The present study was undertaken to know the localization of different enzymes in testicular parenchyma in domestic pig. Neutral mucopolysaccharides and acid mucopolysaccharides were mild in testicular capusule and strong in interstitial tissue of testis. Leydig cells of interstitium showed adundant glycogen. Lipids were localized moderately and strongly in the seminiferous tubules and leydig cells respectively. Moderate to strong reactions for basic proteins were observed in testicular parenchyma of testis. The activities for acid phosphatases and alkaline phosphatases were localized mainly in the interstitial tissue of testis and very mild in seminiferous tubules. The reductase i.e., succinic dehydrogenase enzyme activity was localized strongly in the interstitial tissue of testis.
Swine husbandry is becoming a profitable enterprise due to its high prolificacy and faster growth rate with shorter generation interval. This mainly depends on reproductive traits. The testes are compound tubular glands which exhibit both endocrine and cytogenic functions. Very scanty information regarding enzymes localization studies on testis in pig compared with other domestic animals. The present work has been undertaken to know the histochemical reactions of the testis in adult boar.
Materials and Methods
For this present study, the samples of testes were collected from fourteen adult apparently healthy pigs from local slaughter houses in and around Hyderabad. For histochemistry of carbohydrates and proteins, the tissue pieces collected and fixed in 10% neutral buffered formalin. Paraffin sections of 5-6 µm thickness were cut for Periodic Acid Schiff (PAS) technique for mucopolysaccharides, Combined Alcian blue-PAS (Ab-PAS) technique for acid and neutral mucopolysaccharides, Best’s carmine method for glycogen (Luna, 1968) and Mercury-Bromophenol blue method for basic proteins (Stoward and Pearse, 1985). For lipids and enzymes, tissues were fixed at 4oC in chilled 10% neutral buffered formalin. Frozen sections of 15-20 µm stained for oil red ‘O’ in propylene glycol for lipids (Luna, 1968), Gomori’s Calcium method for alkaline phosphatase activity, Gomoris’ lead method for acid phosphatase activity and Schultz’s method for cholesterol and unfixed fresh frozen tissues were used for the Standard dehydrogenase technique for succinic dehydrogenase activity (Bancroft and Gamble, 2008).
Results and Discussion
Localization of neutral mucopolysaccharides in the testicular parenchyma was evident by the presence of strong, mild to moderate and weak PAS reactions in interstitial tissue, seminiferous tubules and in the capsule of the boar testis respectively (Fig. 1) which is similar to the reports of Bashir et al. (2012) in goat and Pathak et al. (2013) in donkey and mules.
Fig. 1: Photomicrograph of testis showing strong localization of PAS reaction (arrow) in the interstitial tissue. SFT: Seminiferous tubules, IT: Interstitial tissue PAS (Periodic Acid Schiff) X 100
Seminiferous tubules, interstitial tissue and epithelium of rete testis reacted strongly for Ab- PAS whereas septula testis, testicular capsule showed mild reaction which were concur with the reports of Kannan et al. (2008) in quail. Varied, moderate and strong glycogen reaction in capsule, seminiferous tubules and interstitial tissue respectively which was also reported in goat (Sudhakar et al., 2010 and Bashir et al., 2012) and bull (Gofur et al., 2008).
Moderate to strong localization of lipids was observed in the seminiferous tubules, inter tubular tissue and Leydig cells in the present study which was in concurrence with earlier findings of Singh and Bharadwaj (1978) in camel, Singh (1996) in buffalo and Kishore et al. (2007) in ram. Leydig cells contained varying amount of cholesterol in goat and sheep (Sudhakar et al., 2010) whereas Pathak et al. (2013) reported intense green droplets of cholesterol in the interstitium of testis in donkey and a poor reaction in mule.
Bromo phenol blue reaction for basic proteins was moderate in testicular capsule and strong in seminiferous tubules and Leydig cells of boar (Fig. 2). Though such observations were not made earlier, Rao (1994) recorded moderate activity for Millon’s reaction for tyrosine in the testis of domestic duck.
Fig. 2: Photomicrograph of testis showing strong localization of basic proteins (arrow) in the interstitial tissue. SFT: Seminiferous tubules, IT: Interstitial tissue, C- Capsule. Bromo Phenol Blue Stain X 40
Alkaline phosphatase localization was moderate and intense in inter tubular tissue and seminiferous tubules of the boar respectively. It was totally mild and absent in testicular capsule (Fig. 3).
Fig. 3: Photomicrograph of testis showing localization of alkaline phosphatase activity (arrow) in the interstitial tissue. SFT: Seminiferous tubules, IT: Interstitial tissue, C- Capsule. Gomori’s calcium method X 40
Similar findings were observed in the quail (Kannan et al., 2008) whereas the reaction was intense in rete channels of emu (Rajendranath et al., 2013). The testis of boar showed mild to moderate acid phosphatase localization in the basement membrane, interstitial tissue and seminiferous tubule whereas intense acid phosphatase reaction was observed in the rete channels of testis in domestic drake (Rao and Vijayaraghavan, 2000). The interstitial tissue showed strong succinic dehydrogenase (SDH) enzyme localization (Fig. 4), but not so in the capsule and seminiferous tubules of testis of boar which is contrary to the reports of Rao (1994) who demonstrated that SDH activity was moderate and weak in seminiferous tubules and inter-tubular tissue in domestic duck. Leydig cells are plays an important role in the production of testosterone which is necessary for the production of spermatozoa.
Fig. 4: Photomicrograph of testis showing strong succinic dehydrogenase localization (arrow) in interstitial tissue. SFT: Seminiferous tubules, IT: Interstitial tissue. Succinic dehydrogenase activity X 40
The enzymes alkaline phosphatase, acid phosphatase and succinic dehydrogenase were mainly localized in the Leydig cells only. Glycogen, proteins and lipids are more abundant in the Leydig cells of interstitium than in the seminiferous tubules and testicular capsule.
Histochemical studies of testis revealed strong, mild to moderate and weak reactions in interstitial tissue, seminiferous tubule and testicular capsule respectively for PAS and PAS-AB. Glycogen reaction was mild and moderate in seminiferous tubules and interstitial tissue respectively. Strong and moderate reaction were noticed in interstitial tissue and seminiferous tubules respectively for fat and basic proteins. Alkaline phosphatase activity was strong in SFT. Mild to moderate acid phosphatase activities were observed in SFT and interstitial tissue. Interstitial tissue exhibited strong succinic dehydrogenase enzyme activity.