A study was conducted to test the viability of the milt of the African catfish Clarias gariepinus following extension in citrated egg yolk extender and stored in a deep freezer at -20°C. Two male brood stocks of Clarias gariepinus with average age of 14 months, average weight 1.5 kg and average measurement of 41.5 cm length were used. The experiment comprised of two groups via the test and control groups. The test group consisted of seven milt samples extended in citrated egg yolk extender and stored in deep freezer at -20°C while the control group consisted of seven milt samples diluted in 2.9% sodium citrate solution and stored in the fridge at 4°C. Both the test and control groups were stored for seven days and the milt samples were examined daily for percentage motility, percentage live and mass activity. Results showed that motility persisted in both groups up to day seven of the experiment with the test and control having motility of 10% and 20%, percentage live of 10% and 20% and mass activity of +(1) and +(1), respectively as at day seven. There was no statistically significant difference between the mean motility, mean percentage live and mean mass activity of the two groups. The mean percentage motility for the test and control groups were 41.43 ± 68 and 48.57 ± 65, respectively, mean percentage live were 41.42 ± 68 and 48.57 ± 65, respectively and mean mass activity were 1.86 ± 08 and 2.00 ± 10, respectively. It was concluded that citrated egg yolk extender at -20°C was suitable for short term storage of the milt. It was recommended that optimum cryoprotectant agents be established. It was also recommended that field trials be involved to test fertilizability and hatchability after such storage.
Keywords: Citrated egg yolk, Milt, Extender, Catfish
An estimated 73.8 million tonnes of aquaculture production comes from fresh water aquaculture. Aquaculture has been reported as a fast growing sector in India with over 6.5 fold growth rate . With regard to biological properties, the African walking catfish, Clarias gariepinus  is believed to be the most suitable species for aquaculture in Africa . Its distribution is wide, performs excellently well in different environmental conditions (sub-tropical to tropical), it adjusts well to a wide range of water quality conditions primarily due to its breathing ability; it feeds on a wide array of natural prey under diverse conditions; its fecundity is great and it is easily reproduced under captivity . The importance of C. gariepinus as an aquaculture species in Africa was first realized by Hey . Before the mid 1970's, little research on its culture potential was conducted ; research interest in African catfish has grown since the past 10 decades and is now conducted in many parts of Europe and Africa with the most recent being in Nigeria (West Africa). These largely independent but parallel research initiatives have resulted in the rapid development of farming technology  and of techniques for reproduction and rearing . Today, commercial and subsistence farming exist in many African countries. Nigeria, South Africa, Zambia and Ghana are the highest producers (in terms of annual tonnage). In Nigeria, catfish production is currently distributed among few big and a lot of small farms. The annual production is difficult to calculate due to poor records , but the nation is developing in technology (such as re-circulating systems) and hoping to be the largest African catfish producer in the world in the next 10-20 years, with 10-30,000 tons per year .
Catfishes are economically important group of fresh and brackish water fish worldwide. Several species have been successfully introduced in aquaculture  and the African catfish, Clarias gariepinus (formerly C. lazera; ), is perhaps the most important, not only in Africa but also in South East Asia (e.g. Thailand) and in Europe (e.g. The Netherlands). The availability of gametes throughout the year is important to ensure a constant supply of fish all year round. Under captivity (25°C; 12 h light per day), C. gariepinus shows a continuous gametogenesis once sexual maturity is reached . However, whereas females can be stripped of eggs after treatments with carp pituitary extracts cPE ; or gonadotropic hormones ; release of sperm and male reproductive behavior are unspontaneous , regardless of hormone treatment. Storing batches of germ cell or milt by cold storage would significantly improve the reproductive potential of the males. The use of adequate milt extenders that would guarantee long term storage becomes critical to the success of milt cryopreservation. Storage of gametes or germ cells remains a topic of discussion among scientists. Regardless of these efforts, the use of preservation and storage technologies to aquaculture practice is very limited . Freezing, cryogenic storage and thawing of living material is addressed in cryopreservation . Milt is a seminal fluid of fish containing single type of cells of the spermatozoa which are freely suspended in fluid medium (the seminal plasma) and the cells are not subject to growth or division. Production of milt marks onset of sexual maturity in males. Cryopreservation of milt or semen is being used worldwide. It is now applied in horse, pig, sheep and poultry breeding which has resulted in improved gene pool of native stocks . Successful cryopreservation is well established for sperm cells from many fish species, but a standard technique for true cryopreservation of fish ova has proved elusive . Considering the cost of keeping broodstock population, it would provide a method of retaining specific genetic line of fish. It would offer a secondary source of a genetic line in case of broodstock loss. It also enhances the preservation of endangered genetic lines in wild populations .
Diluents maintain inactivity of spermatozoa when semen is diluted before freezing due to their stabilizing potential on the physicochemical properties. Saline and sugar-based extenders have been created and used . The extender is set on buffered physiological saline solutions. This is due to the resemblance of inorganic composition of seminal plasma of spermatozoa with such buffered solution . In a short-term storage experiment, sperm was stored at different dilution ratios (1/10 and 1/50) and temperatures (4 and 20°C) and storage in constant agitation or unagitated. In addition, for the sperm cryopreservation of large volumes (cryovials of 2 and 5 ml), different cooling rates (1 and 3 cm above liquid nitrogen have been tested . To minimize cryoinjuries during cooling and thawing process a large variety of cryoprotective agents of permeating and non-permeating categories are available for use. The widely used cryoprotective agents are DMSO and glycerol. Competency of extenders and cryoprotectants vary from one fish to another. Milt is usually packaged in cryovials, plastic straws of visotubes chilled over liquid nitrogen vapor or they are kept in programmable freezer and stored in liquid nitrogen . Another way of cryopreserving the fish milt is cryopreserving it as pellets on dry ice blocks and then storing in caped cryovials in liquid nitrogen. For cryopreservation various cooling methods had been used successfully on fish sperm. Controlling the size, configuration and location of ice crystal demand a careful manipulation of temperature excursion. For any protocol development, choice and concentration of cryoprotectant and rate of cooling needs to be optimized for each species . One universal protocol cannot be suggested from the current state of art of fish spermatozoa cryopreservation and species differences. This is because the response to cryoprotectant and freezing vary with the different biology. Although some common guides may be used for particular species of fish but effective utilization of the protocol is valuable for each peculiar species. Standardization for the development of any trustworthy protocols for fish milt cryopreservation should always be given priority .
Cryopreservation leads to the generation of reactive oxygen species (ROS), which impair post thaw motility, viability, intracellular enzymatic activity, fertility and other sperm parameters . Antioxidants play an important role in sperm motility, integrity, metabolism and function, by protecting the cells against oxidative damage. Damage to sperm function has been successfully minimized in several mammalian species by the addition of antioxidants to the extender media prior to cryopreservation .
Egg yolk semen extender has performed excellently well in cryopreservation of bovine semen at -196°C. Establishment of a freezing protocol at -20°C using egg yolk extender would go a long way to encourage milt storage among subsistent fish farmers. Deep freezers are easily affordable and can be found anywhere among local farmers. Hence, the justification for suitable milt diluents, motility activators and cold shielding agents for deep freezing storage. This study therefore aims to assess the efficacy of a readily available semen extender (citrated egg yolk) on milt of African catfish, preserved at deep freezing temperature of -20°C by testing the quality of post - thawed milt extended in citrated egg yolk extender, with the objectives of having quality milt available to farmers all year round, in order to reduce the stress of having to search for and transport male brood stocks at each spawning period.
MATERIALS AND METHODS
The study was carried out at physiology Laboratory of Faculty of Veterinary Medicine, University of Abuja which is situated within the Federal Capital Territory (FCT), Abuja (longitude 7009’54”E and 7010’38”E and latitude 8059’13”N and 8059’49”N .
The experimental design involved storage of semen in two experimental groups consisting the test group which was citrated egg yolk extended milt, shared into seven (7) properly labeled sterile containers, preserved at deep freezing temperature of -20°C and the control group which involved milt extended in sodium citrate shared into seven insulin syringes, properly labeled and stored at 4°C, a condition known to preserve semen of the African catfish. The two groups were observed for seven days and percentage motility, percentage live as well as mass activity of spermatozoa in these samples was recorded.
Two male brood stocks of Clarias gariepinus with average age of 14 months, average weight 1.5 kg and average measurement of 41.5 cm length were purchased from a reputable farm (Olompet Holdings) in Abuja for the study. The male brood stocks were conveyed to the Theriogenology Laboratory, in well aerated containers. The choice of males was based on the possession of well vascularized genital papilla.
Preparation of semen extender
Preparation of buffer: About 250 ml of distilled water was heated to boiling point and allowed to cool. The preparation of 2.9% tri-sodium citrate solution was made by dissolving 2.9 g of the salt in 10 ml of distilled water in a flat bottom flask. This was shaken together until the salt dissolved completely. The solution was then made up to 100 ml by adding distilled water.
Preparation of egg yolk component (yolk separation): Two freshly laid eggs were washed and disinfected using 70% alcohol. They were cracked carefully into two, such that the albumen drained out from the crack until very little of it was left with the yolk. The yolk was then carefully dropped on Whartman® filter paper which absorbed what was left of the albumen. The yolks were collected into beakers.
The solution for the study was made using 80 ml tri-sodium citrate solution and 20 ml off egg yolk. This gave a total of 100 ml solution. To this 2 ml of penicillin and 2.5 ml streptomycin were added.
One of the male brood stocks was selected and sacrificed by spinal transection, after which the belly was dissected and the testes were removed. Blood clots and other tissues were rinsed away using tri-sodium citrate solution. The testes were cleaned and dried by placing on a Whartman® filter paper, which absorbed the excess fluid. Physical parameters which included weight, length and width were observed for the right testis. Semen was collected by gentle perforation of the testis with a sterile needle into a 5 ml sterile container. The content of one testis (the right) was extracted for mixture with the extenders.
Post collection examination
Pre-extension motility was determined by:
a) Mixing one drop of fresh milt with one drop of distilled water on a clean slide to check motility and mass activity using the light microscope at x40 (this was done for the two groups).
b) Using an insulin syringe to collect 0.1 ml of fresh milt, this was made up to 1 ml mark with trisodium citrate solution, giving a 1 in 10 dilution. A drop of this mixture was placed on a clean glass slide with a drop of neutral stain (eosin-nigrosin) for observation of percentage live. This was done with a light microscope using x100 objective lens. The color and volume of semen extracted were also observed.
Test group: 1 ml of freshly collected milt was carefully decanted into a sterile container with 4.5 ml of the diluents (citrated egg yolk extender solution). The second half 4.5 ml of the diluents containing 7% glycerol was added at room temperature to the first half constituting 3.5% glycerol in a 9 ml diluents. This mixture gives a dilution rate of 1 in 10 (1:10). The mixture was then gently rocked together to ensure proper mixing and then dispensed at 1 ml mark each into seven (7) 5 ml sterile containers. These containers were labeled accordingly and kept in an ice pack, before transfer to a deep freezer set at -20°C. Daily storage examination was done for motility; mass activity and percentage live for a period of 7 days.
Control group (milt+sodium citrate): A one in ten (1: 10) dilution of milt was made with trisodium citrate. This was carefully dispensed into seven (7) insulin syringes at 1 ml mark each, labeled accordingly and kept in an ice pack before transfer and storage in a refrigerator at 4°C. This was also examined for a period of 7 days for the same parameters.
Data were expressed as means and standard error of mean (SEM) and the differences between groups were analyzed using the Student t-test with SPSS/PC computer programme (version16.0). A confidence value of p<0.05 was considered statistically significant. Moreover, Pearson correlation analysis was also done to establish strength of positive or negative relationship between the two groups.
Testicular parameters measured were weight, length and width and values obtained were 5.5 g, 7.8 cm and 1.5 cm respectively. The pre-dilution findings indicated a pre-dilution motility of 90%, percentage live of 90% and mass activity of +++ (Table 1). Results for percentage live (Table 1) indicated that about 20% and 10% of spermatozoa were viable at day 7 post storage in the control and test groups, respectively (Table 1). Mass activity results (Table 1) showed that in both groups, progressive mass activity of spermatozoa dropped with increasing days of storage. There was no significant difference (P>0.05) although Pearson correlation analysis, showed a strong positive relationship 0.924 for motility and percentage live and 0.904 for mass activity between the two groups (Table 1 and Figures 1-3) correlation was significant at the 0.01 level (2-tailed).
This experiment suggests that citrated egg yolk extenders are suitable for short term preservation of the milt of the catfish when stored in deep freezer. Long term storage of the milt of the catfish in the deep freezer may be a possibility if adequate concentration of cryoprotectant agents like glycerol is added. Citrated egg yolk extender performed excellently well in enhancing survival within the first 24 h, motility recorded was 80% at 24 h post storage. Progressive motility dropped as the duration of storage (days) increased. The test group which involved storage of milt extended with citrated egg yolk at -20°C recorded motility as well as viability of 10% at day 7 post storage. This finding in the test group lends some support to the research published by Adeyemo et al.  who reported the observation of 10% post-thawed motility after one month with milt of Clarias gariepinus stored with 10% egg yolk at -40°C. It is a fact that cryoinjuries can result due to slower or faster than optimal cooling and thawing rates , therefore, the minor variation in motility, mass activity and percentage live observed in the experiment could be ascribed to the slow freezing rate in the deep freezer. Performance of sodium citrate in this work was expected. It has been standardized to preserve milt of Clarias gariepinus when refrigerated [27,28], thus it served as the control group. Results indicated that citrated water at 4°C gave 20% motility and with percentage live of 20% by day 7 of preservation. This observation agrees with the work of Mansour et al.  who observed that at optimal condition when buffered saline solution is used for storage of milt of Clarias gariepinus at 4°C, sperm viability persists longer than 7 days. Lardy and Philips  and Foote  also documented that physiological buffers have the ability to dilute semen and maintain its viability for some days when chilled.
Citrated egg yolk extender has shown potential in short term storage of milt of the African catfish at a temperature of -20°C, but may not be suitable for long term storage at this temperature. Na citrate still remains a suitable buffer for short term storage of milt of African catfish but as used in this research has to be at 4°C. It is recommended that more work should be done on the effect of deep freezing at -20°C on citrated egg yolk extended milt of the African catfish with adequate cryoprotectant agents to explore its long term storage potentials. Finally it is recommended to take this research to field trials to test fertilizability and hatchability using the stored milt.
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