Testicular Morphology and Sperm Motility in Cultured African Catfish ( Clarias gariepinus ) at Different Stages of Development

Testicular morphology and sperm motility were evaluated in cultured Clarias gariepinus (n = 25) purposively assigned to five groups according to their age. The results showed that the testes were paired, elongated, dorso-ventrally flattened structures, situated in the caudal aspects of the body cavity. The mean length of both right and left testes increased linearly with age, being significantly (p < 0.05) higher at 6 months than at 4 and 5 months of age, and also significantly (p < 0.05) higher at 8 months than at 6 months of age, while the mean weight and organo-somatic index of the catfish testes increased linearly until 6 months of age, after which no significant (p > 0.05) increase in the testicular weight and organo-somatic index was observed. Unidirectional progressive movement of spermatozoa was detected in the milt of C. gariepinus at 6, 7 and 8 months of age, but sperm cells were non-motile at 4 and 5 months of age. Histological sections showed seminiferous lobules, whose germinal epithelia were characterized by many cysts enclosing clones of sperm cells. Each cyst enclosed a clone of sperm cells at an identical stage of spermatogenesis. Spermatids and spermatozoa were present in the lumen of the seminiferous lobule. The obtained results indicate that the morphology of the testes of C. gariepinus is similar to the testes of members of the order Siluriformes, but sexual maturity and production of motile spermatozoa may be achieved at 6 months of age in the African catfish.


Introduction
Catfish is a member of the order Siluriformes, which is the most diverse fish order (Van Dyk and Pieterse, 2008) and many species of fish in this order have been introduced into aquaculture.Catfish culture contributes about 60% of the world's supply of protein (FAO, 2007), especially in developing countries of Africa, where the sharp-tooth catfish (Clarias gariepinus) is widely distributed (Nwuba and Aguigwo, 2002).In Nigeria, commercial fish production is a growing enterprise, and catfish farming is the most popular form of fish farming in the country.Understanding the reproductive biology of the catfish is one of the most important factors for improvement of catfish culture, and this is paramount in commercial aquaculture enterprises.
The testes of mature catfish are paired elongated structures located in the dorsal part of the posterior body cavity.The testes may vary in size depending on the age of the fish and the season of the year (Genten et al., 2009), but the left testis is thought to be usually longer than the right one (Van Dyk and Pieterse, 2008).Breeding in catfish is seasonal, and the level of testicular activity and spermatogenesis depend mostly on the light period and temperature (Garg and Sundararaj, 1985).
Hormonal, environmental and genetic manipulations are amongst several strategies developed to influence the sexual maturation of cultured catfish, but there is limited information on the relationship between gonadal development, hormone cycle and spawning (Barrero et al., 2007).
A recent study has suggested that characterization of mature testes and sperm morphology of the fish may have practical applications related to artificial fertilization and sperm preservation (Furbock et al., 2010).However, there is very little information on age-related variations in the morphology of the testes of the African catfish.
The present study investigated the morphological and histological changes in the testes, and sperm motility of the African catfish (C.gariepinus) at different ages during development.

Experimental animals and sample collection
Clarias gariepinus (n = 25) used in the study were all males obtained from a commercial fish farm located at Ozubulu in Ekwusigo Local Government Area, Anambra State, Nigeria.They were measured, weighed and purposively assigned to five groups of five fish each according to their ages.The ages (months) were 4 (mean weight = 112.0± 17.4 g and mean length = 24.10 ± 0.39 cm), 5 (mean weight = 178.0 ± 22.2 g and mean length = 28.98 ± 0.85 cm), 6 (mean weight = 780.0± 118.9 g and mean length = 50.82± 2.71 cm), 7 (mean weight = 856.0± 45.4 g and mean length = 51.76 ± 1.01 cm) and 8 (mean weight = 1467.2± 37.1 g and mean length = 63.17 ± 2.89 cm).The animals were stunned and dissected to obtain the testes used for the morphological studies.The gonadosomatic indices were determined according to the following formula (Roff, 1983): GSI = (testicular weight/total body weight) × 100.

Assessment of sperm motility
Semen collected from each fish was examined under a light microscope for presence of mature spermatozoa.The spermatozoa were visually assessed for unidirectional progressive movement.

Analysis of testes histomorphology
Samples of testes of fish in the five groups were promptly fixed by immersion in Bouin's fluid for 48 h.Later, the fixed specimens were dehydrated in increasing concentrations of ethanol, cleared in two changes of xylene, and embedded in paraffin wax.Then, 5 µm thick sections were cut, mounted on glass slides and stained routinely with haematoxylin and eosin (H & E) for light microscopy.Photomicrographs were captured using a Moticam Images Plus 2.0 digital camera (Motic China Group Ltd.).

Statistical analyses
Data obtained for the quantitative parameters were expressed as mean ± standard error of mean.These were subjected to one-way analysis of variance (ANOVA).Variant means were separated post-hoc using the least significant difference (LSD) method.

Gross anatomy
The testes of C. gariepinus were paired elongated, dorso-ventrally flattened structures situated just beneath the dorsal body wall in the caudal aspects of the body cavity.They were attached to the dorsal body wall by the mesorchium.The medial border of each testis was smooth, while the lateral border exhibited serrated edges (Fig. 1).

Quantitative parameters
Data on mean length of the fish, as well as mean length of left and right testes are shown in Table 1.The mean length of the fish was significantly (p < 0.05) higher at 6 months than compared with that from 4 and 5 months of age, and also significantly (p < 0.05) higher at 8 months compared to 6 and 7 months of age (Table 1).Similarly, the mean length of both right and left testes increased linearly with age, being significantly (p < 0.05) higher at 6 months than at 4 and 5 months of age, and also significantly (p < 0.05) higher at 8 months than at 6 months of age.The right testis was longer than the left testis at 4, 6 and 7 months of age, but at 8 months of age, the left testis was longer than the right testis.
Table 2 shows information on mean weights of the fish and the testes.The mean weight of the fish followed a 282 comparable pattern as the mean length of the fish, so that it showed a significantly (p < 0.05) higher value at 6 months than at 4 and 5 months of age, and also a significantly (p < 0.05) higher value at 8 months compared to 6 and 7 months of age (Table 2).Mean weight of pooled left and right testes increased significantly (p < 0.05) from 4 to 6 months of age, after which no significant difference (p > 0.05) was observed in the mean weight of the testes with age.Moreover, the mean weight of right testes was greater than that of left testes at 4, 5, 6, and 7 months of age, but at 8 months of age, the mean weight of left testes was greater than that of right testes.
Comparison of the gonadosomatic indices revealed that the mean percentage contribution of pooled left and right testicular weight to the body weight of the fish increased from 0.362 ± 0.032% at 4 months to attain a peak value of 0.937 ± 0.047% at 6 months, after which the value decreased to 0.645 ± 0.312% at 8 months of age (Table 2).The mean gonadosomatic index of the right testes was consistently higher than that of the left testis at 4, 5, 6 and 7 months of age, but the mean gonadosomatic index of the right testes was greater than that of the left testes at 8 months of age.

Sperm motility
Mature spermatozoa were observed in the semen obtained from fish in all the five groups studied.However,

Histology
Photomicrographs of the testes of C. gariepinus at 8, 6 and 4 months of age are shown in Figs. 2, 3 and 4 respectively.Each testis was surrounded by a thick connective tissue capsule, the tunica albugenia, which was richly vascularized and exhibited the presence of some adipose cells (Fig. 2).Connective tissue trabeculae from the tunica albugenia penetrated the testicular parenchyma to demarcate compartments, the seminiferous lobules, each of which showed a lumen at its centre.Interstitial tissue composed of connective tissue cells, interstitial cells and blood vessels separated adjacent seminiferous lobules.The germinal epithelium within each seminiferous lobule exhibited several sperm cells at different stages of maturation.The sperm cells were enclosed in several cysts present within the germinal epithelium (Figs. 2,3,4).Each cyst enclosed a clone of sperm cells at an identical stage of spermatogenesis.Thus, some cysts enclosed the spermatogonia, while others enclosed the spermatocytes.The nuclei of the spermatocytes were smaller in size and appeared darker than the nuclei of the spermatogonia.Moreover, the cysts containing the spermatogonia were mostly situated close to the base of the epithelium, while the spermatocyte-containing cysts were located in the adluminal region of the germinal epithelium.Spermatids and spermatozoa were seen in the lumen of the

Discussion
Structural and functional status of the testes and sperm cells are important considerations for successful breeding programs that involve artificial fertilization.Thus, characterization of mature testes and sperm morphology of the catfish may have practical applications in commercial catfish farming.The illustration of paired, elongated, dorsoventrally flattened testes with serrated lateral borders in the C. gariepinus is consistent with the features reported for members of the order Siluriformes, including the Doradidae and the Auchenipteridae families (Meisner et al., 2000).It is apparent from the present study that testicular length may be influenced by the length of the catfish, and both the fish length and length of testes increased linearly with age.Whereas, Ratty et al. (1990) reported that the right testes are larger than the left testes in Thunnus alalunga, Van Dyk and Pieterse (2008) showed that the left testes of fish are usually longer than the right testes.However, the present study demonstrated that the right testes of C. gariepinus were longer than the left testes until 8 months of age, when the left testes grew to be longer than the right testes.This suggests that the dissimilarity in the observations of the previous authors may have resulted from disparity in the ages of the fishes used for their respective studies.
The mean weight and organo-somatic index of the catfish testes increased linearly until 6 months of age, after which no significant increase in the testicular weight and testicular-somatic index was observed.The testes of Clarias gariepinus may have attained optimal weight and peak sperm production potential at 6 months of age, since the weight of an organ may be related to its functional status.Indeed, a positive correlation has been established between testicular weight and sperm production, and the gonadosomatic index has been employed for estimation of reproductive performance of fish (Kumari, 2014).The idea that the catfish testes may have achieved optimal development at 6 months of age is further supported by the finding that spermatozoa with capacity for unidirectional progressive movement were first identified in the milt of C. gariepinus at 6 months of age, an indication of sexual maturity in the fish.Sperm motility is considered the best biomarker of milt quality (Lahnsteiner et al., 2004) and it is thought to have great influence on successful fertilization in fish (Oguntuase and Adebayo, 2014).
Histology of the catfish testes demonstrated that the seminiferous epithelium within the seminiferous lobules contained cysts of clones of sperm cells at identical stages of spermatogenesis.It has been shown that the cyst wall is derived from the sertoli cell associated with the original germ cell from which the clone of sperm cells arise (Santos et al., 2001).Cysts of the more mature sperm cells were located in an adluminal position, from where the resulting spermatids may be released into the lumen of the seminferous lobule.Maturation of spermatids into spermatozoa is thought to take place within the lumen of the seminiferous lobule (Genten et al., 2009), and the asynchronous development of spermatogenesis in the various cysts may support continuous spermaition in cultured C. gariepinus.The apparently larger luminal diameter of the seminiferous lobules, and their content of numerous spermatids and spermatozoa at 6 and 8 months of age than at 4 months of age may be related to our proposition that C. gariepinus attains sexual maturity at 6 months of age.This suggested age at sexual maturity in the catfish is at variance with the reports of some previous authors (Cek and Yilmaz, 2007).However, genetic factors and culture conditions, including nutrition have been shown to influence the age of maturation of cultured fish (Le Bail, 1996).

Table 1 .
Mean length (cm) of C. gariepinus and mean lengths (cm) of right and left testes of the fish at different ages

Table 2 .
Mean weight (g) of C. gariepinus, mean weights (g) and mean gonadosomatic indices (%) of right and left testes of the fish at different ages a, b, c different superscripts in a row indicate statistically significant differences