured follicles considered as degenerating follicles containing cellular debris of nucleated and some non-nucleated cells were encountered as reported previously in thyroid gland of human
(Kelly et al., 1984) and sheep (
Roy and Saigal, 1986 and
Rajalakshmi et al., 2019).
Follicular and parafollicular (light or C) cells were the two cell types that made up the follicular epithelium (Fig 5). A few parafollicular cells were encountered in the follicular epithelium and none of them extended upto the follicular lumen (Fig 5). The follicular lining was predominantly simple cuboidal, low columnar, or squamous type (Fig 4 and 5) of epithelium, depending upon the functional state of the follicles consistent to observations in goats (
Joshi, 2016) whereas,
Hussain and Al-Taay (2009) did not observe columnar-type cells in buffalo. The mean epithelial height of squamous and cuboidal cells recorded in the follicular epithelium was 6.72±0.1 µ and 9.85±0.14 µ, respectively. However,
Peksa et al., (2011) recorded the highest follicular height as 9.42±1.50 µ in bulls whereas,
Igbokwe and Ezeasor (2015a) reported that the mean follicular cell height was 5.53±0.08 µ in adult cattle. A few follicles possessed two types of epithelial cells,
i.e., low columnar and cuboidal or cuboidal and squamous (Fig 5), which could be considered in the transition stage of metabolic activity in conformity with the observations in sheep (
Khaleel and Salih, 2017). A few follicular cells were also found in the interfollicular area. The parafollicular cells resided mainly in the interfollicular areas, mostly solitary and occasional in clusters (Fig 6), as reported earlier in buffalo (
Hussain and Al-Taay, 2009) while no C-cells were observed in camel (
Kausar and Shahid, 2006). The number of parafollicular cells was higher in the deeper part of the gland than the peripheral part of gland
(Okada et al., 1995 in sheep). Parafollicular cells were generally round or oval, with indistinct boundaries, lightly basophilic nuclei and lightly eosinophilic cytoplasm having larger nucleus than those of follicular cells (
Hussain and Al-Taay, 2009). Myoepithelial cells and fibroblasts were also present near the follicular basement membrane.
The isthmus of the buffalo thyroid gland was also glandular and covered by a connective tissue capsule like cattle
(Igbokwe et al., 2015); whereas there was fibrillar appearance of colloid in isthmus of camel (
Abdel-Magied et al., 2000). Parafollicular cells were observed in the interfollicular area, albeit in smaller proportion as compared to lateral lobes (
Khaleel and Salih 2017 in sheep); however,
Okada et al., (1995) reported absence of C-cells in isthmus of sheep.
In current study, ultimobranchial bodies were observed in the thyroid gland of 3 out of 12 buffaloes. These ultimobranchial bodies varied greatly in structure, shape (round to pearl) and size and were mainly found in the deeper part of the glandular parenchyma (Fig 7), surrounded by numerous blood vessels. The capsule of ultimobranchial bodies was made up of stratified squamous epithelium and their lumen contained smooth muscle fibers intermingled with other cell types in consonance to the findings in sheep (
Roy and Saigal, 1986).
A small number of ultimobranchial follicles (Fig 8) having light-colored, foamy colloid containing variable amounts of cellular debris and a stratified epithelium were also observed as documented previously by
Roy et al., (1978) in goat.
Sayed et al., (2005) observed that ultimobranchial body remnant of adult buffalo appeared in the form of irregular and elongated follicles of variable size and shape with predominance of calcitonin cells while
Rajalakshmi et al., (2019) suggested that ultimobranchial follicles may act as a source of follicular cells in sheep.
Histochemical studies
The follicular colloid exhibited a strong PAS-positive reaction (Fig 9) indicating the presence of neutral mucopolysaccharides and glycoproteins as observed in sheep
(Rajalakshmi et al., 2019) and Chabro chicken
(Vishen et al., 2021). The strong PAS reaction in colloid could be suggestive of its synthetic and transformation activities. The connective tissue content, follicular cells, ultimobranchial bodies and blood vessels showed a weak to moderate PAS reaction, whereas parafollicular cells did not show any reactivity consistent with observations in sheep
(Rajalakshmi et al., 2019).
The connective tissue content displayed moderate to strong; follicular cells showed moderate (Fig 9), whereas parafollicular cells and the colloid exhibited negative aclianophilic reaction for the presence of acid mucopolysaccharide reactions whereas,
Sarma et al., (2013) documented very weak alcianophilic reaction in connective tissue components in goat.
Follicular cells exhibited a moderate reaction (Fig 10), while parafollicular cells and colloid expressed a negligible to weak reaction for lipids whereas in earlier report on sheep thyroid there was accumulation of lipids droplets in the follicular cells, C cells and ultimobranchial follicles
(Rajalakshmi et al., 2019).
Follicular cells demonstrated moderate, while parafollicular cells exhibited weak reaction for basic proteins; whereas,
Sarma et al., (2013) reported that the parafollicular cells exhibited weak to strong reaction in goats; however,
Vishen et al., (2021) reported intense reaction for basic proteins in thyroid follicular cells of Chabro chicken. The blood vessels exhibited strong reaction, whereas connective tissue and ultimobranchial bodies showed a mild reaction for presence of basic proteins.
Connective tissue, follicular cells, parafollicular cells and colloid showed no reaction for iron and a negligible to weak reaction for calcium, contrary to the observations of
Joshi (2016) in goat thyroid. Blood vessels of the capsule and parenchyma of the gland showed a weak to moderate presence of iron and calcium.
Ultrastructural studies
Transmission electron microscopy confirmed light microscopy findings. Follicular cells (Fig 11) exhibited euchromatin and heterochromatin, with dilated rER cisternae, as observed in camels
(Atoji et al., 1999). Nuclei were lined by heterochromatin, with the nucleolus as the largest clump
(Singh et al., 2023). Follicular cells appeared low columnar, cuboidal (Fig 11) or squamous with polymorphic, often indented nuclei indicative of cellular activity.
Igbokwe et al., (2015) revealed that younger goats had cuboidal cells while older ones had flattened cells indicating metabolic status. Follicular cells contained secretory vesicles of varying size and electron density, representing colloid droplets
(Igbokwe et al., 2015); electron light vesicles were less active, while electron dense vesicles were more active (Fig 11).
Follicular cells were polarized, with organelles distributed differently between the basal and apical regions. The apical surfaces were surrounded by colloid. rER strands were dispersed in the cytoplasm, some attached to the nuclear membrane and occasional phagocytic vesicles were present (Fig 11). Parafollicular cells (Fig 12) were oval to elongated, with numerous dense granules of variable electron density and size
(Singh et al., 2023) in cattle and camel, respectively. The isthmus parenchyma was loosely packed and richer in connective tissue than lateral lobes.
