Scanning Electron Microscopic Studies on the Development of Epididymis and Vas Deferens in Non-descript Goat (Capra hircus) during Postnatal Period 

Goat is considered as the great future in changing livestock scenario (Ansari et al., 2017). The poor man’s cow-goat has tremendous potential to be projected as the future animal for rural areas under the changing agro-climatic conditions and lack of forages (Jindal et al., 2011). Scanning electron microscopy (SEM) permits observation of structures in three dimensions. With other types of microscopy, these structures must be visualized by the reconstruction of serial sections (Johnson et al., 1978 and Singh et al., 2024). In capitalizing on this advantage, SEM has been employed to observe structures within the epididymis and vas deferens of the non-descript goat. The ductus epididymidis is extremely tortuous and coiled. Macroscopically, the epididymis is divided into a head, body and tail (Getty, 1975). It is surrounded by a thick tunica albuginea of dense irregular connective tissue covered by the visceral layer of the tunica vaginalis (Banks, 1993). Generally, the proximal parts of the duct (head and body) are involved in the maturation process of spermatozoa (Schimming et al., 2012; Rus et al., 2022 and Kour et al., 2023). The tail of the epididymis serves as their main storage place. Spermatozoa leaving the testis are both immotile and infertile, whereas spermatozoa leaving the epididymis have gained motility and fertility. During their passage through the ductus epididymis, spermatozoa undergo a series of morphologic and functional changes that lead to the acquisition of full fertilizing capacity by the time they reach the tail (Dellmann and Eurell, 1998 and Samuelson, 2007). The ductus epididymis, after a sharp bend at the end of the tail, gradually straightens (Getty, 1975) and acquires the histologic characteristics of the ductus deferens. In stallions and ruminants, the ductus deferens unites with the excretory duct of the vesicular gland to form a short ejaculatory duct, which opens at the colliculus seminalis into the urethra. The pseudostratified lining epithelium of the bovine ejaculatory duct contains engulfed spermatozoa. In boars, the ductus deferens and the excretory duct open separately into the urethra. In carnivores, the ductus deferens joins the urethra alone because the vesicular gland is absent. The terminal portion of the ductus deferens is one of the male accessory glands, regardless of whether it forms an ampulla (stallions, ruminants, dogs) or does not (boars, cats); it contains simple branched tubulo-alveolar glands in the propria-submucosa (Dellmann and Eurell, 1998 and Samuelson, 2007).
       
Abundant research has been done on the gross and histomorphology of epididymis and vas deferens in goat and other domestic animals. But a very scanty literature is available on their scanning electron microscopic details in domestic animals and this area is quite unexplored till date. The ultrastructure of the ciliated and non-ciliated cells with their interaction with the mature spermatozoa is vital for accessing the reproductive status of the animal. The present study was carried out to develop a baseline data on the scanning electron microscopic characteristics of different components of tunics of epididymis and vas deferens in non-descript goat at different stages of post-natal life under study.

The present scanning electron microscopic study was conducted on the epididymis and vas deferens of non-descript goats at various stages of post-natal life. The samples were collected from the nearby local slaughter houses in and around Bhubaneswar. The animals were divided into three groups, viz. neonatal (within 1 month), prepubertal (from 1-18 months) and pubertal (above 18 months) with 10 healthy animals in each group (Banerjee, 2019). The age of the animal is known from the data book of the slaughter houses. The samples of epididymis and vas deferens were processed for scanning electron microscopic study (Scanning Electron Microscope, Make: Hitachi and Model: S-3400N) as per the standard methods of Sahu et al., (2021). The samples were subsequently viewed and the photographs were taken in the facility available at Central Instrumentation Facility (CIF), OUAT, Bhubaneswar. The measurements of various parameters of epididymis and vas deferens were also taken at the scanning electron microscopic level by the inbuilt software programming system. The recorded data were subjected to routine statistical analysis (Snedecor and Cochran, 1994).

The present study focused on the scanning electron microscopic details of the tunics of epididymis and vas deferens in non-descript goats at different stages of postnatal life under study.
 
Scanning electron microscopy of epididymis
 
The epididymis was covered with a tough layer of fibrous tissue called as tunica vaginalis. The septae originated from the tunica vaginalis and penetrated deep into the epididymis dividing the organ into a non-regular form resembling lobules (Sharma et al., 2012).  In the connective tissue of the septae or in between the lobules, few small blood vessels were also observed (Singh, 1989; Schimming et al., 2001; Mohamed, 2005 and Sharma et al., 2012). The outer surface of tunica vaginalis was folded. These folds were longitudinal and transverse with the depressions or troughs in between them. Ridges and passages were also noticed in these folds at higher magnification. These ridges and passages were seen in all the age groups (Fig 1), but quite well developed in the pubertal goats (Fig 2). These ridges and folds might be attributed to the increase in surface area (Sharma et al., 2012). The ducts comprised of lining epithelium, peritubular stroma and intertubular stroma. The peritubular stroma known as propria-submucosa surrounded the epithelium and consisted of concentrically arranged smooth muscle fibers separated by connective tissue fibers (Fig 3 and Fig 4). The epididymal lumen contains spermatozoa and fluid, whose composition is constantly changed as the fluid moves from the initial to the terminal segment (Robaire and Viger, 1995). The intertubular stroma was present in between the ducts and mainly comprised of inter-woven connective tissue fibers (Fig 4). The present findings were in agreement to the observations of Schimming et al., (2001) in dog and Sharma et al., (2012) in buffalo foetus.








       
The epididymis was lined by ciliated and non-ciliated types of cells (Fig 5, Fig 6 and Fig 7), which was similar to the finding of Saleem et al., (2018) in the local Hill fowl of Uttarakhand. The ciliated cells showed tufts of cilia (Fig 6), where as the free surfaces of the non-ciliated cells were lined by microvilli (Fig 8). The present observations were in agreement with the findings of Budras and Saucer (1975) in cock, Bakst (1980) in male chicken and turkey and Saleem et al., (2018) in the local Hill fowl of Uttarakhand. However, Aire and Soley (2000) in ostrich observed several non-ciliated cells showing single long cilium which projected into the ductal lumen. The luminal surface of the epididymal duct gave the appearance of a honeycomb (Fig 9) due to the peculiar arrangement of stereocilia projecting from the columnar cells (Murakami et al., 1975). The stereocilia were not uniform in length and distribution as in the case of microvilli of the intestinal absorptive cells, but left a funnel-shaped space (Fig 10) widely opening to the lumen on the center of each cell (Murakami et al., 1975). Individual stereocilia were in general long and slender with almost constant in diameter. The diameter of stereocilia was measured as (267±5.14) nm, (1.35±0.06) µm and (1.71±0.04) µm in neonatal, pre-pubertal and pubertal non-descript goats from base to the tip respectively. Their outer surface seems smooth and without a particular structure. Frequently, small globules were found to adhere to the tip or the shaft of the stereocilia, which might be the artifacts produced during preparation. The functional significance of the brush border was unclear, but probably it conveyed the luminal content forward (Orsi et al., 1998).












       
The luminal surface of epithelium especially the non-ciliated cells of the ducts was studded with apical protrusions and few circular areas giving cobbled appearance (Fig 11) in the prepubertal and pubertal goats (Saleem et al., 2018). These areas might represent the regions from which the apical protrusions had become detached (Sharma et al., 2012). The presence of apical protrusions on the luminal surface of the epithelium in the prepubertal and pubertal animals was indicative of the apocrine secretory activity of the epididymis at these stages (Paunescu et al., 2014). While the process of vesicle shedding via plasma membrane budding was originally thought to represent an artifact caused by poor fixation, these vesicles are now considered to play significant roles in the communication between neighboring cells (Cocucci et al., 2009).


       
Variable numbers of spermatozoa were present in the lumen over the stereocilia, mostly in the pubertal group (Fig 12). The heads of the spermatozoa invaded deep into the epithelium of the duct, especially to the non-ciliated cells indicating a close physical interaction between spermatozoa and the epithelial cells (Paunescu et al., 2014), but it was contradictory to the observations of Murakami et al., (1975) in Japanese Monkey, Schimming et al., (2001) in dog and Saleem et al., (2018) in local Hill fowl of Uttarakhand.  This strong bond was vital in sperm maturation in the epididymis, allowing the transfer of new proteins from epithelial cells to the sperm cells. The extracellular vesicles might be originating either from the fusion of multivesicular bodies with the apical membrane followed by the release of exosomes, or from the budding of larger vesicles from the plasma membrane (Belleannee et al., 2013; Sullivan and Saez, 2013). The presence of a very dense array of stereocilia in principal cells precluded the detection of any exocytotic or fusion event that would take place at the level of the plasma membrane (Paunescu et al., 2014). The morphology of certain sperm cells suggested a distinct kind of interaction with the epididymal epithelium. Increasing the magnification reveals concentric rings on the sperm midpiece (Villalpando et al., 2000), as well as small, vesicle-like structures on the surface of the cytosolic droplet located at the mid-principal piece junction (Paunescu et al., 2014).


 
Scanning electron microscopy of Vas deferens
 
The vas deferens was covered with a tough layer of fibrous tissue known as tunica vaginalis. The outer surface of tunica vaginalis was folded. These folds were longitudinal and transverse with the depressions or troughs in between them. Ridges and passages were also noticed in these folds at higher magnification in all the age groups, but quite well developed in both pre-pubertal and pubertal non-descript goats (Fig 13). These ridges and folds might be attributed to the increase in surface area.


       
The luminal surface of ductus deferens of local Hill fowl showed numerous longitudinal folds. They were raised at regular intervals giving it a wavy appearance (Fig 14) with ridges and grooves (Saleem et al., 2018). Every groove presents in the midline a straight ridge due to the overlapping of the adjacent cell membranes (Orlandini et al., 1979). The higher SEM magnification showed that the lamina epithelialis was made up of irregular polygonal cells with indistinct cell boundaries (Saleh et al., 2020). The apical surface of the epithelial cells appeared slightly dome-shaped (Fig 14). The present finding was in agreement with the report given by Saleem et al., (2018) in local Hill fowl of Uttarakhand.


       
The epithelium of the vas deferens comprised of ciliated and non- ciliated types of cells. The ciliated cells showed tufts of cilia (Fig 15), where as the free surfaces of the non- ciliated cells were lined by microvilli (Fig 16). The present finding was in line with the reports of Saleem et al., (2018) in local Hill fowl of Uttarakhand, Saleh et al., (2020) in Dromedary camel and Kocakoglu et al., (2023) in long horned beetle. Individual stereocilia are in general long and slender with almost constant in diameter from base to the tip. Their outer surface seems smooth and without a particular structure. The functional significance of the brush border was unclear, but probably it conveyed the luminal content forward. It might also help in the movement of the sperms and secretion in and out of the glandular alveoli, located in the lamina propria of the vas deferens (Saleh et al., 2020). The luminal surface of epithelium, especially the non-ciliated cells of the duct was studded with apical protrusions (Fig 17) in the prepubertal and pubertal goats (Brueschke et al., 1974). The presence of apical protrusions on the luminal surface of the epithelium was indicative of the apocrine secretory activity of the vas deferens at these stages. Further, the apical surface of the non-ciliated cells was having microvilli. The microvilli might increase the surface area for water reabsorption and luminal fluids concentration (Saleh et al., 2020). The spermatozoa were demonstrated in contact with the surface epithelium of the vas deferens (Fig 17), mostly in the pubertal group (Saleem et al., 2018 and Saleh et al., 2020). The presence of a large amount of the spermatozoa in tubular lumen was indicative of storage of spermatozoa for a time before ejaculation (Perera, 1974 and Ali et al., 1978). 

The present study would help in developing a baseline data on the scanning electron microscopic characteristics of epithelium, lamina propria, tunica muscularis and tunica serosa of epididymis and vas deferens in non-descript goat at different postnatal stages under study. The appearance of ciliated and non-ciliated cells of epididymis and vas deferens, the orientation of cilia along with the appearance and attachment of mature spermatozoa to the epithelial cells of both epididymis and vas deferens would help in accessing the reproductive status of the animal. The same could be exploited in the selection of goats at specific stages of life for breeding purposes.  

This article is a part of Doctoral Research work of the first author. The Authors are very much grateful to the Dean, CVSc. and A. H., OUAT, Bhubaneswar and In-charge, CIF, OUAT, Bhubaneswar for providing necessary facilities for successful completion of this research work in time.

There is no conflict of interest among the authors.