666 CAN. J . ZOOL. VOL. 51. 1973

Acknowledgments

This investigation was supported by a grant from the National Research Council of Canada. I thank Ms. Nancy Young for her able technical assistance.

BROWN, G. W., JR. 1964. Tpe ,metaboIim of Amphibia. In Phys~olugy of the Arnphlbra, Edited by J. A. Moore. Acadcmrc Press, New York and London. pp. 1-98.

Joms, D. R. 1967. Oxygen consumption and heart rate or several species of anuran Amphibia during sub- mergence. Comp. Biochcm. Phys~ol. 20: 691-707.

N o w s , W. E., JR., P. A. GRANDY. and W. K. D A ~ . 1963. Cornparatrve studies of oxygen consumption of three s w i w of nwtcnic salamanders as influenced by temperature, body size, and oxygen tension. Bid. Bull. {Woods Hole), 125: 523-533.

T o m D. P. 1969. R~spiration and circulation in Amphiltma trldoctyliwn. Ph.D. Thesis, University of British Colambia. Vancouver, B.C.

Toms, D. P., G. SHELTON, and D. J. RANDALL. 1971. Gas tensions in the lungs and major blood vasels of the urodelc amphibian, A mphiuma tridactylrm. J. Ejrp, Biol. 55: 47-61.

WHITFORD, W. G., and V. H. HUTCHISON. 1963. Cuta- neous and pulmonary gas exchange in the spotted salamander, Ambystoma maculatum. Biol. Bull. (Woods Hole), 124: 344-354.

1965a. Effect of photoperiod on pulmonary and cutaneous resviration in the svotted salamander Ambystoma mdculatum. Copeia, pp. 53-58.

1965b. Gas exchange in salamanders. Physiol. ZOO]. 38: 228-242. - 1967. Body size and metabolic rate in sala- manders. Physiol. Zool. 40: 127-133.

WINTERSTEIN, H., F. ALPDOGAN, and M. BASOGLU. 1944. Untersuchungen iiber die alveolarluft des frosches. Istanbul. Univ. Fen. Fak. Mecm. Seri B Tabii Il~mler, 9 : 171-180.

Cryopreservation of echinoderm sperm1

R. S. DUNN AND J. MCLACHLAN Atlantic Regional Laboratory, National Research Council of Canada, Halifax, Nova Scotia

Received November 14, 1972

DUNN, R. S., and J. MCLACHLAN. 1973. Cryopreservation of echinoderm sperm. Can. J. Zool. 51: 666669.

A simple method for the cryopreservation of sea urchin, sand dollar, and starfish sperm is described. Using dirnethyl sulfoxide (DMSO) as the protective agent, sperm were stored up to 30 days at -196°C. These preparations all retained some motility after thawing. Some samples of thawed sea urchin sperm could achieve a level of egg fertilization equivalent to that achieved by fresh, unfrozen sperm, provided that large doses of the thawed sperm were used. Attempts to cryopreserve fucoid sperm by the same method were unsuccessful.

DUNN, R. S., et J. MCLACHLAN. 1973. Cryopreservation of echinoderm sperm. Can. J. Zool. 51 : 666669. On trouvera ici la description d'une mkthode de prkrvation par le froid de sperrne d'oursin de mer,

d*oursin plat ct d'ttoile de rner. Le sperme est gardt, pour une m o d e allant jusqu'h 30jours, B - 196'C. le dimCthylCsulfoxide[DMSO)servant d'ageni protecteur. Aprh le dkgel, on observe uncertain degd de mot11116 dans toutcs Ics preparations. Dans certain5 cas, le spcrme dtpeld d'ounin de mer garde un pouvoir de fertilisat ion Bquivalent a cefu~ de sperme Frais non congel&, mais ce, A condition qu'on ~itil~se de hautes doses du speme dtgelk. Par ailleurs, les tentatives de prHcrver du sperme de fucoYdks au moyen des m&mes m6thodes ont CchouC. r r a d u ~ t par le journal]

As an extension of algal culture techniques currently in use at the Atlantic Regional Lab- oratory, we have attempted to develop methods for cryopreservation of fucoid sperm. Echino- derm sperm were examined initially, out of convenience, since several species are plentiful in our area, and gametes are easily obtained. We had hoped that successful techniques could be extended to algal sperm.

Recently there has been considerable interest in freezing sperm of salmon (HoyIe and Idler 1968; Tmscatt and Idler 1969), cod (Mounib et al. 19681, trout (Ott and Horton 1971), and oyster (Lannan 1971), but we are unaware of attempts to cryopreserve either echinoderm or algal sperm.

Materials and Methods Organisms were collected in Nova Scotia. Sea urchins

(Strongylocentuotus driibachiemis 0. F. Miiller) were INRCC No. 13251.

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obtained by SCUBA at Shad Bay, Inner Sambro Island, and St. Margaret's Bay, Halifax Co., during June, 1972. Starhh (Asterias vulgaris Verrill) were taken from St. Mary's Bay, Digby Co., in July, and sand dollars ( E c h i ~ a c h i u s p m Lamarck) were also collected in July from Shad Bay and Jeddore Harbour, Halifax Co., by divers. The phaeophycean seaweeds (Fucus vesiculosus L. and Aswphyllum nodosum [L.] Le Jol.) were collected repeatedly in the intertidal zone near Sambro Harbour, Halifax Co., during June and July, 1972. Animals and plants were maintained in the lab- oratory at 3OC in membransfiltered seawater, until gametes were obtained from them. None were kept longer than 1 week.

Gametes were obtained from sea urchins by injection of 0.5 M KC1 (see Stephens 1972). Semen was collected in a dry Petri dish on ice. The sea urchin stock-sperm suspension was prepared by adding an equal volume of filtered seawater to the semen, to allow ease in pipetting. Sea urchin eggs were shed into a 200-ml beaker filled with ice cold seawater. The eggs were subsequently washed twice by decantation in 200 ml of seawater, and then placed in 100 ml of seawater and stored at 3OC for use in fertilization experiments on the same day.

Starfish and sand dollars were dissected to remove ripe gonads, which were transferred to 50 ml of filtered sea- water in a Petri dish on crushed ice, where gametes were spontaneously released (Costello and Henley 1971). The sand dollar stock-sperm suspension was clearly much more dilute than those obtained for sea urchins and starfish.

Fucus and Ascophyllum released gametes when ripe conceptacles were partially dehydrated and reflooded with seawater (McLachlan et al. 1971).

Methods used in freezing of sperm were based on techniques described for other organisms (see above- mentioned authors). In most experiments, filtered sea- water was used as the extender. The cryoprotective agent. which was either DMSO (dimethyl sulfoxide) or glycerol, was added to the seawater to make up the required concentration. Of this mixture, 1.5 ml was pipetted into a 5-ml serology ampoule. Using a I-ml disposable syringe with a No. 18 needle, 0.5 ml of the stock-sperm suspension was added to the ampoule and the contents were mixed gently.

In some experiments with glycerol the extender was a sodium chloride medium containing 40 parts 0.4M NaCl with 0.1 M glycine, and 8 parts of 1.3% NaHCO3 (Mounib et al. 1968). Otherwise the procedure was the same. All materials were kept on crushed ice during this procedure. The h a l volume in every case was 2.0 ml, and the ratio of sperm suspension to extender plus cryoprotective agent was 1:3. After mixing, ampoules were stoppered and left on ice to equilibrate for at least 30 min.

Before freezing the contents of the ampoules were again mixed gently, and were placed in a 3 X 3 cm wire basket attached to a 30-cm steel rod secured to a I-m iron stand with adjustable clamps. Freezing of the ampoules was accomplished by lowering the basket into a widemouthed thermos (40 cm high, 5 cm diam) which contained about 10 cm of liquid N2. AS the shaft of the iron stand was calibrated in centimeters, the rate of

lowering of the basket inside the thermos could be controlled accurately. For example, an average rate of temperature decrease of - 5°C /min was equivalent to lowering the basket at a rate of 2 cmlmin. The actual rate of temperature decrease was monitored with a YSIz "500" thermistor probe and YSI model 44 tele- thermometer. The probe was placed in a 5.0 ml ampoule containing 2.0 ml of solution identical with that of the single ampoule being tested. Both ampoules were lowered simultaneously. Once samples reached -45OC (just above the surface of the liquid Nz) they were im- mersed in the liquid Nz for at least ) h. Samples to be kept frozen were transferred to a liquid N2 refrigerator.

Thawing was accomplished by allowing samples to stand at room temperature for about 45min, until the temperature of the fluid rose to 10°C.

As an index of recovery of frozen sperm, we recorded the motility of thawed specimens and compared this with the motility of unfrozen specimens which had been prepared in exactly the same way, save for freezing. Motility was estimated microscopically at 100 X on an arbitrary scale from 0 to 10, where 10 is the motility of the unfrozen control, and 0 is no movement. Motility of thawed specimens of echinoderm sperm did not change appreciably over 3-4 days when samples were held at 3°C. Thereafter, motility decreased rapidly, and we did not observe increased motility after the initial observa- tions were made on freshly thawed specimens.

The ability of cryopreserved sperm to fertilize could be tested only in sea urchins, as a result of the lateness of the collecting times with respect to the spawning season. In these experiments, the 100-ml egg preparation referred to above was gently mixed to suspend the eggs. Then a 0.5-1111 sample was removed, and transferred to 6.0 ml of seawater in a Petri dish at 10°C. Samples of sperm were added by syringe, and gently mixed. Formation of a distinct fertilization membrane, observed at 25 X, was assumed to indicate fertilization.

Mean sperm concentration in the stock suspensions was approximated by counting appropriately diluted samples in a hemocytometer.

Results

Glycerol in concentrations of 5 4 % always caused large reductions in motility of sperm from all of the species tested. With starfish and fucoid sperm, motility ceased immediately on addition of this substance in concentrations even less than 5aj0. Subsequent dilution with seawater did not restore activity. A small proportion of sea urchin and sand dollar sperm (< 10yo) remained active in control samples in 5-20% glycerol, but no motility was observed in the thawed specimens of all species. In our experiments, saline medium as the extender appeared to have no advantage over seawater

2YSI: Yellow Springs Instruments.

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668 CAN. J. ZOOL. VOL. 51, 1973

insofar as postfreeze motility rates were similar; rates of - 1, - 5, and - 10°C /min, and concen- and we used the latter in most of our tests. trations of DMSO and glycerol ranging from

Although algal sperm remained active in 0 to 40y0. DMSO up to 20%, we found no method which We recognize that more intensive study of resulted in postfreeze motility; we tried freezing other cryoprotective agents and specific ex-

TABLE 1 Effect of freezing to -196OC on the motility of echinoderm sperm, with DMSO as

the cryoprotective agent

Concn.* No. of Species of DMSO, % replicates

Sea urchin 0 1 6 1

12 2

Stariish

Rate of freezing, "C/min

-

5 5 5

Immediate 1 5 5

Time at - 196°C

3 h 3 h 3 h 3 h 3 h

25 days 30 days

3 h t h 3 h

Motility? of thawed

sample

0 I 5 3 h - 12 2 5 t h 1 20 2 5 3 h 3 20 2 1 t h 3 20 2 Immediate 4 h -

Sand dollar 0 1 7.5 2

15 2 20 2

*Refers to concentration (v/v) in filtered seawater. tMotilit~ estimates based on an arbitrary scale, where 10 is the motility of an unfrozen control which was

prepared exactly as the expenmental sample.

TABLE 2 Comparative fertility of sea urchin sperm before and after freezing at -196'C

Fraction* Dose, no. % fertilization of

Sperm of sperm % original Ex~ t . ure~aration x 107 DMSO 15 min 120min fertility

1 Stock 17 0 > 95 2 95 Unfrozen control 17 12 50 60 Unfrozen control 85 12 60 90 Cryopreserved 850 12 15 25 1/30

1 Stock 17 0 > 95 > 95 Unfrozen control 17 12 > 95 > 95 Cryopreserved 170 12 90 > 95 1/10

3 Stock 17 0 > 95 > 95 Unfrozen control 17 12 80 85 Unfrozen control 170 12 2 95 > 95 Cryopreserved 425 12 5 5 Cryopreserved 850 12 20 31 1/200

.This value is a ratio of the dose of unfrozen control/dose of frozen sample, at the same percent fertilization.

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NOTES 669

tenders might reverse our conclusions with algal sperm and with glycerol as a cryoprotector in echinoderms.

More positive results were realized with echinoderm sperm (Table I), using DMSO in concentrations from 0 to 40y0 with seawater as the extender. The effects of varying freezing rate, concentration of DMSO, and time in liquid nitrogen are indicated in the table. In the sand dollar a freezing rate of -S°C/min and a 7.5-15Yo DMSO concentration allowed recovery of some postfreeze motility. Concen- trations of DMSO outside this range were ineffective.

For sea urchins the effective range of DMSO concentration was 12-18%, and for starfish, 20-30y0. Concentrations higher or lower caused decreased motility after freezing. Freezing rates of - 1 and - 5°C /min were equally effective in sea urchins and starfish, but immediate freezing at optimum DMSO concentration caused 1Wy0 loss of motility in thawed specimens.

Storage of sea urchin and starfish sperm for 25 to 30 days at - 196OC did not affect postfreeze motility, as compared with samples prepared the same way, but kept frozen for 3 h.

The relatively poor recovery of postfreeze motility in sand dollar sperm may have been influenced by the smaller sperm concentrations obtained on gamete collection. The mean count for sperm at the time of freezing was 1.8 X 104/mm3, whereas starfish (1.4 X lO61mm3) and sea urchin (8.5 X lO6/mm3) sperm concen- trations were 100-fold greater during the freezing process.

The effects of freezing on the ability of sea urchin sperm to fertilize fresh eggs are pre- sented in Table 2. The cryopreserved sperm in these experiments were frozen at -S"CJmin in 12% DMSO, Except for the use of different individual sea urchins as sperm sources, there were no differences in procedure among the three experiments listed. The response of the egg preparation was measured as percent

fertilization at 15 and 120min, and was de- termined for three sperm treatments: the original stock suspension in seawater; the cryopreserved sample; and an unfrozen control identical with the cryopreserved sample, save for the freezing. The fraction of the original fertilizing ability retained after freezing was estimated from the ratio of the dose of the unfrozen control to the dose of the cryopreserved sample, where fertilization rates and incubation times were equal. For purposes of this approxi- mation it is assumed that the relationship between sperm dose and response of egg fertili- zation was linear.

Some of the eggs fertilized with cryopreserved sperm were maintained in culture up to the pluteus larval stage, and development appeared normal.

Acknowledgment We thank Misses Carolyn J. Bird and Taina

Tuominen for assistance in collecting the animals.

COSTELLO, D. P., and C. HENLEY. 1971. Methods for obtaining and handling marine eggs and embryos. Marine Biological Laboratory, Woods Hole, Mass.

HOYLE, R. J., and D. R. IDLER. 1968. Preliminary results in the fertilization of eggs with frozen sperm of Atlantic salmon (Salmo salar). J. Fish. Res. Board Can. 25: 1295-1297.

LANNAN, J. E. 1971. Experimental self-fertilization of the Pacific oyster (Crassostrea gigas), utilizing cryo- preserved sperm. Genetics, 68: 599-601.

MCLACHLAN, J., L. C.-M. CHEN, and T. EDELSTEIN. 1971. The culture of four species of Fucus under laboratory conditions. Can. J. Bot. 49: 1463-1469.

MOUNIB, M. S., P. C. HWANG, and D. R. IDLER. 1968. Cryogenic preservation of Atlantic cod (Gadus morhua) sperm. J . Fish. Res. Board Can. 25: 2623-2632.

Om, A. G., and H. F. HORTON. 1971. Fertilization of steelhead trout (Salmo gairdneri) eggs with cryo- preserved sperm. J. Fish. Res. Board Can. 28: 1915- 1918.

STEPHENS, R. E. 1972. Studies on the development of the sea urchin Strongylocentrotus droebachiensis. I. Ecology and normal development. Biol. Bull. (Woods Hole), 142 : 132-144.

TRuscorr, B., and D. R. IDLER. 1969. An improved extender for freezing Atlantic salmon spermatozoa. J. Fish. Res. Board Can. 26: 3254-3258.

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