Research Report

Effect of Sperm Sac (Testis) in Cryopreservation Protocol of Milt (Spermatozoa) of Clarias Gariepinus  

Ubah S.A.1 , Okere N.C.2 , Nwankwo P.A.1 , Orokpo I.A.3
1 Department of Theriogenology, Faculty of Veterinary Medicine, University of Abuja, Nigeria
2 Fish and wild life unit, Department of Veterinary Public Health and Preventive Medicine, University of Ibadan, Nigeria
3 Department of Physiology and Biochemistry, Faculty of Veterinary Medicine, University of Abuja, Nigeria
Author    Correspondence author
International Journal of Aquaculture, 2017, Vol. 7, No. 14   doi: 10.5376/ija.2017.07.0014
Received: 29 Jun., 2017    Accepted: 31 Jul., 2017    Published: 18 Aug., 2017
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This is an open access article published under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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Ubah S.A., Okere N.C., Nwankwo P.A., and Orokpo I.A., 2017, Effect of sperm sac (testis) in cryopreservation protocol of milt (spermatozoa) of Clarias Gariepinus, International Journal of Aquaculture, 7(14): 94-100 (doi: 10.5376/ija.2017.07.0014)


This study was carried out to assess the adequacy of cryopreservation protocol of milt (spermatozoa) of Clarias garipenus, using the sperm sac (testis) intact with the objectives of evaluating the milt for percentage live, motility and mass activity for a period of seven days at -20°C. Semen samples (milt) were collected from mature male broodstocks of Clarias gariepinus of 14 months old and weighing 1.5 kg. There were two groups comprising test and control groups. Test group comprising of the sperm sac was dipped in Na-Citrate and placed in a properly labelled container and preserved at -20°C while control group comprised of milt extended in Na-Citrate dispensed into seven insulin syringes, properly labelled and stored at 4°C. These were observed for a period of seven days and percentage motility, percentage live as well as mass activity of spermatozoa of these samples were recorded. Student’s t test was used in analyzing the data. Results showed statistically significant difference (p<0.05) in motility, percentage live and mass activity between test and control groups, with test group showing mean percentage motility, percentage live and mass activity of 24.29±76%, 24.29±76% and 1.14±1.8 respectively while the control showed values of 48.57±65%, 48.57± 65% and 2.0±1.0 respectively. It was concluded that use of the sperm sac at -20°C can only be adopted within three days of collection from the fish. It was recommended that farmers adopt the protocol only for a short term period to eliminate the frustration of artificial spawning failure due lack of good semen to complete the synchronous reproductive process.

Sperm sac; Milt; Cryopreservation; Catfish; Artifical spawning

1 Background

The African catfish, Clarias gariepinus (Burchell, 1822) is considered one of best suitable alternatives to tilapia for subsistence for fish farming. African Catfish is a good source of animal protein and has a very tasty delicacy. It is cultured for its high growth rate, disease resistance and its’ tolerant of a wide range of temperature, as well as oxygen and high salinity level (Haylor, 1993). This has attracted a lot of stakeholders whose major problem is to satisfy the increasing demand for the fish; one of the challenges of African catfish farming is making fingerlings available all year round. There is need to research on various methods of milt preservation so as to ensure the possibility of developing effective and easier methods of milt preservation. Proper preservation of milt will ensure an all year round availability of gametes to farmers and easy access to genetically improved milt for African catfish reproduction.


This research work is concentrating solely on a method of preservation and it is believed with no doubt that it will give adequate information on the role of the sperm sac on preserved milt of the African catfish.


This research will give more insight on the adequacy of cryopreservation protocol of milt (spermatozoa) of Clarias gariepinus using the sperm sac (testes) intact with the objectives of evaluating the milt for percentage live, motility and mass activity for a period of seven days at -20°C.


To obtain spermatozoa it is necessary to sacrifice male brood fish (Steyn and Van Vuren, 1987) or surgically remove part of their testes (Bart and Dunham, 1990). Although this method is efficient it jeopardizes selection and genetic improvement. Such reproductive dysfunctions are mainly due to the fact that fish in captivity do not experience the natural conditions of the spawning grounds and as a result, the pituitary fails to release the maturational gonadotropin, luteinizing hormone, LH; (Zohar and Mylonas, 2001). Storing batches of spermatozoa by cryopreservation would significantly improve the reproductive potential of male catfish. African catfish is on high demand in Nigeria. However, producing enough fingerlings poses a major problem. Also, increased productivity is directly linked to increase in the number of offspring and their survival rate (Madu et al., 1998). Obtaining genetically superior males as sperm suppliers would contribute immensely to this. The use of such males could be optimized if there is enough facility to preserve their supplied sperm for longer periods, thereby allowing females to be spawned when and where males are not available. Thus there is need for appropriate cryoprotectants and freezing protocols.


Lack of a totally reliable method of long term preservation of milts creates the need for researches directed towards developing adequate preservation protocols. The aim of the this study was therefore to test the adequacy of a preservation protocol that involves the sperm sac intact with the objectives of evaluating the milt for percentage live, motility and mass activity for a period of seven days at -20°C. 


2 Results

2.1 Table 1

The physical parameters (weight, length and width) of the dissected left and right testes were recorded. Table 1 showed the results obtained. Also in Figure 1 we have a photograph of dissected testes of Clarias gariepinus showing both the left and right testes.


Table 1 Physical characteristics of both testes (sperm sacs)


Figure 1 Photograph of dissected testes of Clarias gariepinus


2.2 Table 2

Table 2 shows the pre-storage milt characteristics of both the left and right testes. Both had the same values except in volume where the right testis was not extracted so the volume (ml) could not be determined.


Table 2 Pre- storage milt characteristics


2.3 Table 3

Table 3 showed sperm motility under various durations. It was discovered that the motility of the test group became less than 50% after 48 hrs while the control group became less than 50% after 96 hrs.


P(0.00) < 0.05. This is statistically significant. However, Pearson correlation analysis shows strong positive relationship (0.9) between the two groups.


Table 3 Sperm motility (%) for test and control groups


2.4 Table 4

In Table 4 it was observed that the percentage % live for the test group became less than 50% after 48 hrs and were all dead by 120 hrs while the control group was less than 50% after 96 hrs.


Table 4 Percentage live (%) for test and control groups


P(0.00) < 0.05. This is statistically significant. However, Pearson correlation analysis shows strong positive relationship (0.9) between the two groups.


2.5 Table 5

In Table 5 the sperm mass activity was observed to drop to ++ after 24 hrs and was 0 after 120 hrs while the control group was ++ after 48 hrs but was still + at 168 hrs.


Table 5 Sperm mass activity for test and control groups


P(0.02) < 0.05. This is statistically significant. Pearson correlation analysis shows strong positive relationship (0.9) between the two groups.


3 Discussions

Pre-storage results obtained in this research observed motility of 90% from milt extracted from both testes. This value is in agreement with the suggested pre-extension motility of 80% and above (Akay et al., 1995; Urbanyi et al., 1999; Adeyemo et al., 2007). The poor result obtained in the test group following storage can be attributed to some factors one of which could be the repeated effect of freezing and thawing. Results obtained here revealed a drastic drop in motility as well as viability from 70% at 24 hrs of storage to 50% at 48 hrs. All the cells were found to be immotile at 120 hrs post storage and there were no viable cells as well. It is known that freezing injuries can be caused by slower or faster than optimal cooling and thawing rates (Vivieros, 2002), hence the sharp decline in motility and viability could be attributed to the accumulating injuries and exhaustion from repeated freezing and thawing process. Another factor that could have caused the poor result is the lack of dilution of the milt with the extender. This assumption is in line with the finding of Gwo et al. (2009) that undiluted gametes are not suitable for freezing and they must be diluted with a suitable extender, which is a solution of balanced salts and sometimes organic compounds.


Another major finding in this research is the performance of Na citrate. It is well known that Na citrate has the ability to preserve milt of Clarias gariepinus when refrigerated (Mansour et al., 2005; Adeyemo et al., 2007), thus it served as the control in this research. Results obtained showed that Na citrate at 4°C gave 20% motility and viability by day 7 post storage. This finding supports the work of Mansour et al. (2005) 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 for > 7 days. Lardy and Philips (1940) and Foote (1980) also reported that physiological buffers have the ability to dilute semen and maintain its viability for some days when refrigerated. It is also noteworthy to state that the result with this experiment confirms the viability of the milt used in this research.


4 Materials and Methods

4.1 Experimental fish

Two male brood stocks of Clarias gariepinus 14months of age, weighing 1.5 kg each, were purchased from a reputable farm (Olompet Holdings) in Abuja for the experiment. The male brood stocks were conveyed to the Theriogenology Laboratory of the Faculty of Veterinary Medicine University of Abuja, in well aerated containers. The choice of males was based on the possession of well vascularized genital papilla (Figure 2).


Figure 2 Photograph of genital papilla of a male brood stock of Clarias gariepinus


4.2 Experimental design

The experimental method adopted in any research study to a large extent is dependent on the nature and objective of the study. There were two groups comprising the test and the control groups. The Test group was the left testis dipped in Na citrate placed in a properly labeled container and preserved at -20°C. The control group included milt extended in Na citrate shared into seven insulin syringes, properly labeled and stored at 4°C, a condition already known to preserve semen of the African catfish. This group served as the control for the experiment. The two groups were observed for a period of seven days and percentage motility, life/dead ratio as well as mass activity of spermatozoa in these samples were recorded. The control group involved taking out a sample out of the seven syringes each day, while test group involved daily thawing of the sperm sac sample to collect and examine milt.


4.3 Milt collection

The male brood stocks was selected and sacrificed by spinal transaction, 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 both testes. Semen was collected by gentle perforation of the testes with a sterile needle into a 5 ml sterile container. The content of one testis (the right) was extracted for mixture with the extender while the other was left intact.


4.4 Pre-storage parameters were determined

(1) By mixing one drop of fresh milt with one drop of distilled water on a clean slide and observed under the microscope for motility and mass activity. (this was done for the two groups)

(2) By 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) to make a semen smear for observing percentage live. Observation was done with a light microscope using × 100 objective lens Figure 3 and Figure 4.

(3) Colour and volume of semen extracted were also observed.


Figure 3 Photomicrograph of (negrosin and eosin) stained milt smear (×100) of Clarias gariepinus with high number of morphologically normal live cells


Figure 4 Photomicrograph of (negrosin and eosin) stained milt smear (×100) of Clarias gariepinus, with high number of morphologically normal but dead cells


4.5 Post collection examination

4.5.1 Test group (testis dipped in sodium (Na) citrate)

The left testes was carefully placed in a sterile container containing trisodium citrate, kept in an ice pack before being transferred to the freezer. The sack was carefully punctured daily to collect a drop of semen to observe motility, mass activity, and life/dead ratio for a period of 7 days. 


4.5.2 Control group (milt + Na citrate)

A one in ten (1 in 10) dilution of milt was made with trisodium citrate. This was carefully aspirated into seven (7) insulin syringes at 0.1 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.


4.6 Statistical method

The statistical tests used for analysis here is the student T-test, pearson correlation analysis. Results were expressed as means and standard error of mean (SEM) data was analyzed using the student T-test with SPSS/PC computer programme, version 16.0. Differences with confidence values of P < 0.05 were considered statistically significant. Pearson correlation analysis was also done to establish strength of positive or negative relationship between the two groups.


5 Conclusions

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. The importance of dilution of milt with a suitable extender before storage cannot be over emphasized as indicated by the result in test group irrespective of the emphasis on the dilution of milt, the test group wasn’t totally a right off as it showed that milt can be preserved in testes after collection, as long as it is at -20°C and does not exceed 3 days. It was recommended that farmers collect the sperm sac (testes) a day or two days before spawning, determine the quality of the semen and store in a deep freezer before injecting the females. A number of times local farmers inject the female brood stocks, do the proper timing, only to sacrifice the male brood stock at the appointed time and it will not have a suitable sperm sac or good semen to complete the synchronous reproductive process. This leads to enormous economic loss and frustration. More efforts should be made towards establishing suitable freezing protocols for the milt of the African catfish.


Authors’ contributions

Nwankwo, P. carried out the milt collection and determination of physical characteristics of samples. Orokpo, I. carried out Pre and Post storage collection parameters examinations. Okere, N. participated in design of study, performed the statistical analysis and drafted the manuscript. Ubah, S. conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.



We wish to acknowledge the Theriogenology lab and Physiology lab of Faculty of Veterinary Medicine, University of Abuja.



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