Morphometric traits, spermatozoa microscopy and stripped eggs fertilization with milt collected from live African Mudfish (Clarias gariepinus)

K O Idahor, A Yakubu, A F Umar, F Yahaya and L A Musa

Department of Animal Science, Nasarawa State University, Keffi, Shabu-Lafia Campus, P.M.B., 135, Lafia, 950101, Nigeria.
omokingida@yahoo.com

Abstract

The African Mudfish (C. gariepinus) has been reared in Africa for many decades yet with poor performance record, attributable to unreliable practical farming conditions with particular emphasis on artificial reproduction strategies. This has resulted in killing the male as the last resort for sustainable expansion in aquacultural productivity. Undoubtedly, this will lead to time, economic and genetic resource wastages. Hence, the need for alternative technique aimed at using syringe to collect milt from live African Mudfish.

To achieve this, 120 males and 6 females were used in three separate investigations and information were gathered on morphometric parameters of the fish, microscopic observation of milt and fertilization of stripped eggs. The results showed that the papilla length varied between 1.31±0.15 and 1.55±0.06cm, pectoral fin length (6.26±0.24 and 7.07±0.86cm), testicular depth (2.60±0.17 and 3.04±0.35cm) and the testicular length ranged from 4.03±1.05 to 5.08±1.01cm. Also, matured and motile spermatozoa were microscopically observed in the milt collected from 3 (representing 10.0%) of the fish investigated. More significantly, fertilization (13.3%) of the stripped eggs with the milt collected occurred across the treatment groups, irrespective of the syringe insertion point and the testes (left or right).

Based on these findings, it may be possible to collect milt with syringe for breeding in African Mudfish which will certainly end the killing of male fish. Nevertheless, the fish restriction technique, syringe insertion angle, possibility of spermatozoa deformation, short needle with small hollow centre and slanted end could be what led to the little or no milt harvested cases recorded. These without doubt posed some confines to the present search for alternative artificial reproduction technique. Hence, more detailed studies would be needed to elucidate the adoption or otherwise of this propounded novel artificial reproduction strategy in African Mudfish.

Keywords: breeding technique, catfish morphometry, spermatozoa motility

Introduction

The African Mudfishes have been described based on morphological, anatomical and biographical studies leading to different identified species. The large species are of interest for aquacultural production, widely distributed across the Pan African regions even in Minor Asian countries. They have elongated cylindrical body covered with smooth skin (without scales) which has spiny pectoral fins, dorsal fins as well as anal fins that is extremely long reaching the caudal fin. The head is flat and highly ossified with four pairs of barbels that are not branched. Generally, they possess supra-brachial organ composed of paired pear-shaped air chamber containing two arborescent structures. These are supported by cartilage and covered by highly vascularised tissue that can absorb oxygen from atmospheric air (Moussa 1956). The accessory air breathing organ allows the fish to survive for many hours or weeks out of water or in muddy marshes. The sex of African Mudfishes can easily be identified with a distinct urogenital papilla in males located just before the anus but less developed in the females. They thrive well in calm waters ranging from lakes, streams, rivers, swamps to flood plains (Clay 1979). They are classified as omnivores or predators that mainly feed on aquatic and terrestrial insects, fish, mollusk, higher plant debris and fruits (Bruton 1979). It is worthy of note that feed composition meant for African Mudfishes changes as they grow bigger with diptera predominating the diet of small sizes while zooplankton predominates the diet of larger fish (Groenewald 1964).

In natural reproduction, African Mudfishes show seasonal gonadal maturation processes, speculated to be associated with rainy season and are influenced by water temperature and photoperiod. The final triggering of spawning is caused by a rise in water level due to rainfall (de Graaf et al 1995). In captivity, the African Mudfishes do not spawn spontaneously due to environmental factors like rise in water level and inundation of shallow areas. According to FAO (1996), there have been several techniques developed for the artificial reproduction in African Mudfishes since the early 1970s. Stimulating the events that occur during rainy season has been discovered to trigger mating and spawning processes but with as low as 1 – 2 fingerlings/m². Also, hormone induced reproduction has been reported with attendant severe ulcers on the injected fish. Although artificial propagation under more controlled conditions such as stripping of eggs, spermatozoa collection followed by fertilization has been developed (Hogendoorn and Vismans 1980), de Graaf et al (1995) reported that the males are killed and the milt then directly mixed with the stripped eggs. Consequently, the aim of the present study was to investigate the possibility of using syringe to harvest milt without killing the male.

Materials and Methods

Description of the experimental site

The investigation was carried out in the months of December and January at the Hatchery Unit, Department of Fisheries, Faculty of Agriculture, Nasarawa State University, Keffi, Shabu-Lafia Campus Nigeria. Lafia is located between latitude 8^o 35ʹ N and longitude 8^o 32ʹ E on altitude 181.53m above sea level with temperature range of 32 – 34^oC, relative humidity ranging from 40 – 60% and mean day light of 11hrs (NIMET 2013).

Experimental design, data collection and analysis

A total of 120 male African Mudfish at 6 – 12mths of age and weighing between 0.6 and 1.2kg as well as 6 females (as source of stripped eggs only) were sourced from Lafia and its environs. The males only were randomly distributed accordingly to the treatment groups in three separate studies as follows:

Study I

A total of 30 males were used to obtain some morphometric measurements in 4 – 6mths, 7 – 9mths and 10 – 12mths old fish as the treatment groups. The papilla and pectoral fin length values were recorded using measuring tape (Butterfly Brand®). The distance between the mouth and caudal fin base and the end of the caudal fin were measured using measuring tape to obtain the standard body length and total body length respectively (Viveen et al 1986). Thereafter, all the fish were sacrificed and the distance between the outer abdominal region and the paired testes were considered as the testicular depth using measuring tape. Also, measuring tape was placed on the caudal end to the cranial end of each of the paired testes to obtain the testicular length values. SPSS (2012) statistical software package was used in analyzing the data collected and where there were statistical differences, Duncan multiple range tests of the same software package was used in the mean separation.

Study II

The outcome of study I provided a guide to access the paired testes in situ. Thus, three syringe insertion points were chosen based on the end of the pectoral fins (left and right) which served as the starting point (Δ90^o vertically). The paired testicular length and depth gave an insight to the choice of Δ45^o caudally and Δ45^o cranially. Hence, 10 males were allotted each to Δ45^o caudally, Δ90^o vertically and 45^o cranially as the treatment groups. This was further replicated in 5 fish per treatment based on the testes (i.e. left and right). Each fish was restrained in ventral (faceup) position prior to syringe insertion and the milt supposedly collected from Δ45^o caudally, Δ90^o vertically and 45^o cranially was emptied into a test tube containing 1ml of saline water prepared by dissolving 9g of table salt in 1ltr of distilled water as described by de Graaf (1989) to obtain diluted milt. Though this method was targeted at high egg hatchability, in the present study, it was rather aimed at spermatozoa low density and high motility on the microscope slide for near accurate observation in the microscope. An applicator was used to take a drop of the diluted milt on a microscopic slide for observation at x10, x40 and x100 magnifications in the microscope (Olympus Microscope® Tokyo, Japan) as described by Egbunike (1995). The observations were recorded as No or Yes for spermatozoa observed. All the values generated were subjected to simple descriptive statistics prescribed by Adesoye (2004).

Study III

A total of 60 males were distributed randomly to the treatment groups (Δ45^o caudally, Δ90^o vertically and Δ45^o cranially) such that each had 20 fish with 10 replicate per treatment based on testes (i.e. left and right). Also, 6 females were used but only as source of stripped eggs that were used in the fertilization test. In each female, spawning of eggs was induced with hormone (Ovaprim: Syndel Laboratories Ltd., Canada), the eggs were stripped and divided into 10 separate spawning bowls. In the males, syringe was inserted vertically (Δ90^o) at the end of the pectoral fin on the abdominal region. Taken this as the starting point, the syringe was also inserted caudally at Δ45^o and cranially at Δ45^o to access the testes for milt collection in situ. The milt so-collected was diluted with physiological brine as described by de Graaf (1989). In each of the spawning bowls containing stripped eggs, the diluted milt was added, mixed gently and the incubation as well as hatching of the eggs were monitored as illustrated by FAO (1996) and the observations were recorded as No or Yes for eggs fertilized. All the observed values gathered were simply analyzed statistically as frequency and percentage of occurrence according to Adesoye (2004).

Results

The morphometric measurements in male African Mudfish are shown in Table 1. It was observed that the values of all the parameters measured increased with age of the fish. There were no statistical differences (P>0.05) in the values recorded in left pectoral fin, standard body length, total body length and right testicular lengths across the treatment groups. Similarly, the papilla length values were not significantly different (P>0.05) between the fish in 4 – 6mths old group (1.31±0.05cm) and those at 7 – 9mths (1.37±0.29cm) group. However, these values recorded in both groups were statistically lower (P<0.05) than 1.55±0.06cm recorded in 10 – 12mths old fish.

On the other hand, the values of right pectoral fin length, left and right testicular depth did not differ statistically (P>0.05) in fish at 7 – 9mths and 10 – 12mths old groups. But, both were nonetheless significantly higher (P<0.05) than 6.26±0.24cm, 2.61±0.21cm and 2.6±0.17cm recorded in this order for right pectoral fin length, left and right testicular depth in fish at 4 – 6mths old. The left testicular length value (4.76±1.11cm) recorded in 7 – 9mths old fish was neither significantly superior (P>0.05) to 4.36±0.70cm (4 – 6mths old fish) nor 5.08±1.01cm recorded in 10 – 12mths old fish.

Presented in Table 2 is the microscopic observation of spermatozoa collected from live African Mudfish (C. gariepinus).

In a total of 30 fish used in the investigation, only in 3 representing 10.0% that spermatozoa were recorded. At Δ90^o vertically inserted point, spermatozoa were observed in milt harvested from both testes (1 each representing 3.3%). Similarly, spermatozoa were present in milt harvested at Δ45 ^o cranially inserted point from the left testis (1 representing 3.3%) only. See Plates A – D and none was found at Δ45^o caudally inserted syringe point.

Plate A. Milt collection at Δ45o caudally	Plate B. Milt collection at Δ90o vertically	Plate C. Milt collection at Δ45o cranially	Plate D. Spermatozoa observed at x100

Fertilization of stripped eggs with milt collected from live African Mudfish is expressed in Table 3.

Out of a total of 60 replicates, only 8 (representing 13.3%) were fertilized with the milt collected with syringe from the intact fish (See Plates I – IV).

Plate I. Milt collection	Plate II. Spawned eggs	Plate III. Incubation and hatching	Plate IV. Fertilized eggs observed

It was observed that fertilization occurred across the treatment groups with the highest occurrence (6 representing 10%) in Δ90^o vertically collected milt. In Δ45^o caudally and cranially collected milt, a case of fertilization each (1 representing 1.7%) was recorded.

Discussion

Although, there were evidences of the fish still growing as revealed by the increased values of parameters with age (Liem et al 2001), the observed value similarity could be an indication that at 4 – 12mths of age, African Mudfish probably presented morphometric features of maturity. This observation partially lent credence to 7mths of age reported by FAO (1996) as a criterion for male catfish brood stock maturity. Therefore, milt containing matured spermatozoa could be harvested from African Mudfish at 4 – 12mths of age.

The little or no spermatozoa observed in the milt viewed in the microscope, could be largely due to little or no milt harvested using syringe, the fish restriction method used, dilution of the milt (de Graaf 1989) before it was viewed in the microscope and partly due to low magnification power of the microscope used. Nevertheless, since spermatozoa were observed in the study, it may be possible to collect milt for fish breeding without killing the male.

The observed supremacy recorded in Δ90^o vertically syringe insertion fish group, could be adduced probably to mid of the testis where milt may seemingly concentrate. Interestingly, the stripped eggs fertilizations were recorded in both testes (left and right) at any syringe insertion point reflecting that, it could be possible to collect milt from the male fish without killing it. This observation apparently contradicted the report of de Graaf et al (1995), de Graaf, (1989) and Jansen (1985) that males should be killed to get the milt for artificial reproduction.

Conclusion

• The results revealed that at 4 – 12mths of age, African Mudfish may possess morphometric characteristics that apparently indicated maturity. It was also shown that matured and motile spermatozoa were observed microscopically in the milt harvested from African Mudfish at 4 – 12mths old in both testes. Taken into cognizance the end of pectoral fin at the abdominal region, milt were however sparingly harvested at Δ45^o caudally, Δ90^o vertically and Δ45^o cranially syringe insertion points. More significantly, it was discovered that the syringe harvested milt apparently contained spermatozoa with viability potentials that could be able to fertilize stripped eggs without necessarily killing the fish.
  
  
•  It is however pertinent to note that fish restriction technique during milt collection, syringe insertion orientation, likelihood of spermatozoa distortion, short needle with small hollow centre and slanted end perhaps resulted in the little or no milt harvested. These in no small measure, posed some limitations to the current investigation. Hence, more detailed studies would be needed to elucidate the adoption or otherwise of this novel propounded artificial reproduction technique in African Mudfish.

References

Adesoye PO 2004 Practical Guides to Statistical Analysis for Scientists. A Primer edition. Debo Prints, Ibadan, 189 pp.

Bruton M N 1979 The food and feeding behaviour of Clarias gariepinus (Pisces, Claridae) in lake Sibaya, south Africa, with its emphasis on its role as a predator of cichlids. Trans. Zool. Soc. London, Volume 35:47-114.

Clay D 1979 Population biology, growth and feeding of the African catfish, Clarias gariepinus , with special reference to juveniles and their importance in fish culture. Arch. Hydrobiol., Volume 87 (4): 453-482.

de Graaf G J, Galemoni F and Banzoussi B 1995 The artificial reproduction and fingerling production of the African catfish Clarias gariepinus (Burchell 1822) in protected and unprotected ponds. Aquaculture Res., Volume 26: 233-242.

de Graaf GJ 1989 La reproduction artificielle et l'alevinage de Clarias gariepinus au centre de production d'alevins de Loka en Côte-d'Ivoire, Rapport d'une mission effectuée du 28/10/1989 au 10/11/1989. Project du développement de la pisciculture rurale, FAO/IVC/87/007.

Egbunike G N 1995 A toast for spermatozoa: One half the story of mammalian life. An Inaugural Lecture Series, University of Ibadan, Ibadan Nigeria.

FAO 1996 Handbook on the artificial reproduction and pond rearing of the African catfish Clarias gariepinus in sub-Saharan Africa. Food and Agricultural Organization, Fisheries Technical Paper 362, Rome.

Groenewald A A V J 1964 Observations on the food habits of Clarias gariepinus Burchell, the South African freshwater Barbel (Pisces: Claridae) in Transvaal. Hydrobiol., Volume 23: 267-273.

Hogendoorn H and Vismans M M 1980 Controlled propagation of the African catfish, Clarias lazera (C&V). II. Artificial reproduction. Aquaculture, Volume 21: 39-53.

Jansen J A L 1985 Elevage du poisson-chat Africain Clarias lazera (C&V) en République Centraficaine. I. Propagation artificielle. FAO project GCD/CAF/007/NET. Document technique No. 20, 37pp.

Liem K F and Warren F W 2001 Functional anatomy of the vertebrates: An evolutionary perspective. Harcourt College publishers, 277pp.

Moussa T A 1956 Morphology of the accessory air breathing organs of the Teleost Clarias lazera (C&V). J. Morph., Volume 98:125-160.

Received 5 May 2014; Accepted 22 July 2014; Published 1 August 2014

Go to top