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Cryogenic Preservation of Sperm fromStriped BassJerome Howard Kerby aa North Carolina Cooperative Fishery Research Unit, Box 5577North Carolina State University , Raleigh , North Carolina ,27650 , USAPublished online: 09 Jan 2011.

To cite this article: Jerome Howard Kerby (1983) Cryogenic Preservation of Spermfrom Striped Bass, Transactions of the American Fisheries Society, 112:1, 86-94, DOI:10.1577/1548-8659(1983)112<86:CPOSFS>2.0.CO;2

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Trar•actions o• the American Filheries Society 112: 86-94, 1983

Cryogenic Preservation of Sperm from Striped Bass

JEROyIE How^m) North Carolina Cooperative Fishery Research UniP

Box 5577, North Carolina State University Raleigh, North Carolina 27650

Abstract

Spermatozoa of striped bass Morone saxatilis were successfully cryopreserved, retaining fertil- izing capacity for up to 2 years. Spermatozoa in 14 extenders, in combination with three con- centrations of four cryoprotectants at various sperm: medium ratios, were frozen at different rates and tested. Best results were obtained with extender "OH-189" (7.30 g NaC1, 0.38 g KCI, 0.23 g CaC12'2H20, 5.00 g NaHCO•, 0.41 g NaH•PO4' H•O, 0.23 g MgSO4.7H•O, 5.00 g fruc- rose, 7.50 g lecithin, and 5.00 g mannitol in 968.95 g distilled H,) combined with 5.0% dimethyl- sulfoxide and mixed in a 1:4 sperm: medium volume ratio. The highest fertilization percentage obtained with cryopreserved semen was 87.7. No fertilization was obtained when glycerol, eth- ylene glycol, or propylene glycol were the cryoprotectants. Mean freezing rates faster than 5 C/ minute were more effective than slower rates.

Received November 17, 1981 Accepted September 9, 1982

The striped bass Morone saxatilis is a warm- water, anadromous species of recreational and commercial importance along both Atlantic and Pacific coasts. It is also a highly desired sport fish in freshwater impoundments where it has been stocked. Because of its popularity, several hatcheries for artificial propagation of the species now operate. Managers at some striped bass hatcheries have difficulty procuring suffi- cient males throughout the spawning season. This provided the impetus ['or the research re- ported here, directed toward cryopreservation of striped bass spermatozoa.

Preservation of viable gametes of fishes has been of interest to fishery scientists since de Quatrefages (1853) began his investigations. Early studies were directed mainly at prolong- ing the viability of eggs and sperm in a non- frozen state, whereas more recent studies have [bcused on techniques for maintaining the vi- ability of frozen gameres (Horton and Ott 1976).

Although cryopreservation of mammalian and fowl sperm is used widely for selective breeding in animal husbandry, techniques for fish have not evolved yet to the point that they

•The North Carolina Cooperative Fishery Re- search Unit is jointly sponsored by the North Caro- lina Wildlife Resources Commission, North Carolina

State University, and the United States Fish and Wildlife Service.

are routine. Adequate methods of cryopreserv- ing fish sperm would facilitate animal-husband- ry techniques that are now difficult or impos- sible (Sneed and Clemens 1956; Graybill and Horton 1969; Horton and Ott 1976; and oth-

ers). For example, cryopreserved sperm would allow hybridization of species with temporal spawning differences, provide greater ease in carrying on selective breeding programs for disease resistance and stock improvement, ex- pedite sperm transfer from one hatchery to another, allow sperm from desirable individu- als to be used after their death, enhance prop- agation where there is a deficiency of males, and reduce the number of males maintained by hatcheries.

Attempts to cryopreserve sperm have not been as successful for fish as for higher verte- brates, although advances have been made in recent years, particularly with some cold-water and marine species. Blaxter (1953) was the first to successfully store fish sperm in a frozen condition; slices of the testes of spring-spawn- ing Atlantic herring Clupea harengus harengus, frozen in glycerol and seawater and stored for 6 months in solid carbon dioxide (-79 C), yielded 80 and 85% fertilization when mixed with fresh ova. Mounib et al. (1968) obtained a mean fertilization of 36% after preserving sperm from Atlantic cod Gadus morhua for periods up to 60 days in liquid nitrogen (-196 C). More recently the mean fertilization rate has been in-

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CRYOPRESERVAT1ON OF STRIPED BASS SPERM 87

creased to 59% with a different extender

(Mounib 1978). Pullin (1972) had comparable success with plaice Pleuronectes platessa after 315 days of storage, and Chao et al. (1975) was somewhat successful with sperm from striped mullet Mugil cephalus.

Most sperm-preservation experiments with cold-water fish have involved salmonid species. Varying degrees of success have been reported for rainbow trout Salmo gairdneri, brown trout S. trutta, Atlantic salmon S. salar, brook trout

Salvelinus f ontinalis, grayling Thymallus thymal- lug, and the five west coast salmon species, chi- nook Onchorhynchus tshawyscha, pink O. gorbus- cha, coho O. hisutch, kokanee (lacustrine sockeye) O. nerha, and chum O. hera (Hodgins and Ridg- way 1964; Hoyle and Idler 1968; Truscott et al. 1968; Graybill and Horton 1969; Ott and Horton 1969, 1971 a, 197 lb; Truscott and Idler 1969; Ott 1975; Mounib 1978; Stein and Bayrle 1978; Stoss et al. 1978; Zell 1978; Erdahl and Graham 1980; Kurokura and Hirano 1980; Le-

gendre and Billard 1980; Stoss and Holtz 1981). Several cryoprotectants, especially glycerol, propylene glycol, ethylene glycol, and dimeth- ylsulfoxide (DMSO), have been used in com- bination with many extenders, most of which have been modified from the Cortland salt so-

lution. Different species have been reported to have different extender requirements, but DMSO has been the most successful cryopro- tectant (Ott and Horton 1971a, 1971b; Ott 1975; Stein and Bayrle 1978; Stoss et al. 1978; Erdahl and Graham 1980; Stoss and Holtz 1981). A comprehensive review of salmonid work was published by Scott and Baynes (1980).

Successful cryopreservation of sperm for one cool-water species, northern pike Esox luciu.s, has been reported (de Montalembert et al. 1978; Stein and Bayde 1978); sperm samples were frozen in pellets on solid COs.

Few attempts to cryopreserve sperm from warmwater fishes have been reported. Early at- tempts to store sperm of common carp Cyprinu•s carpio resulted in sperm motility, but fertiliza- tion attempts fhiled (Sneed and Clemens 1956; Kossman 1973; Stein and Bayde 1978). How- ever, Moczarski (1976, 1977) successfully fer- tilized eggs with cryopreserved sperm from both grass carp Ctenopharyngodon idella and common carp. Motility of sperm cells from white bass Motone chrysops and channel catfish Ictalurus punctatus also has been preserved, but no fer-

tilization tests were conducted (Laflin 1968; Guest et al. 1976).

Methods

Experiments were conducted at the South Carolina and Virginia striped bass hatcheries. Adult striped bass were collected with electro- shocking equipment. Ripe males were wiped with a damp towel to avoid contamination of semen with water or mucus, and semen samples from individual males were manually expressed (stripped) into dry 30-ml glass jars that were then loosely capped. No samples that contained blood or excretory products (other than small amounts of urine) were used. Sperm subsam- ples were activated with hatchery water and ex- amined (magnification, 1,000x) to ensure that cells were highly motile. Semen samples were then mixed in 1:1, 1:3, or 1:4 ratios (volume: volume) with previously prepared media (ex- tender with cryoprotectant in the desired con- centration) at temperatures (15-18 G) corre- sponding to hatchery water temperatures. Cryoprotectants included glycerol (10.0-23.8%), ethylene glycol (10.0%), propylene glycol (10.0%), and DMSO (5.0-25.0%). The cryopro- tectant was added to the extender immediately beibre it was mixed with semen. Aliquots (1 ml) of the extended semen were introduced into

1-ml glass ampoules (1976) or 2-ml polypro- pylene A/S NUNC screw-capped vials (1977- 1979) obtained from Union Carbide Incorporated 2 and frozen (a) by immersing the ampoules directly into liquid nitrogen, (b) by suspending the ampoules 4 to 5 minutes in the vapor above liquid nitrogen before immersion, or (c) with a Linde BF4/6 Biological Freezing System. When the latter apparatus, which al- lows some control of the freezing rate, was used, a thermocouple was inserted in one sample and temperature was monitored with a Model TT- 1 indicating pyrometer (Almac Cryogenics, In- corporated). All samples were stored in liquid nitrogen until they were used for fertilization experiments.

After various periods of storage, samples of cryopreserved sperm were thawed and tested with fresh eggs to determine fertilization rates. Female striped bass were injected with human

• Use of brand names does not imply government endorsement.

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T ̂ BLE 1.--Chemical composition of extenders for cryopreserving striped bass spermatozoa (grams/liter). a

Extender NaCI KCI CaCI•' 2H20 NaHCO• NaH2PO4' H20

OH- 129 b 7.30 0.38 0.23 5.00 0.41 OH- 134 • 7.30 0.38 0.23 5.00 0.41 OH- 141 • 7.30 0.38 0.23 5.00 0.41 OH- 164 • 7.30 0.38 0.23 7.50 0.41 OH- 189 t• 7.30 0.38 0.23 5.00 0.41 OH-210 t• 7.30 0.38 0.23 5.00 0.41 OH-235 t• 8.50 5.00 OH-251 t' 8.50 5.00 OH-275 • 8.50 3.75 Cortland • 7.25 0.38 0.23 1.00 0.41 Modified Cortland • 1.88 7.20 0.23 1.00 0.41 Alsever's a 4.00 Sodium chloride medium • 19.48 2.17 Hfk 10 • 6.04 1.64 0.14

Grams of solute per liter total solution. Ott and Horton (1971a, 1971b). Truscott et al. (1968).

Hodgins and Ridgway (1964). Mounib et al. (1968). Truscott and Idler (1969).

chorionic gonadotropin (about 280 Interna- tional Units per kilogram of fish) to induce ovu- lation (Stevens 1966). Ripe eggs were taken by stripping. Approximately midway through the stripping procedure, a 30-ml sample was placed in a dry glass jar. Egg subsamples (0.3 ml), con- sisting of about 300 eggs, were placed in 300- ml glass preparation dishes with fresh or thawed sperm, and about 100 ml of hatchery water were added and swirled to ensure complete mixing. Frozen sperm in ampoules usually were thawed rapidly in a warmwater bath (50-60 C), as rec- ommended by Horton and Ott (1976), but a few were thawed in hatchery water (15-18 C). Fresh sperm was added to the control dishes at the rate of 0.2, 0.25, or 0.5 ml per dish, de- pending on the sperm: medium ratios of the experimental samples, so that about the same volume of semen was present in control and experimental dishes. Blanks (which contained only eggs) were maintained with each experi- ment to determine whether or not any parthe- nogenetic development occurred. After about 5 minutes, the sperm-water mixture was de- canted, the eggs were rinsed in clean water, fi-esh water was added, and the dishes were placed in a bath with running hatchery water for 3 to 8 hours. Eggs then were examined microscopi- cally to determine fertilization percentages and preserved in 5% buffered formalin. Eggs fer- tilized with cryopreserved sperm in two exper- iments were allowed to develop through hatch- ing.

Additional procedural details are included with the following accounts of specific experi- ments.

Sperm Motility

Preliminary experiments during 1976 and 1977 included ibur extenders: modified Cort-

land, Alsever's, Hfx 10, and sodium chloride medium (Table 1) in varying sperm:medium ratios and cryoprotectant concentrations. Pre- served sperm were thawed and examined mi- croscopically at intervals for motility. No motil- ity was observed in any of the thawed samples containing ethylene glycol, propylene glycol, or glycerol, and only occasionally were motile cells seen in samples protected with DMSO. Similar results were noted for samples preserved in 1978 and 1979, and when motility was observed, the cells seldom moved vigorously. DMSO inhibit- ed motility in nonfrozen samples, whereas non- frozen samples containing any of the other three cryoprotectants retained a high degree of' mo- tility betbre they were frozen. Increasing con- centrations of DMSO increased inhibition of

motility.

Fertility

Several samples stored in 1976 and 1977 were used in fertilization tests during 1978. Positive results were obtained with only two. These had been preserved in Hfx 10 containing 10% DMSO, with a sperm: medium ratio of 1:4, and

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CRYOPRESERVATION OF STRIPED BASS SPERM 89

TABLE 1.--Extended.

MgSO4- 7H20 Fructose Lecithin Mannitol Glucose Na3C6H507' 2H20 Glycine

0.23 1.00 5.00

0.23 1.00 7.50 0.23 1.00 7.50 1.00 0.23 1.00 7.50 2.50 0.23 5.00 7.50 5.00 0.23 1.00

0.23

0.23

0.22 0.60

5.00 15.00 7.50

1.00

1.00

20.50 8.00 7.51 6.26

6.00

frozen in the vapor immediately above liquid nitrogen. Fertilization percentages were 5.9 and 9.1 alter 2 years of storage. No fertilization was obtained with samples that were immersed di- rectly in liquid nitrogen or with samples that contained a cryoprotectant other than DMSO.

Experiments subsequent to 1977 relied pri- marily on fertilization tests for indication of sperm viability. Fourteen extenders, combined

with 5.0, 7.5, or 10.0% concentrations of the

four cryoprotectants and mixed in a 1:4 sperm: medium ratio were tested after freezing in the Linde system. As in the 1976 and 1977 exper- iments, DMSO was the only cryoprotectant that resulted in motility of, or fertilization with, fro- zen-thawed sperm. Of the DMSO concentra- tions tested, 5.0% normally produced the high- est fertilization percentages. Extenders OH-189,

TABLE 2.--Mean percent fertilization of striped bass' ova with sperm J•ozen and stored in liquid nitrogen (-i 96 C). Cryoprotectant was' dimethy•-ulJbxide (DMSO). Mean freezing rate (C/minute) was calculated from +10 to -40 C. Sperm:medium ratio was' 1:4 (volume:volume). Each sample lot consis'ted of semen from an individual male. Time frozen represents the range of time individual samples' were frozen before fertilization trials.

Num- Range in % fertilization (mean _+ SE, ber of mean freez- Num- Range in range in parentheses)

% sample ing rates bet time frozen Extender DMSO lots (CJminute) of trials (days) Cryopreserved sperm Control sperm

OH-129 5.0 2 18.8, 30.6 7 0.1-351 1.2 _+ 0.3 (0-2) 72.7 _+ 7.6 (33-93) OH-134 5.0 13 2.4-18.8 69 0.2-736 17.3 _+ 1.9 (0-71) 69.9 _+ 2.4 (11-96)

7.5 1 16.7 6 0.6-737 6.1 _+ 4.6 (0-29) 72.6 -+ 8.9 (33-90) 10.0 2 18.2, 18.8 8 0.3-731 0.0 80.0 -+_ 3.9 (59-92)

OH-141 5.0 1 25.0 5 0.4-33 1.5 _+ 1.3 (0-7) 83.4 _+ 3.8 (74-94) 10.0 1 25.0 3 0.4-2 0.0 83.4 _+ 5.8 (74-94)

OH-164 5.0 I 18.2 5 0.3-33 1.4 _+ 0.9 (0-5) 73.9 _+ 8.5 (41-90) 10.0 1 18.2 4 0.3-33 1.8 -+ 1.7 (0-7) 71.6 -+ 10.6 (41-90)

OH-189 5.0 10 2.4-17.0 48 0.2-734 23.6 _+ 2.8 (0-88) 64.6 _+ 3.1 (11-96) OH-210 5.0 2 16.7, 30.5 6 0.1-19 0.1 _+ 0.1 (0-1) 65.1 _+ 10.7 (33-93) OH-235 5.0 13 2.4-17.3 68 0.1-736 13.7 _+ 1.9 (0-71) 63.5 -+ 2.5 (11-93)

7.5 1 16.7 5 0.6-737 1.5 -+ 0.8 (0-5) 69.2 • 10.0 (33-86) OH-251 5.0 4 5.9-17.3 23 0.1-718 4.4 _+ 1.3 (0-22) 70.5 _+ 3.7 (33-93)

7.5 1 17.1 5 0.2-10 3.3 _+ 2.2 (0-12) 75.0 -+ 5.6 (59-93) OH-275 5.0 10 2.4-17.0 53 0.1-733 14.9 _+ 2.6 (0-80) 66.1 _+ 2.9 (11-96) Hfx 10 5.0 2 16.7-18.8 7 0.1-350 7.4 _+ 3.8 (0-27) 62.3 ñ 8.6 (33-94)

7.5 1 16.7 3 0.7-11 2.3 _+ 2.0 (0-6) 56.5 _+ 13.0 (33-77) Modified Cortland 5.0 2 4 0.8-352 0.1 -+ 0.1 (0-<1) 43.2 -+ 6.2 (33-57)

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T^BI•E 3.--Mean freezing rates (C/minute) that produced the highest.[bur mean and maximum fertilization percentages' for each of the extenders listed. Cryoprotectant was 5.0% dimethylsul•bxide; sperm: medium ratio was 1:4 (volume: volume). Rates tested ranged from 2.4 to 18.8 C/minute.

Ranking

1 2 3 4

Extender Mean Maximum Mean Maximum Mean Maximum Mean Maximum

OH-134 17.0 7.4 7.4 17.0 7.6 8.4 13.0 18.2 OH-189 7.4 7.4 13.0 13.0 7.6 8.4 8.4 7.6 OH-235 8.4 8.4 13.0 13.0 7.6 17.3 17.3 7.6 OH-275 7.4 7.4 7.6 7.6 13.0 13.0 17.0 3.5

OH-134, OH-275, and OH-235, with 5.0r/• DMSO, provided the highest fertilization rates. Mean percentages were 23.6, 17.3, 14.9, and 13.7, respectively (Table 2). Maximum fertiliza- tion obtained was 87.7%, with OH-189. No pos- itive results were obtained with Cortland, A1- sever's, or sodium chloride extenders.

Freezing Rates

The controlled-rate freezing apparatus pro- vided some control of freezing rates after 1977, but precise repeatability was not possible be- tween freezes and, because a recorder was not available, freezing-rate curves were not ob- tained. Therefore, the mean freezing rate be- tween +10 and -30 C was calculated for each

experiment. Freezing rates through the phase change (liquid to solid) and at other times may have varied considerably •i'om the mean.

Experiments with extenders OH-134, OH- 189, OH-235, and OH-275 (for which there were the largest number of fertilization tests and the greatest variation in freezing rates) produced mixed results (Table 3). Mean freez-

T ̂ BI•E 4.•Mean fertilization percentages of sperm samples tested after storage periods in liquid nitrogen ranging from a few hours' to 2 years'. Control percerztages provide a relative measure of egg quality.

ing rates ranged from 2.4 to 18.8 C/minute. Samples frozen at a mean rate of 7.4 C/minute produced the highest mean fertilization for OH- 189 and OH-275 and the second highest for OH-134. This rate also produced the maximum l•rtilization values for three of the four extend-

ers (OH-134, OH-189, and OH-275). However, more rapid freezing rates also produced rela- tively high percentages. Freezing rates less than 5 C/minute were less effective than the more

rapid rates.

Preservation Time

Samples preserved during 1978 and 1979 were stored in liquid nitrogen for periods rang- ing from less than 1 day to approximately 2 years before fertilization tests were conducted. Fertilization capacity of stored sperm probably did not degrade in the first year, but some deg- radation apparently occurred in the second year (Table 4). Mean control fertilization percent- ages were similar throughout the period. How- ever, because of the limited number of obser-

T ̂ m•E 5.--Fertilization percentages' of sperm samples thawed in a 50•50 C water bath (rapid) and samples thawed in a 15-18 C water bath (slow).

Cryopreserved sperm Control Extender Number Number

Years Mean % of' t•rtil- Mean % of tl•rtil- of fertil- ization fertil- ization

storage ization tests ization tests

0 18.3 118 65.1 118 1 20.5 85 67.3 85 2 10.3 37 69.9 37

% fertilization

Cryopreserved sperm Sample Control

lot Rapid Slow (fresh number thawing thawing sperm)

OH-134 1 11.8 21.9 75.2 2 4.9 3.8 53.6

OH-235 3 5.4 2.8 92.9 3 3.2 1.6 72.9

OH-251 4 2.4 0.9 92.9 4 0.9 2.2 92.9

5 0.0 0.6 72.9

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CRYOPRESERVAT1ON OF STRIPED BASS SPERM 91

vations for samples frozen for 2 years, the results should be further substantiated.

Thawing Rate

Ott (1975) reported that rapidly thawed sal- monid sperm yielded better fertilization per- centages than those thawed more slowly. In the present study, identically treated samples were thawed rapidly (in a 50-60 C water bath) or slowly (in a 15-18 C bath) and paired tests were conducted with the same controls to determine

if differences in thawing rates affected fertil- ization percentages. Because the fertilization percentages usually were similar (Table 5), the rapid method was adopted as standard proce- dure.

Progeny

In two experiments, eggs that were fertilized with stored sperm were allowed to hatch and the prolarvae were examined microscopically. All appeared to be normal and active individ- uals. Subsequent to these experiments, over 80,000 young (30-73 mm fork length) were reared in ponds stocked with larvae produced with cryopreserved sperm. These fish also ap- peared normal and healthy and subsequently were stocked into South Carolina reservoirs.

Assessment

Several uncontrollable variables made anal-

yses of the data difficult. The most important were freezing rate and egg quality. Freezing rates could not be precisely controlled and were not precisely repeatable, and the lack of a tem- perature recorder prevented accurate deter- mination of rates through the phase change, when heat of fusion is released. Variations in

fertilization capacity between samples frozen si- multaneously and tested with high-quality eggs (>85% control fertilization) may be due to dif- ferential cooling from inadequate circulation of liquid nitrogen vapors in the freezing chamber or to differences in thawing rates. Additional work with more sophisticated freezing and thawing systems will be necessary to adequately determine effects of these variations.

A confounding variable that has made it dif- ficult to separate effects of freezing rate, ex- tender, and freezing duration is egg quality (determined by fertilizing eggs from each fe- male with fresh sperm). This control fertiliza- tion is also a function of sperm quality, but be-

cause sperm quality always appeared to be "good" (>95% highly motile), and fertilization percentages almost always exceeded those ob- tained in South Carolina's production hatchery (where at least two males normally were used), sperm quality was assumed not to be a signifi- cant factor. Control fertilization ranged from 10.9 to 95.7%, indicating large variations in egg quality among females. Because egg quality could not be determined in advance, many ex- periments were conducted with eggs of low ini- tial quality, making it difficult to compare re- sults from stored sperm. Results of experiments with lower-quality eggs were not eliminated be- cause fertilization percentages with stored sperm were sometimes better than for tests in which

egg quality was much better. There appeared to be no correlations between control and ex-

perimental fertilization percentages, except that higher-quality eggs normally produced higher experimental percentages.

Other variables included storage duration, thaw rate, extender type, and percentage of cryoprotectant. Storage duration for 1 year did not appear to affect sperm viability, but some degradation may have occurred during the sec- ond year. Ott (1975) [bund that storage of coho salmon sperm for 28 days did not affect viabil- ity, and other investigators (Pullin 1972; Mou- nib 1978; Kurokura and Hirano 1980) success- fully preserved fish sperm for about 1 year. Although Ott (1975) found that rapid thawing resulted in higher fertilization percentages, thawing rates tested in the present study did not appear to affect sperm fertilizing capacity.

Gregory and Summers (1974) pointed out that methodology developed to cryopreserve bull semen often results in nearly 100% fertilization, and that subsequent attempts to use the same methods, extenders, and cryoprotectants [br fish sperm were not successful. It is increasingly ev- ident that wide variations exist in extender and

cryoprotectant requirements fbr different fish species. The most effective cryoprotectant ex- amined to date is DMSO, but glycerol, ethylene glycol, and propylene glycol have all resulted in post-freeze motility or fertilization with dif- ferent species (Blaxter 1953; Mounib et al. 1968; Truscott and Idler 1969; Pullin 1972; Guest et al. 1976; Erdahl and Graham 1980). The most effective DMSO concentrations normally have been 7 to 10% of the medium. Horton and Ott

(1976) recommended 10% on the basis of ex-

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tensive work with salmonids, whereas Erdahl and Graham (1980) found 7% a highly effective concentration. However, 5% DMSO provided better results with striped bass sperm; other cryoprotectants--including glycerol•were in- effective. Ott and Horton (197 lb) and Ott (1975) found differences in "cryopreservativeness" among salmonids and reported that each species required its own extender for best results. For example, extenders developed for other salm- on and trout species were ineffective when used for sperm from kokanee. The present study confirms their results, because several extend- ers suitable for other species were not satisfac- tory fbr striped bass sperm.

A review of the literature indicates that sperm from saltwater fish may be more easily stored than sperm from freshwater fish because rela- tively simply media and procedures seem to be generally successful (Blaxter 1953; Mounib et al. 1968; Pullin 1972; Mounib 1978; Kurokura and Hirano 1980; Scott and Baynes 1980). Al- though the striped bass is a marine and estua- fine species, it is anadromous and has adapted to a landlocked existence. Like the salmonid

species, it apparently requires different media and procedures than do marine species. How- ever, it is also apparent that sonhe extenders developed for other fish species can be used successfully for striped bass.

Sperm motility has been used in many studies as the criterion fbr sperm viability, but it may not be a good indicator of fertilizing ability. Al- though sonhe authors reported a rough positive correlation between motility and fertilization capacity (Mounib et al. 1968; Truscott et al. 1968; Ott 1975), others found little or no re- lationship between the two (Terner and Korsch 1963; Fribourgh 1966; Truscott and Idler 1969; Kossman 1973; Stein and Bayrle 1978). For ex- ample, Terner and Korsch (1963) reported that "iramotile" rainbow trout sperm retained their fertilization capacity, whereas thawed motile sperm of common carp were unable to fertilize fresh eggs (Kossman 1973; Stein and Bayrle 1978). Thawed striped bass spermatozoa exhib- ited very low or no motility and movement was not vigorous, but fertilization percentages ranged as high as 87.7%. However, one cannot conclude that nonmotile cells are capable of fertilizing eggs. Microscopic examination of sperm cells fbr motility is imperfect at best, be- cause even freshly collected, unfrozen sper-

matozoa are motile for only about 60 seconds after activation with hatchery water. Gonse- quently, tinhe available to search a slide for ac- tive cells is limited. Furthermore, a large per- centage of the cells are killed. Yet, because of the large number per unit volume, a low per- centage of live, active cells still could result in high fertilization rates.

Optimal freezing rates also appear to vary for different species but available data are insuffi- cient and too inaccurate to adequately describe their effects. Although Mounib (1978) reported successful preservation of Atlantic cod and At- lantic salmon sperm after direct immersion in liquid nitrogen, extremely rapid freezing is normally lethal to fish spermatozoa (Hoyle and Idler 1968; Ott 1975; present study). In gen- eral, moderately rapid freezing rates are pref- erable to slower ones. Mounib et al. (1968) re- ported that a 5 C/minute freezing rate was better than a 1 G/minute rate for Atlantic cod sperm. Graybill and Horton (1969), Ott and Horton (197 la, 197 lb), and Ott (1975) preferred a more rapid cooling rate obtained by suspending the ampoules in liquid nitrogen vapor. They re- ported the freezing rate was approximately 30 G/minute, but did not define whether it was an overall mean or the rate through the freezing point. Other investigators generally reported mean rates, which must have varied consider- ably at different phases of the freezing process, but which were not precisely described. I was unable to approach a 30 G/minute rate using similar procedures, and found that rates varied from one trial to the next. However, available data suggest that rates between 5 and 20 G/ minute may be more effective than rates below 5 G/nfinute for striped bass sperm.

The large variability apparent in my results is a phenomenon common to this type of' re- search. Several investigators (Bfiyfikhatipoglu and Holtz 1978; de Montalembert et al. 1978; Stoss et al. 1978; Kurokura and Hirano 1980) have commented on it and it is evident in much

of' the other published work. Even though an extender system may be satisfactory (that is, it often results in fertilization greater than 70%), there is much that is not understood about the

freezing and thawing processes; small alter- ations apparently can cause highly variable re- sults. Additional, more sophisticated research is necessary if the potential for cryopreservation of fish sperm is to be realized fully.

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CRYOPRESERVATION OF STRIPED BASS SPERM 93

Acknowledgments

Support for this project was provided in part by a faculty research grant from North Caro- lina State University, and in part by contracts 14-16-0008-2146 and 14-16-0009-79-132 from

the United States Fish and Wildlife Service. Ad-

ditional support and assistance were provided by the South Carolina Wildlife and Marine Re- sources Department.

The work was conducted at the South Car-

olina and Virginia striped bass hatcheries, and special thanks are due to Jack D. Bayless and Reginal M. Harrell (South Carolina Wildlife and Marine Resources Department) and to Kenneth Mitchell and David K. Whitehurst (Virginia Commission of Game and Inland Fisheries) for their cooperation and assistance. I thank the following members of the Young Adult Con- servation Corps (United States Fish and Wild- life Service) for their assistance: S. Craig Brad- ley, Eric A. Long, Gregory A. Marlow, M. Deryl Pickett, Jeff M. Phillips, and M. Allyson Rob- ertson. Additional assistance and cooperation were provided by Tony Thornley and Richard Woods (South Carolina Wildlife and Marine Resources Department). Marsha Shepherd pre- pared computer programs. Norma Walker and Dorothy Wright typed the manuscript. Jack D. Bayless, Melvin T. Huish, and Reginal M. Har- rell reviewed earlier drafts of the manuscript.

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