16, 448-460 (1979)
CRYOBIOLOGY
Cryopreservation
of Spermatozoa of the American Crassostrea virginica Gmelin
SUSAN R. ZELL,*j’ *Department
of Zoology,
MARK H. BAMFGRD,*
AND
HERBERT HIDUt
University of Maine, Orono, Maine 04473, and TDepartment University of Maine, Orono, Walpole, Maine 04573
Techniques for cryopreserving oyster spermatozoa would enhance the potential for controlled breeding of oysters, especially since oysters are protandric hermaphrodites. After sex reversal the eggs of a selected oyster could be fertilized with its own cryopreserved sperm. Self-fertilization or back crosses would increase the rate of inbreeding for valuable traits such as rapid growth, disease resistance or better shape, texture or flavor. Frozen spermatozoa could also provide a ready supply of male gametes for crossing populations that breed asynchronously or for shipment to different locations. Since the original success in preserving mammalian spermatozoa, scientists have frozen the spermatozoa of many different mammals (26), teleosts (6), and a few invertebrates, including echinodermata (9), the American oyster (12), and the Pacific oyster (15, 29). Fertility of frozen oyster spermatozoa ranged from 0% for the American oyster to 33% for the Pacific oyster, with storage for 24 hr at -170°C (29). This paper contains the report of a technique for cryopreserving spermatozoa of the American oyster in liquid nitrogen (- 196°C). After 68 days of storage cryopreserved spermatozoa had a fertility of 91% compared to 92% for fresh spermatozoa.
Oyster,
of Oceanography,
MATERIALS
Gametes
Gametes were obtained from American oysters (Crassostrea virginica Gmelin), conditioned about 2 weeks in seawater that had been filtered through l-pm pores. The filtered seawater had a pH 8.0 and a salinity of 20 ppt. The supply of sexually mature oysters was limited, but whenever possible, gametes from two or more oysters of the same sex were combined. Each lot of spermatozoa or eggs, containing gametes from one or more oysters, was numbered consecutively (i.e., s-l through s-9 or e-l through e-9). Gametes from any one oyster were used in only one lot. Initially gametes were extracted from oysters that had been spawned the preceding day. Occasionally egg lots had low fertilities and contained many eggs that looked immature. Diluent
The diluent used for successfully freezing spermatozoa was a 2.6x Hanks’ phosphate-buffered salt solution (Grand Island Biological Supply Co.), to which 80 mM glycine, 55 mM NaHCO, and 8% DMSO were added. The pH was adjusted to 8.0 (Orion Model 801A, digital pH meter), equal to that of the seawater and higher than the optimal buffering capacity of the diluent. A second solution of the same diluent was prepared and tested as a check of both the recipe and the sensitivity of the sperReceived February 20, 1979; accepted June 11, matozoa to random variations in the dil1979. uent. Concentrations of the principal ions, ’ Currently Faculty Associate, College of the Atlantic Bar Harbor, Maine 04609. sodium, potassium, and chloride, and sa448 001l-2240/79/050448-13$02.00/O Copyright 0 1979 by Academic Press, Inc. All rights of reproduction m any form reserved.
CRYOPRESERVATION
OF AMERICAN TABLE
OYSTER
449
SPERM
1
Ionic and Osmotic Concentrations of Spermatic Fluid from the American Oyster Crassostrea virginica, Seawater, and the Cryoprotective Diluent Concentration (mM) Solution Spermatic fluid” 1 2
Na+
Kf
425 422
16 17
Salinity (PPt)
Osmolality (mOsm)
496 477
31.V 30.6
913’ 877’ 832’
Cl-
Seawater Diluent 1 2
-
-
4.52d
29.0
397 413
14 15
379 375
24.3* 24.1*
Spermatic Fluid > Diluent’
N.S.
N.S.
P < 0.005
1990 2180
P < 0.005
” Each sample of spermatic fluid was pooled from two to three different oysters. b Salinity was calculated from the measured Cl- concentration. (’ Osmolality was calculated from the osmotic coefficient for a NaCl solution, equal in molarity to the measured Cl- concentration. d Chloride concentration was calculated from measured salinity. p Hypothesis that spermatic fluid > diluent; tested by normal t distribution with 2 dJY
linity and osmolality of the two diluent solutions are shown in Table 1. Corresponding values for seawater and spermatic fluid are presented in the same table. Spermatic fluid was the supernatant fluid formed when the contents of extracted gonads were centrifuged for 2-3 min. in a Beckman Spinco Microfuge. Sodium and potassium concentrations were measured by flame photometry (Instrument Laboratories, Inc., Model 343, F. M.). Chloride concentrations in the diluent and spermatic fluids were measured coulometrically (Buchler Cotlove Chloridometer). In seawater, chloride concentration was calculated from its measured salinity, according to the following relationship; salinity (o/o0)= 0.03 + 1.805 (chlorinity) (O/00) (3). According to the same equation, salinities of the diluents and spermatic fluids were calculated from their measured chloride concentrations. Osmolality of the diluent was measured by freezing point depression (Halbmikro Osmometer, Knauer Type M No. 9001) after the diluent, which was too concentrated for the range of osmolalities stan-
dardized on the osmometer, was diluted 1: 1 with distilled, deionized water. Osmolality of spermatic fluids and of seawater was calculated from the measured chloride concentrations and the osmotic coefficient of a 0.4-0.5 molal solution of sodium chloride (25). The difference between molality and molarity for a sodium chloride solution of 0.4-0.5 M is negligible (35). A complete atomic absorption scan (Garrel-Ash No. 750; Atomcomp. Fisher Scientific) was run on the diluent solutions. Magnesium was 2.5 mM in both; calcium was 0.86 mM in the first and 1.6 mM in the second. Traces of arsenic, tin, and boron were observed in both solutions. No other elements were detected. METHODS
Collecting and Diluting Gametes Extracted gametes. After the top shell of an oyster was removed, the epithelium overlying the gonadal tissue was pierced with a pipet and the gonadal contents were aspirated into the pipet. Undiluted spermatic extract was cooled briefly in a watchglass on ice at 0°C before dilution with di-
450
ZELL,
BAMFORD,
AND
HIDU
luent at 0-2°C. Approximately 2 ml of spermatic extract was mixed with 10 ml of diluent for a l/6 dilution. The diluted spermatozoa were placed in a 15-n& capped, plastic test tube (16-mm diameter) on ice at 0°C for 20 min before being added to eggs or frozen. Two lots of spermatozoa that were frozen and stored were handled differently from the preceding description. Diluted spermatozoa from lot s-3 were held at 0°C for 75 min before being added to eggs or frozen. Undiluted spermatozoa from lot s-4 were held in a IS-ml capped test tube at 0°C for 120 min before being used. A sample was added to eggs; the rest was diluted and held for an additional 20 min before being frozen. Extracted eggs and accompanying fluid were added to seawater of 23 -26°C. In early experiments eggs from one female were diluted into 2-3 liters of seawater (e-l, e-2) for a final concentration of 15-50 eggs/ml. In later experiments the extracted eggs were first diluted into 50 ml of seawater and then rediluted into 150-450 ml for a final concentration of 250-900 eggs/ml. Spawned gametes. In one experiment spermatozoa were frozen after they had been spawned into seawater. An oyster was stimulated to spawn by the presence of fluid into which another male had previously spawned. The milky stream of freshly emitted spermatozoa, together with surrounding seawater, was collected in a pipet, cooled, and mixed with diluent. In one experiment a female (e-7) was induced by the presence of egg fluid extracted from another female to spawn into 17 liters of seawater.
an effort was made to freeze the spermatozoa at -5”CYmin. The recorded cooling rate between 0 and -20°C varied from -5 to -7.5”Clmin. From -20 to -80°C the rate of cooling was erratic, but the average cooling rate for each run varied between -5 and -13.5Vmin. When latent heat of fusion was released between - 10 and -2O”C, the probe registered a warming of as much as 23°C or as little as 1 or 2°C. The temperature rise, which was generally about 7 to 12”C, was followed by a retooling at -S”C/min. After reaching -80°C in the chamber, samples were plunged into liquid nitrogen (- 196°C) and stored submerged in liquid nitrogen. Storing. Samples, frozen only to -5, -20, -40, or -8O”C, were held in the freezing chamber as long as the straw temperature remained constant, but never for more than 2 min. They were then thawed. Samples frozen to - 196°C were stored in liquid nitrogen from 5 min to 68 days. Each time stored samples were removed from the storage Dewar flask, all the remaining straws were briefly lifted out of the liquid nitrogen. Thawing. Straws were thawed by submersion in a 55-60°C water bath. Straws that had been frozen to one of the intermediate temperatures were held in the water bath for up to 10 set, or just until the diluent in the straws had visibly thawed. Spermatozoa frozen to -196°C were thawed by submersion for 10 sec. Immediately after thawing, before the sperm solution could warm more than a degree or two above 0°C the thawed spermatozoa were diluted into the egg-containing seawater.
Cryopreserving Spermatozoa Freezing. Diluted spermatozoa were aspirated into 0.25-ml plastic straws, which were then placed in a 0°C chamber of a biological freezer (Linde BF-4- 1). They were then cooled with liquid nitrogen vapors to either -5, -20, -40, or -80°C. A temperature probe had been placed in a 0.5~ml plastic straw containing diluent and
Fertilizing Eggs In most experiments about 0.25 ml of either fresh or cryopreserved spermatozoa (i.e., 1.5 ml of spermatic extract mixed with diluent) was added to 30-200 ml of seawater containing 200-900 eggs/ml. In the final experiment (Trial 6: s-4/e-9 and s-9/e-9) only 0.12 ml of spermatozoa was added to 160 ml of seawater containing about 400
CRYOPRESERVATION
OF AMERICAN
eggs/ml. The beaker was swirled gently and allowed to sit undisturbed at 23-26°C. Measuring Fertility of Cryopreserved Spermatozoa Development of eggs into larva. Spermatozoan fertility was tested by the addition of spermatozoa to a suspension of eggs, but it was measured indirectly, as the percentage of eggs that developed into larvae. Thus the measured fertility of cryopreserved spermatozoa was influenced by the fertility of eggs as well as the fertility of spermatozoa before freezing. Consequently three different controls were done. Controls. (1) Eggs were fertilized with fresh spermatozoa 20 min after the spermatozoa were diluted and stored at 0°C. Any loss in fertility due to treatment before freezing could thus be distinguished from the effects of freezing and thawing. (2) Eggs from the same females as those that were fertilized with cryopreserved spermatozoa were fertilized with fresh, unchilled, and undiluted spermatozoa. When the fertility of cryopreserved spermatozoa was tested on the same day they were frozen, control and cryopreserved spermatozoa came from the same males, and the percentage of eggs that developed into larvae after fertilization with fresh spermatozoa indicated the maximum fertility one could expect with eggs from the same lot and the cryopreserved spermatozoa. When cryopreserved spermatozoa were tested after I or more days of storage, fresh and cryopreserved spermatozoa had to come from different males, and fertility of the fresh spermatozoa was used as an estimate of the maximum fertility of a given lot of eggs. (3) A 60- to 500-ml sample from each batch of unfertilized eggs was left undisturbed at 23-26°C and was later checked for larval development. Occasionally unfertilized eggs were first exposed to the diluent and then left undisturbed. Thus the possibility was tested that eggs may have been contaminated with spermatozoa or that they may have developed parthenogenetically,
OYSTER
SPERM
451
either spontaneously or because of stimulation by the diluent or handling. Evaluation of larval development. A sample from each egg solution was pipetted into a Sedgewick Rafter cell, and each egg and embryo was counted under 100x magnification. Duplicate l-ml samples of eggs from lot e-l and triplicate 2-ml samples of eggs from lot e-2 were counted. At least 400 eggs/sample from every other lot of eggs were counted. Abnormal eggs or larvae were counted as undeveloped eggs, except in the unfertilized, control samples, where any development was counted as an embryo. About 30-60 min after fertilization, eggs began extruding their first polar body, but depending on the position of an egg in the microscopic field, the polar body may have been obscured from view. After about 5 hr, the developing larvae became ciliated and motile. In replicate samples analyzed at 4, 7, and 10 hr or 2, 4, and 5 hr after fertilization, the measured percentage development was reproducible. Motile gastrulae, however, were difficult, if not impossible to count, and after they were fixed in formalin they could be confused with eggs. Hence all samples were counted 2-3 hr after fertilization. When rechecked after up to 7 hr, all solutions with high percentages of development contained dense suspensions of swimming larvae and few remaining eggs. Larval growth. In one experiment (Trial 4: s-4/e-7) 0.625 ml of cryopreserved spermatozoa was added to 17 liters of seawater in which a female oyster had spawned naturally in the complete absence of spermatozoa. On the same day naturally spawned eggs from a different female were fertilized according to standard hatchery procedure with naturally spawned spermatozoa. The resulting larvae were reared for 11 days. Observing Spermatozoa Motility. Fresh spermatozoa were observed under 100x magnification in either spermatic fluid, seawater, cryoprotective diluent, or an alternative diluent that, for
ZELL, BAMFORD,
452
AND HIDU
TABLE 2 Fertility of Oyster Spermatozoa Minutes after They Were Cooled to Different Subzero Temperatures in One of Two Separately Prepared Replicate Diluents Low temperature P-3
Trial 1: s-l/e-l”,* No. eggs/ml
Undiluted sperm +23
-
Diluted sperm 0 -5 -20 -40 -80 -196
43 40 48 45 47 -
Trial 2: s-2/e-2
Trial 3: s-3/e-3
% Embryos
No. eggs/ml
% Embryos
No. eggs/ml
% Embryos
-
16
81
262
85
95 97 94 87 76 -
(Diluent 1) 22 72 26 92 11 86 29 93
(Diluent 1)
(Diluent 2) 716 100 467 96 871 97 689 81
fl In Trials 1 and 3, one l-ml sample was counted: in Trial 2, triplicate 2-ml samples were counted and averaged for each temperature. Based on a binomial distribution, there is no significant difference between any of the individual samples in Trial 1 or 2 or the -20 and -80°C samples of Trial 3. The other samples in Trial 3 are each significantly different from the others (P i 0.01). b Abbreviations: s-l/e-l = sperm lot-l/egg lot-l (s-l, s-2, e-2, and e-3 came from one oyster each; s-3 and e-l came from two.each).
oysters, had a high pH (9.0) and a low osmolality (425 mOsm). This test solution consisted of a basic Tris-citrate buffer (Poulik’s gel buffer), modified by the addition of 10% polyvinylpyrrolidone, 85 mA4 glycine, 56 mM NaHCO,, and 36 mM KC1 (36). Egg-sperm
interactions. Egg-sperm interactions were observed within minutes after fertilization with either undiluted, fresh spermatozoa; diluted, fresh spermatozoa that were chilled to 0°C; or cryopreserved spermatozoa.
0.005).
RESULTS
Fertility
of Cryopreserved
being diluted and cooled to 0, -5, or -20°C spermatozoa had 95, 97, or 94% fertility. In Trial 2 none of the measured fertilities differed significantly from any of the others. In Trial 1, however, spermatozoa that were frozen to -80°C had a significantly lower fertility than spermatozoa in the -40°C sample (P < O.OS),and in both samples, spermatozoa had a significantly lower fertility than in the 0, -5, or -20°C sample (P < 0.05). In Trial 3 fertility in the - 196°Csample was significantly lower than in any of the other four samples (P
CRYOBIOLOGY
Cryopreservation
of Spermatozoa of the American Crassostrea virginica Gmelin
SUSAN R. ZELL,*j’ *Department
of Zoology,
MARK H. BAMFGRD,*
AND
HERBERT HIDUt
University of Maine, Orono, Maine 04473, and TDepartment University of Maine, Orono, Walpole, Maine 04573
Techniques for cryopreserving oyster spermatozoa would enhance the potential for controlled breeding of oysters, especially since oysters are protandric hermaphrodites. After sex reversal the eggs of a selected oyster could be fertilized with its own cryopreserved sperm. Self-fertilization or back crosses would increase the rate of inbreeding for valuable traits such as rapid growth, disease resistance or better shape, texture or flavor. Frozen spermatozoa could also provide a ready supply of male gametes for crossing populations that breed asynchronously or for shipment to different locations. Since the original success in preserving mammalian spermatozoa, scientists have frozen the spermatozoa of many different mammals (26), teleosts (6), and a few invertebrates, including echinodermata (9), the American oyster (12), and the Pacific oyster (15, 29). Fertility of frozen oyster spermatozoa ranged from 0% for the American oyster to 33% for the Pacific oyster, with storage for 24 hr at -170°C (29). This paper contains the report of a technique for cryopreserving spermatozoa of the American oyster in liquid nitrogen (- 196°C). After 68 days of storage cryopreserved spermatozoa had a fertility of 91% compared to 92% for fresh spermatozoa.
Oyster,
of Oceanography,
MATERIALS
Gametes
Gametes were obtained from American oysters (Crassostrea virginica Gmelin), conditioned about 2 weeks in seawater that had been filtered through l-pm pores. The filtered seawater had a pH 8.0 and a salinity of 20 ppt. The supply of sexually mature oysters was limited, but whenever possible, gametes from two or more oysters of the same sex were combined. Each lot of spermatozoa or eggs, containing gametes from one or more oysters, was numbered consecutively (i.e., s-l through s-9 or e-l through e-9). Gametes from any one oyster were used in only one lot. Initially gametes were extracted from oysters that had been spawned the preceding day. Occasionally egg lots had low fertilities and contained many eggs that looked immature. Diluent
The diluent used for successfully freezing spermatozoa was a 2.6x Hanks’ phosphate-buffered salt solution (Grand Island Biological Supply Co.), to which 80 mM glycine, 55 mM NaHCO, and 8% DMSO were added. The pH was adjusted to 8.0 (Orion Model 801A, digital pH meter), equal to that of the seawater and higher than the optimal buffering capacity of the diluent. A second solution of the same diluent was prepared and tested as a check of both the recipe and the sensitivity of the sperReceived February 20, 1979; accepted June 11, matozoa to random variations in the dil1979. uent. Concentrations of the principal ions, ’ Currently Faculty Associate, College of the Atlantic Bar Harbor, Maine 04609. sodium, potassium, and chloride, and sa448 001l-2240/79/050448-13$02.00/O Copyright 0 1979 by Academic Press, Inc. All rights of reproduction m any form reserved.
CRYOPRESERVATION
OF AMERICAN TABLE
OYSTER
449
SPERM
1
Ionic and Osmotic Concentrations of Spermatic Fluid from the American Oyster Crassostrea virginica, Seawater, and the Cryoprotective Diluent Concentration (mM) Solution Spermatic fluid” 1 2
Na+
Kf
425 422
16 17
Salinity (PPt)
Osmolality (mOsm)
496 477
31.V 30.6
913’ 877’ 832’
Cl-
Seawater Diluent 1 2
-
-
4.52d
29.0
397 413
14 15
379 375
24.3* 24.1*
Spermatic Fluid > Diluent’
N.S.
N.S.
P < 0.005
1990 2180
P < 0.005
” Each sample of spermatic fluid was pooled from two to three different oysters. b Salinity was calculated from the measured Cl- concentration. (’ Osmolality was calculated from the osmotic coefficient for a NaCl solution, equal in molarity to the measured Cl- concentration. d Chloride concentration was calculated from measured salinity. p Hypothesis that spermatic fluid > diluent; tested by normal t distribution with 2 dJY
linity and osmolality of the two diluent solutions are shown in Table 1. Corresponding values for seawater and spermatic fluid are presented in the same table. Spermatic fluid was the supernatant fluid formed when the contents of extracted gonads were centrifuged for 2-3 min. in a Beckman Spinco Microfuge. Sodium and potassium concentrations were measured by flame photometry (Instrument Laboratories, Inc., Model 343, F. M.). Chloride concentrations in the diluent and spermatic fluids were measured coulometrically (Buchler Cotlove Chloridometer). In seawater, chloride concentration was calculated from its measured salinity, according to the following relationship; salinity (o/o0)= 0.03 + 1.805 (chlorinity) (O/00) (3). According to the same equation, salinities of the diluents and spermatic fluids were calculated from their measured chloride concentrations. Osmolality of the diluent was measured by freezing point depression (Halbmikro Osmometer, Knauer Type M No. 9001) after the diluent, which was too concentrated for the range of osmolalities stan-
dardized on the osmometer, was diluted 1: 1 with distilled, deionized water. Osmolality of spermatic fluids and of seawater was calculated from the measured chloride concentrations and the osmotic coefficient of a 0.4-0.5 molal solution of sodium chloride (25). The difference between molality and molarity for a sodium chloride solution of 0.4-0.5 M is negligible (35). A complete atomic absorption scan (Garrel-Ash No. 750; Atomcomp. Fisher Scientific) was run on the diluent solutions. Magnesium was 2.5 mM in both; calcium was 0.86 mM in the first and 1.6 mM in the second. Traces of arsenic, tin, and boron were observed in both solutions. No other elements were detected. METHODS
Collecting and Diluting Gametes Extracted gametes. After the top shell of an oyster was removed, the epithelium overlying the gonadal tissue was pierced with a pipet and the gonadal contents were aspirated into the pipet. Undiluted spermatic extract was cooled briefly in a watchglass on ice at 0°C before dilution with di-
450
ZELL,
BAMFORD,
AND
HIDU
luent at 0-2°C. Approximately 2 ml of spermatic extract was mixed with 10 ml of diluent for a l/6 dilution. The diluted spermatozoa were placed in a 15-n& capped, plastic test tube (16-mm diameter) on ice at 0°C for 20 min before being added to eggs or frozen. Two lots of spermatozoa that were frozen and stored were handled differently from the preceding description. Diluted spermatozoa from lot s-3 were held at 0°C for 75 min before being added to eggs or frozen. Undiluted spermatozoa from lot s-4 were held in a IS-ml capped test tube at 0°C for 120 min before being used. A sample was added to eggs; the rest was diluted and held for an additional 20 min before being frozen. Extracted eggs and accompanying fluid were added to seawater of 23 -26°C. In early experiments eggs from one female were diluted into 2-3 liters of seawater (e-l, e-2) for a final concentration of 15-50 eggs/ml. In later experiments the extracted eggs were first diluted into 50 ml of seawater and then rediluted into 150-450 ml for a final concentration of 250-900 eggs/ml. Spawned gametes. In one experiment spermatozoa were frozen after they had been spawned into seawater. An oyster was stimulated to spawn by the presence of fluid into which another male had previously spawned. The milky stream of freshly emitted spermatozoa, together with surrounding seawater, was collected in a pipet, cooled, and mixed with diluent. In one experiment a female (e-7) was induced by the presence of egg fluid extracted from another female to spawn into 17 liters of seawater.
an effort was made to freeze the spermatozoa at -5”CYmin. The recorded cooling rate between 0 and -20°C varied from -5 to -7.5”Clmin. From -20 to -80°C the rate of cooling was erratic, but the average cooling rate for each run varied between -5 and -13.5Vmin. When latent heat of fusion was released between - 10 and -2O”C, the probe registered a warming of as much as 23°C or as little as 1 or 2°C. The temperature rise, which was generally about 7 to 12”C, was followed by a retooling at -S”C/min. After reaching -80°C in the chamber, samples were plunged into liquid nitrogen (- 196°C) and stored submerged in liquid nitrogen. Storing. Samples, frozen only to -5, -20, -40, or -8O”C, were held in the freezing chamber as long as the straw temperature remained constant, but never for more than 2 min. They were then thawed. Samples frozen to - 196°C were stored in liquid nitrogen from 5 min to 68 days. Each time stored samples were removed from the storage Dewar flask, all the remaining straws were briefly lifted out of the liquid nitrogen. Thawing. Straws were thawed by submersion in a 55-60°C water bath. Straws that had been frozen to one of the intermediate temperatures were held in the water bath for up to 10 set, or just until the diluent in the straws had visibly thawed. Spermatozoa frozen to -196°C were thawed by submersion for 10 sec. Immediately after thawing, before the sperm solution could warm more than a degree or two above 0°C the thawed spermatozoa were diluted into the egg-containing seawater.
Cryopreserving Spermatozoa Freezing. Diluted spermatozoa were aspirated into 0.25-ml plastic straws, which were then placed in a 0°C chamber of a biological freezer (Linde BF-4- 1). They were then cooled with liquid nitrogen vapors to either -5, -20, -40, or -80°C. A temperature probe had been placed in a 0.5~ml plastic straw containing diluent and
Fertilizing Eggs In most experiments about 0.25 ml of either fresh or cryopreserved spermatozoa (i.e., 1.5 ml of spermatic extract mixed with diluent) was added to 30-200 ml of seawater containing 200-900 eggs/ml. In the final experiment (Trial 6: s-4/e-9 and s-9/e-9) only 0.12 ml of spermatozoa was added to 160 ml of seawater containing about 400
CRYOPRESERVATION
OF AMERICAN
eggs/ml. The beaker was swirled gently and allowed to sit undisturbed at 23-26°C. Measuring Fertility of Cryopreserved Spermatozoa Development of eggs into larva. Spermatozoan fertility was tested by the addition of spermatozoa to a suspension of eggs, but it was measured indirectly, as the percentage of eggs that developed into larvae. Thus the measured fertility of cryopreserved spermatozoa was influenced by the fertility of eggs as well as the fertility of spermatozoa before freezing. Consequently three different controls were done. Controls. (1) Eggs were fertilized with fresh spermatozoa 20 min after the spermatozoa were diluted and stored at 0°C. Any loss in fertility due to treatment before freezing could thus be distinguished from the effects of freezing and thawing. (2) Eggs from the same females as those that were fertilized with cryopreserved spermatozoa were fertilized with fresh, unchilled, and undiluted spermatozoa. When the fertility of cryopreserved spermatozoa was tested on the same day they were frozen, control and cryopreserved spermatozoa came from the same males, and the percentage of eggs that developed into larvae after fertilization with fresh spermatozoa indicated the maximum fertility one could expect with eggs from the same lot and the cryopreserved spermatozoa. When cryopreserved spermatozoa were tested after I or more days of storage, fresh and cryopreserved spermatozoa had to come from different males, and fertility of the fresh spermatozoa was used as an estimate of the maximum fertility of a given lot of eggs. (3) A 60- to 500-ml sample from each batch of unfertilized eggs was left undisturbed at 23-26°C and was later checked for larval development. Occasionally unfertilized eggs were first exposed to the diluent and then left undisturbed. Thus the possibility was tested that eggs may have been contaminated with spermatozoa or that they may have developed parthenogenetically,
OYSTER
SPERM
451
either spontaneously or because of stimulation by the diluent or handling. Evaluation of larval development. A sample from each egg solution was pipetted into a Sedgewick Rafter cell, and each egg and embryo was counted under 100x magnification. Duplicate l-ml samples of eggs from lot e-l and triplicate 2-ml samples of eggs from lot e-2 were counted. At least 400 eggs/sample from every other lot of eggs were counted. Abnormal eggs or larvae were counted as undeveloped eggs, except in the unfertilized, control samples, where any development was counted as an embryo. About 30-60 min after fertilization, eggs began extruding their first polar body, but depending on the position of an egg in the microscopic field, the polar body may have been obscured from view. After about 5 hr, the developing larvae became ciliated and motile. In replicate samples analyzed at 4, 7, and 10 hr or 2, 4, and 5 hr after fertilization, the measured percentage development was reproducible. Motile gastrulae, however, were difficult, if not impossible to count, and after they were fixed in formalin they could be confused with eggs. Hence all samples were counted 2-3 hr after fertilization. When rechecked after up to 7 hr, all solutions with high percentages of development contained dense suspensions of swimming larvae and few remaining eggs. Larval growth. In one experiment (Trial 4: s-4/e-7) 0.625 ml of cryopreserved spermatozoa was added to 17 liters of seawater in which a female oyster had spawned naturally in the complete absence of spermatozoa. On the same day naturally spawned eggs from a different female were fertilized according to standard hatchery procedure with naturally spawned spermatozoa. The resulting larvae were reared for 11 days. Observing Spermatozoa Motility. Fresh spermatozoa were observed under 100x magnification in either spermatic fluid, seawater, cryoprotective diluent, or an alternative diluent that, for
ZELL, BAMFORD,
452
AND HIDU
TABLE 2 Fertility of Oyster Spermatozoa Minutes after They Were Cooled to Different Subzero Temperatures in One of Two Separately Prepared Replicate Diluents Low temperature P-3
Trial 1: s-l/e-l”,* No. eggs/ml
Undiluted sperm +23
-
Diluted sperm 0 -5 -20 -40 -80 -196
43 40 48 45 47 -
Trial 2: s-2/e-2
Trial 3: s-3/e-3
% Embryos
No. eggs/ml
% Embryos
No. eggs/ml
% Embryos
-
16
81
262
85
95 97 94 87 76 -
(Diluent 1) 22 72 26 92 11 86 29 93
(Diluent 1)
(Diluent 2) 716 100 467 96 871 97 689 81
fl In Trials 1 and 3, one l-ml sample was counted: in Trial 2, triplicate 2-ml samples were counted and averaged for each temperature. Based on a binomial distribution, there is no significant difference between any of the individual samples in Trial 1 or 2 or the -20 and -80°C samples of Trial 3. The other samples in Trial 3 are each significantly different from the others (P i 0.01). b Abbreviations: s-l/e-l = sperm lot-l/egg lot-l (s-l, s-2, e-2, and e-3 came from one oyster each; s-3 and e-l came from two.each).
oysters, had a high pH (9.0) and a low osmolality (425 mOsm). This test solution consisted of a basic Tris-citrate buffer (Poulik’s gel buffer), modified by the addition of 10% polyvinylpyrrolidone, 85 mA4 glycine, 56 mM NaHCO,, and 36 mM KC1 (36). Egg-sperm
interactions. Egg-sperm interactions were observed within minutes after fertilization with either undiluted, fresh spermatozoa; diluted, fresh spermatozoa that were chilled to 0°C; or cryopreserved spermatozoa.
0.005).
RESULTS
Fertility
of Cryopreserved
being diluted and cooled to 0, -5, or -20°C spermatozoa had 95, 97, or 94% fertility. In Trial 2 none of the measured fertilities differed significantly from any of the others. In Trial 1, however, spermatozoa that were frozen to -80°C had a significantly lower fertility than spermatozoa in the -40°C sample (P < O.OS),and in both samples, spermatozoa had a significantly lower fertility than in the 0, -5, or -20°C sample (P < 0.05). In Trial 3 fertility in the - 196°Csample was significantly lower than in any of the other four samples (P
