Cryopreservation Of Sperm Of The Mekong Giant Catfish, Pangasianodon Gigas Chevey

K. Mongkonpunya, T. Pupipat, S. Pholprasith, M. Chantasut,

R. Rittaporn, S. Pimolboot, S. Wiwatcharakoses, And M. Chaengkij

Abstract

Spermatozoa of an individual male giant catfish, Pangasianodon gigas Chevey, were successfully cryopreserved in liquid nitrogen (LN), retaining fertilizing capacity for up to 18 months. Spermatozoa were extended ( 1:3 milt:medium ratio) in extender "Sl6 " (1.25 g KHCO3 8.55 g sucrose, and 0.30 g reduced glutathione in 100 g distilled water combined with dimethylsulfoxide [8 % final concentration]) within two types of containers (1 ml glass ampules, and in half broken artificial insemination [AI] catheters) and then frozen by suspending the containers of extended milt for 15 minutes in the vapor above LN before immersion. After 2, 360, and 540 days of storage, samples of cryopreserved sperm were thawed (20 seconds in 70°C-80°C water bath) and tested with fresh eggs of Pangasianodon gigas, Pangasius sp. (Mekong origin), and Pangasius sutchi to determine fertilization rates. Mean fertilization rates of sperm cryopreserved in AI catheters were comparable to those of controls, fresh sperm, but significantly greater than those preserved in ampules. Fertilization rates (%) using sperm preserved in AI catheters were (mean _ SE), 67.7 _ 7.1, 67.7 + 0.8 and 34.7 + 5.7 (controls were 79.0 :: 1.4, 64.6 + 0.6 and 59.2 + 9.8) for eggs of Pangasianodon gigas, Pangasius sp., and Pangasius sutchi, respectively).

Introduction

Cryopreservation techniques offer considerable potential for fish culture and have been explored for a variety of species (Horton and Ott 1976, Mounib 1978, Erdahl et al. 1984, Kerby 1983). However, most of the work was done on a small-scale experimental basis. Recently, Kerby et al. (1985) designed their experiments to expand the scope of investigation. They examined the growth, survival, and health of larval striped bass hatched from eggs fertilized with cryopreserved sperm. They produced and harvested thousands of normal fish 43-47 days after stocking. In Thailand, a monsoon country, spermatozoa preservation has not been practiced in fish hatcheries because high quality milt is available almost year-round. However, there has been an increasing awareness of the benefits of cryopreserved sperm, which would contribute to the controlled breeding and husbandry of some uncultivated fishes, e.g., Pangasius snithwongsei and Pangasianodon gigas. Hatchery production of P. gigas has been obstructed by the death of males and/or females while being kept alive waiting for the opposite sex. Preservation of spermatozoa with relatively high viability should be an excellent solution for this problem. Thus, techniques of chilled storage (0-5°C) and cryopreservation have been developed in Puntius gonionotus (Markman 1984) and in Pangasius sutchi (Pupipat 1988, unpublished data). It was found that by using medium "S16" (Table 1) with 0.25 ml straws and 1-ml glass ampules, fertility of the cryopreserved spermatozoa was maintained. However, 0.25 ml straws are too small for large-scale production. We also found that frozen glass ampules containing cryopreserved sperm often explode dangerously during the thawing process.

TABLE 1 Ingredients of Extenders

EXT	KHCO3	SUC	GLUT	MP	DMSO	CH3OH
				%
S16	1.25	8.55	0.30	-	8
S17	-	-	-	5	-	5
S18	-	-	-	10	-	10
S19	-	-	-	15	-	5
S20	-	-	-	15	-	10

 

Notes: SUC= sucrose, GLUT= reduced glutathione, MP= milk powder.

In this study, we examined the use of 1 ml sterile AI catheters (by cutting them into two pieces of equal length), in comparison with 1 ml glass ampules. We also used fresh ova of Pangasius sp. for testing of fertilizing capacity of P. gigas cryopreserved spermatozoa.

Materials And Methods

Fresh milt (about 100 ml) used for cryopreservation in this study was collected from an individual male Mekong giant catfish, which weighed 173 kg and was caught in the 1989 spawning season (April-May). Abdominal stripping was done 20 hr after an injection of Domperidone (5 mg/kg), plus Ovaprin (5 mg/kg). Only creamy white semen was collected into a 250 ml beaker. Motility or mass movement of the whole sperm was examined immediately after activation with an equal volume of distilled water. It was found that motility of the milt sample was excellent. Then, every 100 ml of semen was diluted in a milt:medium ratio of 1:3 with an extender (Table 1) of 1.25 g KHC03, 8.55 g sucrose, and 0.30 g reduced glutathione in 100 g distilled water. The final concentration of dimethylsulfoxide (DMSO) in extended milt was 8%. This extender was chosen based on our previous work, which indicated that it provided the best result.

Approximately 0.8 ml of extended semen was placed into 1 ml glass ampules or into half-broken AI catheters. Then, all samples were frozen by suspending them in the vapor above liquid nitrogen for 15 minutes before immersion.

After varying periods of storage (2, 360, and 540 days), samples of cryopreserved sperm were thawed rapidly by swirling for 20 sec in a 70-80°C water bath and tested with fresh eggs to determine fertilization rates.

For fertilization testing, fresh eggs from a female P. gigas were available two days after sperm preservation and were used for this purpose. Since fresh eggs of P. gigas were not available during the 1990 spawning season, or for about 360 days after the sperm preservation, we used fresh eggs of a female Pangasius sp. of Mekong origin for the fertilization test. Another subsequent fertilization test using the same batch of cryopreserved sperm was attempted with fresh eggs from six female Pangasius sutchi at 540 days after storage. It must be mentioned that hybridization was not a purpose of this study. The Pangasius sp. eggs were used only because P. gigas eggs were not available.

The females were induced to spawn by two hormonal injections using human chorionic gonadotropin (hCG) (100 + 100 i.u./kg) for Pangasius sp., and Domperidone plus Ovaprim (7.5 mg/kg + 25 ug/kg) for P. gigas. These treatments resulted in spawning 6-20 hr after the second injection. Details of the investigation on hormonal-induced spawning will be reported separately.

When a female was stripped, the eggs were split into two containers. One, used for normal production, was also used as the control and received fresh sperm from a male. The second container was further split into 0.5 ml aliquots (ca. 500 eggs) and each aliquot was fertilized by pouring 0.8 ml of nearly thawed semen into the containers, adding hatchery water, and stirring the mixture gently. The step of rinsing was repeated a few times to remove debris and excess sperm. After that, the presumptive fertilized eggs were placed in one lifer plastic bowls full of water to incubate. About 18 hr post-fertilization, the percentages of fertilization were determined. This assay of semen fertility was based on the percentage of embryos that developed to the eyed stage.

An analysis of variance was used to test differences between control (fresh semen) and frozen semen, and also between the two types of containers. Eggs from a single female were used to compare fertility of semen between the treatment groups.

Results And Discussion

Spermatozoa activity (mass movement) was checked for the diluted semen left in the beaker. This was done to ensure that the sperm were still alive at the end of the 3-fur freezing procedure. Sperm motility was found to be as good as fresh whole semen.

The motility of post-thawed sperm was also determined for those left in the ampules and catheters. However, this motility determination was done 5-10 min after thawing. Probably due to time delay, sperm motility was generally poor or unobservable. This indicated that the post-thaw quality of the sperm was deteriorating rapidly. From our experience, sperm activity lasted about 30-60 sec after being activated. Thus, in doing fertilization tests, time is a critical factor. It was our practice to swirl the ampule or the catheter containing cryopreserved sperm in hot water (70-80°C) for 20 sec and then the neck of the ampule or the end of the catheter was cut off and sperm were shaken out and mixed with fresh eggs. The time elapse between thawing and adding hatchery water was a matter of 1-2 min or less.

Fertilization rates examined at 18 hr are shown in Table 2. Due to a limited amount of available semen in Pangasius sp. (Mekong origin), the sample was preserved only in half-broken AI catheters, and a comparison between container types was not possible.

Fertilization percentages of cryopreserved sperm under these experimental conditions were quite inconsistent. They ranged from 8 to 14% (Table 2) for glass ampules and 26 to 77% for halfbroken AI catheters. Thus, our results are comparable to those reported by many others. However, the later set of data showed no significant difference from those of the controls (fresh semen of the same species). Of the containers tested, glass ampules provided sperm with a significantly lower fertility percentage. Besides, quite often the glass ampules exploded or broke while thawing. Therefore, using glass ampules for sperm cryopreservation is not recommended. In addition, both types of containers held approximately 0.8 ml of diluted semen. There was, however, a difference in the diameters of the containers, with the glass ampules having a greater diameter. Thus, larger diameters of glass ampules prevented an even rate of freezing and thawing of semen samples within the ampules, and resulted in lower sperm fertility. Half-broken AI catheters were smaller in diameter and provided higher sperm fertility. The capacity and configuration of the catheters are similar to those of 1 ml French straws. We preferred 1 ml French straws over the catheters, but the straws were not available. Since the spawning season of 1991 (Apr-May) we have been using 4.5 ml cryotubes (Nunc, Inc.) for holding 4 ml diluted semen or about 1 ml fresh semen with a dilution ratio of 1:3. This container is believed to be practical and applicable for hatchery production, since its capacity is large. However, we found that the rates of cooling and thawing used in this experiment were not suitable for the cryotubes, as the fertility of the preserved sperm could not be maintained (data not shown).

TABLE 2 Fertilization Percentages¹ of Sperm Cryopreserved in Medium S16 Examined 18 hrs after Insemination

Ova Source²	Container		Control
	Ampule	AI Catheter
Pangasius gigas	11.7 + 2.5	67.7± 7.1	79.0 i 1.4
	(9-14)	(53-77)	(76-80)
Pangasius sp.	-	67.7±0.8	64.6 ± 0.6
		(66-69)	(64-65)
Pangasius sutchi	10.4 ± 1.1	34.7±5.7	59.2 ±9.8
	(8-12)	(26-45)	(42-95)

 

¹Fertilization percentages were based on the percentage of embryos which developed to eyed stage. ²Eggs from one each of P. gigas and Pangasius sp. were used, but from six of Pangasius sutchi.

³Eggs from a single female were used to compare fertility of semen between treatment groups. Within each treatment, there were three incubations of about 500 eggs.

Since P. gigas is an uncultivated and endangered species, the eggs and semen, if available, are seriously needed for hatchery production. The harvesting season for this species has a very short duration of about two weeks between the end of April and May. During our study period it was not possible to obtain P. gigas eggs for intraspecies fertilization trials. Thus, interspecies fertilization, using eggs of Pangasius sp., was considered the best option for testing the fertility of the cryopreserved sperm of Pangasianodon gigas.

The fertilizing capability of the cryopreserved sperm with Pangasius eggs was comparatively lower than those with P. gigas eggs (Table 2). However, this difference probably was not due to incompatibility of the eggs and sperm, because the control data (fresh eggs x fresh sperm of the same species) also showed the same trend. Therefore, these differences may be a reflection of poor egg quality or other unknown factors.

The large variability in the fertilizing capability of the cryopreserved sperm in our study seems

to be a phenomenon common to this type of research. Apparently, small alterations in the thawing process can cause highly variable results. If the potential for cryopreservation is to be realized, precise standards must be established and used consistently.

Results from this study clearly indicated that, when working with scarce and endangered

species of fishes, cryopreserved sperm represent a valuable tool, and interspecies/intergeneric fertilization may be a useful tool for testing the fertility of the cryopreserved spermatozoa.

Acknowledgments

Support for this work was provided, in part, by the Office of the Science and Technology

Development Board offered through the Division of Inland Fisheries, and, in part, by Kasetsart University. The work was conducted, in part, at the Chainat Inland Fisheries Station and special thanks are due to Mr. Sontipun Pasukdee for his cooperation and assistance. The authors also thank the Office of Research, USAID, for funding the network meeting and the publication of this paper.

References Cited

Erdahl, A. W., D. A. Erdahl, and E. F. Grahm. 1984. Some factors affecting the preservation of salmonid spermatozoa. Aquaculture 43: 348-350.

Horton, H. F. and A. G. Ott. 1976. Cryopreservation of fish spermatozoa and ova. J. Fish. Res. Board Can. 33: 995-1000.

Kerby, J. H. 1983. Cryogenic preservation of sperm from striped bass. Trans. Am. Fish. Soc. 112: 86-94.

Kerby, J. H., J. D. Bayless, and R. M. Harrell. 1985. Growth, survival, and harvest of striped bass produced with cryopreserved spermatozoa. Trans. Am. Fish. Soc. 114: 761-765.

Markman, N. 1984. A preliminary study on cryopreservation of fish spermatozoa. Faculty of Science, Kasetsart University, Bangkok. M.Sc. Thesis. (in Thai.)

Mounib, M. S. 1978. Cryogenic preservation of fish and mammalian spermatozoa. J. Reprod. Fertil. 53: 13-18.