Externally, the liver was surrounded by a thick layer of white fibrous connective tissue, the Glisson’s Capsule formed of primarily collagen fibres, few elastic and reticular fibres, fibrocytes etc. as reported by
Dellmann (1993) and
Thakur et al., (2019) in buffalo. The capsule was composed of collagen and reticular fibres in sheep and goat
(Mandal et al., 2002) and in buffalo foetus
(Doley et al., 2006). The interlobular connective tissue was scarce. However when compared qualitatively between males and females, the connective tissue and particularly the collagen fibres were comparatively more in males (Fig 1) as compared to female. Earlier
Nishimura et al., (2011) in chicken and
Mik et al., (2018) in pigs had reported more amount of connective tissue and specifically collagen fibres in male pigs as compared to female. The incidence of hepatic fibrosis is largely male dominating
(Shimizu et al., 2007). Mik et al., (2018) in pigs also measured quantitatively and observed that connective tissue content was largest in quadrate lobe and minimum in left medial lobe however no difference in distribution of connective tissue in different lobes of male and female liver was observed in present study. No interlobular septa were observed and the hepatic cords of one lobule merged into the adjacent lobules. The plates of hepatocytes and sinusoids in each hepatic lobule appeared to spread outward from the liver lobule’s central vein to its periphery (Fig 2), as observed earlier by
Thakur et al., (2019) in buffalo.
The parenchyma of the liver was comprised of hepatocytes which were arranged as hepatic cords around central veins as also reported earlier by
Thakur et al., (2019) in buffalo and
Cıraklı and Kuloğlu, (2024) in goose liver and
Kalita et al., (2023) in Pati ducks. Hepatocytes had polyhedral shapes with clearly defined borders. They had a centrally placed spherical nucleus with nucleolus. Mostly the hepatocytes were mononucleated but binucleated hepatocytes (Fig 3) were also observed. Different shapes of hepatocyte like small, murky and hazily were observed. This might be because of the animal’s physiological condition at the time of sampling. Many vacuoles were seen in hepatocytes. These empty vacuoles could be lipid inclusions which may appear empty after processing of tissues. Reticular fibres formed a network in the hepatic parenchyma (Fig 4)
Dellmann (1993) reported that the hepatocytes have lots of lipid and glycogen inclusions which gives them a foamy or honeycomb look and that cytoplasm of hepatocytes exhibit a range of morphologies in response to dietary and cellular alterations.
Chen et al., (1996) reported bilirubin granules in the cytoplasm of hepatocytes, which could not be observed in the current investigation. The cytoplasm of hepatocytes was acidophilic. The micrometrical observations on hepatocytes in different lobes of male and female livers have been summarized in Table 1, 2 ,3 ,4. In present study it was noted that the size of hepatocytes in perivenous and periportal area was numerically higher in male than the female but the difference was not significant (p>0.05). Similarly the size of hepatocytes in different lobes of males and females did not vary significantly (p>0.05).
Singh (2019) reported the average size of hepatocyte as 15.072±0.26 µm whereas size of hepatocyte in perivenous area was 12.36±0.25 µm and size in periportal area was 15.18±0.28 µm while
Sasan et al., (2017) reported 12.86±0.49 µm in pig.
From the data on average size of nucleus of hepatocytes in different lobes it was concluded that size of nucleus did not vary significantly (p>0.05) between different lobes and between male and female liver.
The average number of hepatocytes were calculated per 78073.92 µm
2 in periportal and perivenous region in different lobes of male and female livers. It was recorded that the number of hepatocytes was higher in females as compared to males, but the variation was not statistically significant.
Marcos and Correia-Gomes (2015) reported higher hepato cellularity in female rats with higher number of binucleated cells as compare to male rats. The average number of hepatocytes also did not vary significantly (p>0.05) between different lobes of male and female liver.
It was observed that the number of binucleated cells in different lobes of liver vary significantly (p<0.05) between male and female and the number of binucleated cells was significantly higher (p<0.05) in periportal region in different lobes of liver in male and female as compared to the perivenous region (Fig 5). It was observed that female liver had significantly (p<0.05) higher number of binucleated cells as compared to male liver. This can be related to the reason that these binucleated cells act as an important reservoir of cells that helps in generation of mononucleated hepatocytes rapidly by amitotic cytokinesis as reported by
Gandillet et al. (2003). A nuclear protein called proliferating cell nuclear antigen (PCNA) is most abundant in the S phase of proliferating and transformed cells. In the present study, the mitotically active hepatocytes showed immunoreactivity with PCNA antibody (Fig 5).
Theocharis et al. (1994) reported that the majority of the time, PCNA was expressed by big hepatocytes with a large nucleus, prominent nucleoli and microvesicular fatty cytoplasm, as well as binucleated ones with a lot of pale cytoplasm. In periportal regions, PCNA expression was more noticeable (Fig 5).
It was unclear exactly why there were more mononuclear and binuclear hepatocytes in the periportal, however
Junatas et al., (2017) opined that these areas of the liver lobes were the oldest in terms of development and so had the most significant polyploidy cells. Binucleated or cells with tetraploid nuclei are created when mitosis or cytokinesis do not take place. Hepatocytes pile up first as a result of proliferation, then swiftly rearrange themselves to produce the well-known cords. Additionally, it was noted that the quantity of binucleated cells rose following hepatectomy. This demonstrated that mature hepatic cells participated in regeneration and that these cells divide to produce daughter cells with increased ploidy levels. This increased number of binucleated hepatocytes in the periportal region might be involved in the liver’s regeneration process.
Hepatocyte cell membranes that were directed toward the portal triad region generated a distinct surface known as the limiting membrane and Space of mall (Fig 6) was observed between the limiting membrane and the portal traid area as also reported by
Dellmann (1993) in domestic animals.
Kupffer cells
The liver macrophages known as kupffer cells were intra sinusoidally situated (Fig 7). These are effective inflammatory response mediators that secrete a variety of bioactive substances and are crucial for immune defence
(Bouwens et al., 1992). In the present study, Kupffer cells were found to be stellate or occasionally oval-shaped phagocytic cells with a large nucleus and cytoplasm similar to those observed by
Pareek (2000) in sheep,
Mahata et al., (2003) in spotted deer,
Sasan et al., (2017), Kalita et al., (2019) and
Singh (2019) in pig. From the micrometrical data on Kupffer cells it was concluded that in comparison to male goats, female goats had a higher number of Kupffer cells and the difference was statistically significant (p<0.05). However the number of Kupffer cells was higher in the periportal region than perivenous region of different lobes of liver in male and female, but the variation was not significant. The more number of Kupffer cells in females might be related to more estrogen levels as
Marcos and Correia-Gomes (2015) observed that oestrogen causes a five-fold increase in Kupffer cell proliferation in rat.
Colantoni et al., (2000) also noted that juvenile female rats had higher pathology scores, more severe neutrophil infiltration and greater endotoxin levels than males, which ultimately led to a stronger activation of Kupffer cells. The presence of Kupffer cells in the sinusoidal compartment was confirmed by
Neyrinck et al., (2000) who also concluded that these cells played a significant role in the regulation of numerous hepatocytic functions and in the immune biology of the liver under both healthy and pathological circumstances (
Parker and Picut, 2012). According to
Kalita et al., (2019), Kupffer cells in pigs were prominently triangular in active phagocytic cell form and flattened in inactive form. These cells provided first line of defence in the liver. Kupffer cells may urge HSC to make matrix, boost cell proliferation and release retinoids by utilising cytokines and reactive species (
Usynin and Panin, 2008). In this study, the Kupffer cells expressed immunoexpression for CD68 antibody (Fig 8).
Ito cell (Hepatic Stellate cells HCs)
HSCs were primarily found in the Space of Disse which is subendothelial area between hepatocytes and sinusoids. HSCs were oval, ellipsoidal, or round cells with spherical nucleus as also reported by
Singh (2019) in pig liver. It was observed that the number of HSCs was more in females as compared to male and the difference was significant (p<0.05). However no significant difference was observed in periportal and perivenous regions of different lobes in males and females. According to
Krishna (2013), in the human liver, these cells were strongly connected to tiny nerve terminals and were crucial for the regulation of sinusoidal blood flow, fibrogenesis and vitamin A storage. These cells are well established to play a significant part in cirrhosis, hepatic fibrogenesis and regeneration
(Fehrenbach et al., 2001). However, according to
De Bleser et al. (1991), stellate cells were more frequently found in periportal areas than in pericentral areas in man. Hepatic stellate cells, also known as Ito cells, were studied by
Grigoraş et al. (2016), who reported that these cells mobilised and stored vitamin A, which was crucial for the progression of fibrosis in chronic hepatic pathology, regeneration, metabolism regulation and modulation of local inflammatory and immune responses.
Lymphocytes/Pit cells
A unique population of large granular lymphocytes associated with the liver is represented by pit cells. These lymphocyte-looking cells could be found in a number of non-lymphoid organs, including the liver and circulate in the blood
(Bouwens et al., 1987). In the current study, it was observed that one to two lymphocyte aggregations were present in the liver parenchyma. Small random foci of lymphocytes were frequently noted without any toxicological significance as reported earlier by
Swindle et al., (2012). In addition to this, a few lymphocytes were also seen in the portal traid area’s connective tissue. Pit cells have been used to describe these lymphocytes by
Malarkey et al. (2005). Subpopulations of lymphoid cells associated with the liver play a crucial role in antigen presentation. The lymphocyte population of a healthy liver is primarily found in the portal tract, while it can also be dispersed throughout the parenchyma. Therefore, it is not unexpected that immune-mediated destruction caused by a variety of pathogenic xenobiotic and tumor-originating agents targets hepatocytes and biliary epithelial cells (BECs) as described by
Kimura et al., (2005). The liver’s unusual capacity to share immunological information continually is explained by its anatomical position and specific vasculature. For unclear reasons, many liver-resident lymphocytes differ from circulating lymphocytes phenotypically and functionally.
Central vein, Sinusoids, portal vein and hepatic arteriole
The sinusoids were thin walled blood vessels which were lined by endothelium. The sinusoids receive blood from branches of hepatic artery and portal veins. So there is mixed blood from hepatic artery and portal vein. The blood from sinusoids drain into central veins. It was observed that size variations of sinusoids in different area of lobes in male and female was not significant (p>0.05). This small discrepancy might be due to variation in sizes of hepatocytes as sinusoids adjust their size according to space available between hepatocytes. The sinusoids were lined by endothelial cells as reported by
Mahata et al. (2003) in spotted deer,
Bamaniya (2013) in Marwari goat and
Singh (2019) in pig.
The central vein was also lined by endothelium. The difference in size of central vein and portal vein in different lobes in male and female liver was insignificant (p>0.05%). The portal vein was lined with simple squamous epithelium as had thin wall as reported earlier by
Sasan et al., (2017) and
Singh (2019) in pigs. Portal vein showed immunoreactivity with vimentin antibody (Fig 9). Vimentin is a major intermediate filament in the endothelial cells which are of mesenchymal origin
(Liu et al., 2010).
The interlobular branches of the hepatic artery known as the hepatic arterioles either joined the capillaries in the portal region or enter the sinusoids directly. The difference in size of hepatic arteriole in different lobes and between male and female was not found to be significant in this study.
Singh (2019) reported slightly higher size of hepatic arteriole as 33.14±1.14 µm in pig liver. Hepatic artery also showed immunoreactivity with vimentin antibody (Fig 9).
Bile ductules and Bile canaliculi
The bile ducts were lined by cuboidal cells with round nuclei (Fig 10) Occasionally two to three bile ducts could also be observed in portal area. The epithelium in larger bile duct was columnar. A few number of goblet cells were also detected sometimes within the duct epithelial cells. These findings were in concurrence with findings of
Pareek (2000) in sheep,
Bamaniya (2013) in Marwari goat and
Singh (2019) in pigs. The difference in size of bile ductules in different lobes of male and female was not significant. Bile canaliculi could be seen as expanded intercellular space between apposing hepatocyte cell membranes.
Lymphatics and Nerve fibres
In addition to the portal vein, hepatic artery and bile duct, the portal triad area also contained nerve fibres and thin-walled lymphatic vessels (Fig 10). The lymph vessels were lined by simple squamous epithelium encircled by loose connective tissue from the portal triad region as reported earlier by
Singh (2019) in pig liver. According to
Magari et al., (1979), the area of Disse in dogs, pigs, calves and rabbits did not directly communicate with lymphatics. They also reported presence of thick bundles of collagen fibres which may play a crucial role in absorption and movement of lymphatic.
