Histochemical changes during development of Harderian gland in chicken

The Harderian gland is the major exocrine paraocular gland of the domestic fowl. It lies in the orbit ventral and postero-medial to the eyeball. It is a peripheral lymphoepithelial organ (secondary immune organ) which, together with the spleen, the bursa of Fabricius and the caecal tonsils forms a system of avian organs that determines both general and local immunity (Fix and Arp, 1991 and Shirama et al., 1996). It also represents an important part of the immune barrier CALT - Conjunctiva Associated Lymphoid Tissue (Khan et al., 2007, Pawar et al., 1998 and Payne, 1994). Harderian gland of chicken possesses macrophages, lymphocytes, granulocytes in the subepithelial layer and lumina of the lobules for the local immunity of the eye orbit (Baba et al., 1990). It is an actively secreting structure with abundant of plasma cells. Therefore, present study was planned to elucidate the histochemical changes during pre and post hatched development of Harderian gland of chicken.

The present study was conducted on 64 samples of Harderian Gland of Chicken (32 pre hatched embryonated eggs and 32 post hatched chicks) procured from Poultry Farm of Nagpur Veterinary College, Nagpur. Eight samples each of incubated fertile eggs were collected on 14^th, 16^th, 18^th and 20^th day of incubation.  The post hatched birds were divided into four age groups viz 3^rd day, 10^th, 17^th and 24^th day with 8 chicks in each group. Due to very small size of the Harderian gland up to 16^th day of incubation, head was separated and processed for serial transverse section.  However, grossly an appreciable size of the gland was seen on the 18^th day of incubation on the ventro-medial aspect of the eyeball near the ventral obliqus muscle. The gland was dissected on 18^th day and 20^th day of incubation and processed for histological sectioning. The post hatched birds were sacrificed by cervical dislocation and glands were carefully dissected out from both sides of eyeball. After collecting, the samples were fixed in 10% neutral buffered formalin, Bouin’s fluid, Helley’s fluid and Orth’s fluid for further processing. The histochemical studies were conducted by using following techniques:
 
A.   General carbohydrates or glycogen were demonstrated by using Mc Manus’s Periodic acid Schiff (PAS) technique (Bancroft et al., 1996). In this procedure, a positive reaction is indicated by the appearance of magenta colouration resulting from the reaction between aldehydes liberated from 0.5% periodic acid and the decolourized solution (leucofuchsin) of Schiff’s reagent.
B.    Acid and strongly sulphated mucopolysaccharides were demonstrated by using the Alcian blue method (Luna,1972). By this method, acid mucins exhibit blue stainabilities.
C.   AB/PAS for acidic and neutral mucosubstances (Bancroft et al., 1996).
D.  Sudan black B method for lipid (Luna, 1972).
       
Thereafter, the stained tissue sections were examined under the Zeiss photomicroscope supplied by Axiocam ERc 5s camera and photomicrography was performed.

In early embryonic stage, the Harderian gland was in the developing stage and appears only as aggregation of primordial cells. There was no well defined parenchyma, septa and capsule.  In the present study, it was noted that PAS, AB, AB/PAS and Sudan black B activity was not seen in any of the component of developing Harderian gland up to 16 days of incubation.
 
Glycogen
 
On 18^th day of incubation, gland appeared as branched tubular structure and had contorted lumina delineated by epithelial cells. Very weak PAS positive activity was seen in the secretory granules in the cytoplasm of epithelial cells and luminal surface epithelium of tubule. At few places, PAS positive material was confined to the apex of tubule (Fig 1). By the 20^th day of incubation, Harderian gland was more developed and seen to be a branched tubuloacinar type surrounded by thin layer of connective tissue. Mild PAS positive activity was observed towards the free border of luminal surface of few acinar epithelial cells and ducts. This indicates that the PAS activity increases with the development of connective tissue component of Harderian gland with the advancement of age.
 

        
The observations of the present study indicated that the Periodic acid schiff’s reaction in the capsule and septa of the Harderian gland in 3 and 10 day old chicks was moderate (Fig 2), which subsequently became intense in 17 day old chick and reduced to mild to moderate with the advancement of age in 24 day old birds.
 

        
It was noted that moderate PAS positive activity is observed in the acinar epithelium and intense PAS positive activity in the tubular epithelium of the Harderian gland at 3, 10, 17 and 24 days of age. PAS activity was abundant in the epithelium of the secondary tubules along with tertiary tubule (Fig 3). In agreement with the findings of the present study, Wight et al., (1971) in domestic fowl, Kozlu et al., (2010) in osprey and Kozlu and Altunay (2011) in quail, reported that PAS positive material was present in the epithelium and lumina of the corpus glandulae and tubules. They further stated that many epithelial cells of the tertiary tubules contained PAS positive material, which appear to accumulate towards the apical border and then get extended in to the lumina.
 

        
In concurrence with the observations reported by Burns and Maxwell (1979) in fowl, Boydak and Aydin (2009) in geese, Mobini (2014) in Ross broiler, El-leithy (2015) in rabbit and Frahmand and Mohammadpour (2015) in ostrich regarding the presence of PAS positive material in the apical cytoplasm of the epithelial cells of the main collecting duct of Harderian gland, it was noted during the present study that the luminal surface epithelium of acini showed accumulated PAS positive material in all different age groups of chicken which were more than 3 days of age. It was also observed that the cytoplasm of the epithelial cells of the main duct showed secretion towards the free border of cells which was strongly PAS positive (Fig 4).
 

        
The findings of the present study regarding presence of intense PAS positive activity in the plasma cells and Russell bodies in the interstitial connective tissue of the Harderian gland at 17 and 24 days of age (Fig 5) coincides with the observations reported by Wight et al., (1971) in domestic fowl and Bejdic et al., (2014) in laying hens.
 

 
Acidic mucopolysaccharides
 
During the present study, it was observed that the capsule, septa and epithelial cells of the glandular unit and duct showed AB positive activity wherein interstitial cells appeared pink in color with the AB pH 1.0 staining.
       
In the developing embryonic Harderian gland, weak AB positive activity was noted in the cells of acini and tubule on 18^th and 20^th day of incubation of chick.
       
AB (pH 1.0) reaction in the epithelial lining of glandular unit and duct of the Harderian gland in 3 and 10 day old chicks was moderate, which became moderate to intense in 17 and 24 day old chicks (Fig 6).
 

 
By the AB pH 1.0 staining, it was noted that the cytoplasm of all epithelial cells stained blue which indicates the presence of acidic and strongly sulphated mucopoly saccharides as well as presence of plasma cells in the interstitial tissue.  The present findings were in parallel with the reports of Wight et al., (1971) in domestic fowl. Similarly Reshag et al., (2016) observed that the cytoplasm of the epithelial lining of secretory unit and ducts stained blue which indicates the presence of acid mucoploysaccharides in the Harderian gland of domestic pigeon.
 
Acidic and neutral mucopolysaccharides
 
In the present study, it was noted that on 18^th day of incubation, the connective tissue fibers of the capsule and interacinar space showed mild AB positive activity. Epithelial cells of the acini and tubules, showed mild PAS-positive activity. By the 20^th day of incubation, Harderian gland was more developed and seen to be a branched tubuloacinar type surrounded by thin layer of connective tissue capsule. A mild to moderate AB positive activity was seen in the capsule and parenchymal connective tissue strands. Epithelial cells of the acini and tubule were weak or mild PAS positive while in the tubule, PAS positive material was seen to accumulate towards the apical border (Fig 7). This indicates that the AB/PAS activity increases with the development of glandular epithelium of Harderian gland with the advancement of age.
 

       
AB/PAS activity in the epithelial lining of tubule and acini of the Harderian gland in 3 and 10 day old chicks was moderate, which subsequently became moderate to intense in 17 day old chick (Fig 8) and intense with the advancement of age in 24 day old birds. Intense activity of the alcian blue was noted towards free border of the tubular epithelial cells of main duct (Fig 9).
 

 

 
In agreement with the findings of the present study regarding AB/PAS activity Wight et al., (1971) reported that the Alcian blue color reaction was confined to the apical cytoplasm of secondary and main central tubules of Harderian gland of fowl. Kozlu et al., (2010) in osprey reported that epithelial cells of the secretory units and duct showed AB-positive cells after staining with AB/PAS-staining where granules of the secretory cells stained a light blue color with less extent a distinct red color. Similarly Boydak and Aydin (2009) in geese, Mobini (2012) in chicken and Frahmand and Mohammadpour (2015) in ostrich observed AB-PAS activity in the all epithelium lining and duct as well as apex of the glandular units of the Harderian gland.
       
The findings of the present study regarding presence of intense Schiff’s reaction after AB/PAS treatment in the plasma cells and Russell bodies in the interstitial connective tissue of the Harderian gland at 17 and 24 days of age (Fig 10) coincides with the observations reported by Wight et al., (1971) in domestic fowl.
 

 
Lipid
 
The present observation noted that small amount of lipid droplets were aggregated towards the lumen of tubule epithelium on 18^th and 20^th day of incubation of chicken.
       
Sudan black activity in the epithelial lining of tubule and acini of the Harderian gland was mild to moderate at 3 days of age and moderate at 10 days to 24 days of age (Fig 11). The luminal surface epithelium of collecting duct showed intense activity with the advancement of age (Fig 12). However, the capsule and septa of Harderian gland showed weak or negligible activity for presence of lipid with Sudan black B staining.
 

 
              
 
These findings of the present study were in accordance with the findings of Wight et al., (1971) in fowl. They noted lipid granules in the cytoplasm of epithelial cells of the acini and tubules of Harderian gland. Similarly in agreement with the observations of present study, Hussein et al., (2015) in guinea pig observed Sudan black positive activity in the intracellular cytoplasmic vacuoles and luminal secretion of Harderian gland.

The present study concluded that PAS, AB/PAS, Sudan black B activity in the epithelial lining of tubule and acini as well as in connective tissue of the Harderian gland increased with advancement of age. All epithelial cells of the glandular unit of Harderian gland contain both neutral and acidic mucins. Most of the epithelial cells of the secretary units and ducts stained blue with Alcian blue that indicates the presence of mostly acid mucopolysaccharides. The Plasma cells and Russell bodies in the interstitial tissue of the Harderian gland showed strong PAS positive reaction. The luminal surface epithelium of collecting duct and secretory materials inside the lumen showed intense activity of Sudan black B with advanced age. It showed that lipid was mostly confined to the cells lined the tubules.