histomorphological studies on the vomeronasal organ in domestic pig ( Sus scrofa domesticus ) #

studies on


__________________________________________________________________________ Nikhila et al.
The nasal cavity is the first segment of the respiratory tract and plays an important role in filtering, warming and moistening inspired air.Additionally, it contributes to the sense of olfaction.The vomeronasal organ enhances olfactory function, aiding in the discrimination of odours (Kumar, 1991).The vomeronasal organ in pig is a paired tubular structure that is situated on the ventral aspect of the median nasal septum on either side of the vomer bone.The organ is extended from the level of papilla incisiva to the level of fifth palatine rugae (Kumar et al., 2016).The vomeronasal duct is an epithelial tissue forming a hollow tube with sensory and non-sensory epithelium on the walls of the duct.This intricate structure plays a crucial role in detecting chemical signals and enhancing olfactory capabilities in domestic animals (ICVGAN, 1994).The olfaction holds a significant importance for most mammals, particularly for those whose young were born with underdeveloped visual and auditory abilities.In such cases, the detection of specific pheromones became crucial for the offspring to identify their mothers (Teicher et al., 1984 andHalpern, 1987).The "Flehmen response" is a distinct facial movement observed in various mammals, particularly felids and ungulates aimed at directing inhaled substances to the vomeronasal organ (Abbasi and Khosravinia, 2002, Besoluk et al., 2001and Kostov, 2007).The literature available on vomeronasal organ in domestic pig is very scanty.Hence the present study is undertaken to elucidate the histomorphological studies on vomeronasal organ in domestic pigs.

Materials and methods
For histomorphological studies, the vomeronasal organ was collected from domestic pigs after slaughter in AICRP on pigs, SVVU, Tirupati, at the level of papilla incisiva and first, third and fifth palatine rugae of hard palate.The specimens were washed in normal saline and were fixed in 10% Neutral Buffered Formalin.Further, the tissue samples were subjected to routine tissue processing techniques and embedded in paraffin wax (58-60 °C).The sections of 5-6 µm thickness were obtained on clean adhesive smeared glass slides with the help of Leica semi-automatic microtome (Leica RM2125RTS) and were subjected to Haematoxylin and Eosin method for routine histological examination, Masson's trichrome for collagen fibres, Verhoeff's method for elastic fibres, Gomori's method for reticular fibres, Bielschoswky method for nerve fibres (Singh andSulochana, 1997, Bancroft andGamble, 2008).Micrometrical studies on the mean thickness of the epithelium, propria submucosa (medial and lateral wall) and cartilage of the vomeronasal organ was measured statistically and analysed by using the one-way Anova (Snedecor and Cochran, 1994).

Results and discussion
At the level of papilla incisiva, the vomeronasal duct had a narrow elliptical lumen rostrally which became wider caudally.The medial and lateral walls of the duct were lined by stratified squamous non-keratinized epithelium (Fig. 1).Similar observations were reported by Vaccarezza et al. (1981) in rats.However, Salazar et al. (1997) in pigs, cows and horses observed stratified cuboidal or columnar epithelium.The epithelial height of the medial wall (58.5 ± 5.45 µm) was greater than the lateral wall (47.4 ± 2.08 µm).The stratified squamous nonkeratinized epithelium contained three layers namely stratum basale, stratum spinosum and stratum granulosum arranged from outside to the luminal side (Fig. 2).The propria submucosa of the vomeronasal organ had dense irregular connective tissue with collagen, elastic and reticular fibres as reported by Ganganaik et al. (2006) in sheep.Lymphocytic infiltration was also observed in the sub-epithelial connective tissue around the opening of glandular ducts.The vomeronasal glands were seromucous in nature and appeared more at the dorsal aspect of the duct which opened into the lumen of the vomeronasal duct on both walls.Apparently, the thickness of the medial propria submucosa (1047.79± 76.89 µm) was more than the lateral propria submucosa (398.51 ± 70.05 µm) (Fig. 1).The propria submucosa of both sides was continuous with each other towards the dorsal and ventral sides of the vomeronasal organ.The cartilage was C-shaped as it was incomplete in the dorsolateral region as reported by Moawad et al. (2017) in goats.Contrary to present study, the cartilage was U-shaped in sheep (Abass et al., 2012) and cartilage was absent in camel (Karimi et al., 2014).
At the level of first palatine rugae, the entire medial and ventral part of the lateral walls were lined by stratified squamous nonkeratinized epithelium.Occasionally, the dorsal half of the lateral wall was lined by pseudostratified ciliated columnar epithelium with goblet cells (Fig. 3) as reported by Kumar et al. (2016) in young pigs.However, Ganganaik et al. (2006) in sheep reported that both the walls were lined with pseudostratified ciliated columnar epithelium with goblet cells.The lateral and medial walls of the vomeronasal duct were lined by respiratory and olfactory epithelia, respectively according to Kassab and El-Shafey (2012) in buffalo, Besoluk et al. (2001) in Angora goats and Abbasi and Khosravania (2002) in Lori sheep.The pseudostratified ciliated columnar epithelial lining contained four different cells namely basal cells, ciliated columnar cells, non-ciliated columnar cells and goblet cells (Fig. 4).The epithelium lining the lateral wall of the vomeronasal duct showed an undulating appearance and contained short crypts along its length, while the medial wall epithelial lining was continuous in a linear fashion without any crypts as reported by Abbasi (2007) in buffalo and Abbasi and Khosravinia (2002) in Lori sheep.The medial wall epithelial lining dipped down at several places to form crypts lined by mucous-producing cells in sheep (Kratzing, 1971).The epithelial height of lateral wall (57.27 ± 12.12 µm) was comparatively greater than that of medial wall (56.66 ± 8.511 µm).Diffused lymphocytic infiltration was noticed in the underlying connective tissue.The propria submucosa of vomeronasal organ was made up of loose connective tissue composed of connective tissue fibres viz., collagen (Fig. 5), a few elastic (Fig. 6) and reticular fibres (Fig. 7).On contrary to this, Kumar et al. (2016) in young pigs and Ganganaik et al. (2006) in sheep reported that the propria submucosa was made of dense irregular connective tissue.The collagen fibres were densely packed towards the cartilage and they were wavy, arranged parallel nearer to the outer surface of the glands (Fig. 5).The medial propria submucosa (787.26 ± 87.94 µm) was comparatively larger than the lateral propria submucosa (499.65 ± 31.882 µm) with more seromucous glands in addition to different sizes of blood vessels and venous caverns.The C-shaped hyaline cartilage surrounded the vomeronasal organ except at the dorsolateral region which was in accordance with the observations reported by Kratzing (1971) in sheep, Samuelson (2007) in domestic animals and Siraj et al. (2020)in dogs.However, cartilage was absent in camel (Karimi et al., 2014).The connective tissue fibres especially elastic and collagen fibres were abundant closer to the perichondrium of the hyaline cartilage.
At the level of third palatine rugae, the vomeronasal duct showed a narrow vertical lumen with epithelial lining of two different types.The medial wall was lined by olfactory epithelium while the lateral wall was lined by respiratory epithelium (Fig. 8).Three different cells namely basal cells, olfactory cells and supporting cells lined the olfactory epithelium (Fig. 9).These observations were concurrent with the findings of Kratzing (1971) in sheep, Hare (1975) in dogs and pigs, Besoluk et al. (2001) in Angora goats, Abbasi and Khosravinia (2002) in Lori sheep and Abass et al. (2012) in Alawasi Iraqi sheep.Contrary to the present study, both the walls were lined by respiratory epithelium in sheep (Ganganaik et al., 2006) and camel (Karimi et al., 2014) whereas, both the walls were lined by olfactory epithelium in young pigs (Kumar et al., 2016).The epithelial height was more towards the lateral wall (56.66 ± 4.91 µm) than the medial wall (51.93 ± 13.13 µm).The lateral wall showed an undulated appearance and presented several short and long crypts at which the ducts of the glands were opened.The propria submucosa of the vomeronasal organ had loose connective tissue as reported by Kumar et al. (2016) in young pigs whereas Ganganaik et al. (2006) in sheep and Abass et al. (2012) in Alawasi Iraqi sheep stated that it had dense irregular connective tissue.The medial propria submucosa (536.93 ± 66.91 µm) was comparatively thinner than the lateral propria submucosa (875.93 ± 8.95 µm).Lymphocytes were found in varied sizes and shapes.Numerous nerve bundles with unmyelinated axons were observed in the deeper portions of medial propria submucosa (Fig. 10) as reported by Salazar et al. (1996) in cats, Kumar et al. (2016) in young pigs and Kratzing (1971) in sheep.The medial propria submucosa contained fewer glands compared with the lateral propria submucosa.Few glands and glandular ducts were found to be open into the lateral wall.The vomeronasal organ was completely lined by the cartilage except in the dorsal aspect which had a split-like opening.The shape of the cartilage was round to oval.Similar observations were reported by Kumar et al. (2016) in young pigs.Contrary to this, Ganganaik et al. (2006) in sheep stated that C-shaped cartilage enclosed the vomeronasal organ which was incomplete dorsomedially.However, pear-shaped cartilage enclosed the vomeronasal organ in buffalo (Abbasi, 2007).Karimi et al. (2014) in camel and Moawad et al. (2017) in goats found that J-shaped cartilage enclosed the vomeronasal organ.The medial limb (446.39 ± 24.33 µm) of the cartilage was thicker than the lateral limb (358.38 ± 13.02 µm) and extended more dorsally whereas, the lateral limb terminated at the lower portion leaving a small space between the two limbs as reported by Kumar et al. (2016) in young pigs.
At the level of fifth palatine rugae, both walls of the vomeronasal duct were lined by olfactory epithelium (Fig. 11) which was in accordance with the observations reported by Kumar et al. (2016) in young pigs and Karimi et al. (2014) in camel, whereas both the walls were lined by respiratory epithelium in sheep (Ganganaik et al., 2006).Vaccarezza et al. (1981) in rats and Salazar et al. (1996) in cats stated that columnar epithelium lined both the walls of the vomeronasal organ.The epithelial height of the medial wall (25.68 ± 1.45 µm) was comparatively greater than the lateral wall (24.59 ± 0.667 µm).The epithelial crypts were pocket-like and they were noticed in the walls of both sides (Fig. 11).The propria submucosa of vomeronasal organ at the lateral wall (857.85 ± 21.10 µm) was comparatively thicker than the medial wall (684.04 ± 16.89 µm) and had loose irregular connective tissue in the subepithelial portion as reported by Kumar et al. (2016) in young pigs.The collagen, a few elastic (Fig. 12) and reticular fibres (Fig. 13) were present along with the connective tissue cells and microvessels.The deeper portions contained a greater number of mucous acini along with blood vessels of different sizes and venous caverns as reported by Kumar et al. (2016) in young pigs.Numerous nerve bundles with unmyelinated axons were seen across the medial propria submucosa and also in the dorsalwall of the vomeronasal duct.The connective tissue fibres especially collagen fibres became denser close to the cartilage (Fig. 14).The vomeronasal organ was completely surrounded by the cartilage except at the dorsal opening.The shape of the cartilage was oval or elliptical which was in accordance with findings of Kumar (1991) in goats and Kumar et al. (2016) in young pigs.The medial limb was thicker at the extremities (502.49± 45.24 µm), thinner in the middle (386.45 ± 12.38 µm) and it was projected beyond the level of lateral limb.On contrary, Kumar (1991)  that the medial limb was thicker in the middle than at the extremities.

conclusion
The vomeronasal organ plays an important role in olfaction and maintenance of sexual behaviour by detecting the pheromones.The cilia in the epithelium help to facilitate the mixing of mucous fluid along the lumen of vomeronasal duct and improve good contact between the molecule of pheromones and the olfactory cells.The undulating feature of the vomeronasal duct serve in the distension of lumen that help in drawing more mucous fluids containing pheromones from incisive canal into the lumen of vomeronasal duct that acts as a part of mechanical pumping system.

Fig. 13 .Fig. 14 .
Fig. 13.Photomicrograph showing the reticular fibres (Red Arrow) in the stroma of glands (G) and around the basement membrane of epithelium at the level of fifth palatine rugae in pig.V-Vein.Gomori's Reticulum method X 100 Fig. 14.Photomicrograph showing the distribution of dense collagen fibres (Arrow) near to the cartilage (C).V-Vein, VC-Venous caverns.Masson's Trichrome method X 100