c~iIYoI~:oI,o::Y
12, 89-92
( 1975)
BRIEF COMMUNICATIONS The Ability of Cryopreservatives to Prevent Motility Loss and Freeze-Thaw Damage to the Acrosome of Chicken Spermatozoa1 F. D. WESTFALL
AND
G.
C.
HARRIS, JR.
Department of Animal Sciences, Univarsity of Arkansas, Fayetieuil~e,
Since the discovery that gIycero1 would preserve the motility of frozen chicken spermatozoa (9), much interest has been shown in the development of techniques for the preservation of semen from several species. Although glycerol was effective in protecting the motility of spermatozoa, the fertility of nonfrozen or frozen chicken semen treated with greater than 2% glycerol was greatly reduced (10). It was possible to attain a fertility level as high as 407% (4), if the glycerol was removed by dilution and centrifugation prior to insemination. Extensive membrane damage (5) and IOSS of proteins (6) from the chicken spermatozoon into the extraoellular media occurs after rapid freezing by direct immersion in liquid nitrogen ( - 198°C ) , Calcium45 uptake studies with chicken spermatozoa showed that a glycerol level of 12% was needed to provide some protection against the rigors of fast (direct immersion in liquid nitrogen) or slow (l”C/min) freezing (12). GIycerol at levels of 8 or IS% was unable to protect the membrane ultrastructure against damage during fast freezing by direct immersion in liquid nitrogen (5). However, partial protection of the
spermatozoon could be attained at lower gIycero1 concentrations with a sIow (1°C per min to -B”C)-fast (85°C per min to -196°C) freezing procedure. Two other cryopreservatives, dimethylsulfoxide (DMSO) and ethylene glycol, have received considerable attention as substitutes for glycerol. However, a deleterious response was obtained with DMSO when chicken semen was fast frozen (5, 12). Ethylene glycol was also Iess effective than glycerol for preserving the motility of frozen chicken spermatozoa (10). This cryopreservative at levels between 10 and 20% reduced the fertility of unfrozen spermatozoa. The acrosomes of chicken spermatozoa contain trypsin-like enzymes (7) which are essential for penetration of the egg (8, 14). A loss of these enzymes such as occurs as a result of freeze-thaw damage to the acrosome (13) would render the spermatozoon incapable of ovum penetration. Since the spermatozoa must also be motile to traverse the vagina and possibly to penetrate the ovum (2), spermatozoa which ‘are damaged either in the acrosome or in some structure which might cause loss of motility could not become involved in the fertilization process. In the present study three cryopreservatives (glycerol, DMSO, and ethylene
Received June 20, 1974. 1 PubIished with the approval of the Director of the Arkansas Agricultural. Experiment Station. 89 Copyright 0 1975 by Academic Press. Inc. All rights of reproduction in any form reserved.
Arkansas 72701
WESTFALL
90 TABLE EFFxxTs OF GLYCEROL CHICKEN
AND HARRIS, JR.
1
ON ho%L’x AND SPERMATOZOA~.~
drop.
Treatment
Nonfrozen
Frozen
0 2 4 8 12 16 24 0 2 4 8 12 16 24
SE
98s 97a 978 97” 9w 9% 8i-P
97Sb 99a 97”b 96”b 94&b” 9kw 96”b
Oh 33fg 43ef 54”e 53d* 63cd 32s
gp:de gfpfde
zk4
8@
87d” 8W 93abcd 93mbcd f2
1Each mean is the average of four replicate samples. 2Means with different superscripts are significantly different (P < 0.05) ; a-g indicates differences between means using the Duncan’s Multiple Range Test (see Materials and Methods).
gIyco1) were evaluated as to their ability to prevent motility loss and damage to the acrosome as measured by the membrane digestion technique. MATERIALS
Aliquots
(0.1 ml)
were removed and diluted 1:lO into Verona1 acetate buffer (pH 3.5). Small drops of this freshly diluted semen were placed on one end of the fixed gelatin membrane and smeared, Proteolytic digestion was evaluated as described in a previous paper (13). The data were anaIyzed by analysis of variance and the means were compared by Duncan’s MultipIe Range Test (Xl).
NUNFROZKN before and after freezing
AND METHODS
The effects of glycerol (0, 2, 4, 8, 12, 16, 24% ), dimethylsulfoxide ( DMSO > (0, 2, 4, 6, 8, 10, 12, 14% ), and ethylene glycol (0, 4, 8, 10, 12, 16, 24% > on proteolytic digestion and motility of chicken spermatozoa before and after freezing were examined. The semen was collected (2 f and diluted ( 1:lO) into monosodium glutamate solution before the cryapreservatives were added to give a final volume of 1.0 ml. The diluted semen was held at 5°C for 1 hr. Motility was estimated, before and after freezing in crushed dry ice for 1 hr by microscopic examination of a hanging
RESULTS
Glycerol at levels up to 16% (Table I) did not significantly affect motility before freezing, A further increase in glycerol level up to 25% significantly decreased the motility below that obtained with lower glycerol levels. Freezing significantly reduced motility at every level of glycerol. The motility of spermatozoa frozen in 16% glycerol was significantly higher than that obtained with glycerol levels of 0, 2, 4, or 24Gjo. The highest motility of spermatozoa after freezing was observed at the 16% glycerol level. No significant differences were noted between the motility obtained with the 16% glycerol level and either 8 or 12% levels. Motility of spermatozoa after freezing in a11 levels of glycerol was significantly higher than the (control) glutamate dihrent alone. No motility was noted for spermatozoa frozen in this diluent which did not contain any glycerol Glycerol had, no effect on proteolytic digestion of a membrane before freezing (Table 1 ), Freezing caused a significant reduction in proteolytic membrane digestion by spermatozoa frozen in the diluents containing 0, 2, 4, and 8% glycerol Membrane digestion by spermatozoa frozen in 12, 16, and 24% glycerol was not significantly affected by freezing. DMSO at levels of 8% or higher significantly depressed motihty of the spermatozoa before freezing (Table 2), After freezing, only the motility of the spermatozoa in the 8% level of DMSO was significantly
CRYOPRESERVATIVES
AND ACROSOMAL
higher than the motility of spermatozoa frozen without DMSO. DMSO had no effect on the number of spermatozoa capable of completely digesting the membrane before freezing (Table 2). Membrane digestion by spermatozoa frozen in 12 or 14% DMSO diluents was significantIy lower than at the 0, 2, or 4% DMSO IeveIs. Ethylene glycol (TabIe 3) at the 24% level significantly reduced the motiIity of unfrozen spermatozoa. After freezing, motility of spermatozoa in 46% ethylene glycol was significantly higher than the motility of spermatozoa frozen in the diIuent containing no ethylene glycol. However, motility was significantly reduced by freezing at all levels tested. Ethylene glycol had no effect on membrane digestion (Table 3) by unfrozen TABLE EFFECTS
OF
Nonfrozen
Frozen
SE
2
DMSO
ON FROZEN AND NONFROZEN CHICKKN SPF.I~M,\T~Z~.L~*~ Jlotile spermatozoa apermatoaoa cepshle of membrane t%) digestion (%)
0 2 4 6 8 10 12 14
94= 94= 908 82*b 64°C 580 47cd 38”e
97’L 95a’b 93=‘b0 91ab” 92*“0 94*b 9Zsbc
i’6d”’ 716’E 69 fg 64g *7
TABLE EFFECTS
91 3
OF ETHYLENE GLYCOL ON FROZEN NONFROZEN SP~RMATOZOA~J
Treatment
Level uf ethylene &ml (%“o)
Motile eparmatozoa (%)
APFD
Spermatoeos caprrble of membrane 0 d’“???“=
Nonfrozen
0 4 8 10 12 16 24
87s 87a 8P 83” 83a 77” 27b
1OtP 97ab loos 1OoB 998 9W 96abo
Rosen
0 4 8 10 12 16 24
oc 27b 33b 43b 43b 43b 22bc
90Cd 93b= 93b’: 87d 86d 7d 79
SE
zk.8
*2
1Each mean is the average of three replicate samples. 1 Means with different superscripts are significantly different (P < 0.05) ; a-g indicates differences between means using the Duncan’s Multiple Range Test (see Materials and Methods),
spermatozoa. After freezing, digestion by spermatozoa frozen in 16 or 24% Ievels of ethylene glycol was significantly lower than by spermatozoa frozen without added ethylene glycol.
@j&cd
szbcde &jnbcd szbcde slcdef
0 2 4 6 8 10 12 I4
DAMAGE
*4
1 Each mean is the average of five replicate samples. 2 Means with different superscripts are significantly different (P < 0.05) ; a-g indicates differences between means using the Duncan’s Multiple Range Test (see Materials and Methods).
DISCUSSION
Although freezing and thawing caused a significant reduction of the abiIity of chicken spermatozoa to completely digest a protein membrane, this reduction cannot fully explain the severe reduction in fertiIity caused by freezing these spermatozoa. It is more likely that either the loss of motility observed after freezing in all levels of these cryopreservatives or a general reduction in the stabihty of the spermatozoa is a more justifiable reason for decreased fertility of frozen spermatozoa. In view of the protective action of 16% glycerol on proteolytic digestion of the
WESTFALL
92
AND HARRIS, JR.
membrane and the preservation of motility also observed at this level, glycerol at high levels shouId be useful in preserving chicken spermatozoa, The observations that glycerol at 16% had no effect on motility or membrane digestion before freezing indicate that the Ioss of fertility which occurred (1) when unfrozen spermatozoa containing high glycero1 levels were inseminated into the vagina may be caused by an interaction between glycerol and the vagina which prevents the spermatozoa from moving up the female reproductive tract. This hypothesis is supported by the observation that unfrozen spermatozoa containing 15% glycero1 produce high fertility (1) when they are deposited in the uterus. Under the conditions employed, glycerol was the best cryopreservative tested for freeze-preservation of chicken spermatozoa. SUMMARY
Proteolytic membrane digestion and motility were used to determine the effects of cryopreservatives and freezing on acrosomal damage and survival of chicken spermatozoa. None of the cryopreservatives, glycerol, DMSO, or ethyIene glycol caused a decrease in proteolytic membrane digestion by the chicken spermatozoon before freezing. After rapid freezing, high levels (16 and 24% ) of glycerol prevented significant freeze-thaw reductions in proteolytic digestion. High levels of ethylene gIyco1 ( 16 and 24% ) and DMSO ( 12 and 14% ) sign%cantIy reduced the ability of frozen spermatozoa to compIeteIy digest the protein membrane. MotiIity after freezing was highest ( 63% ) in 16% glycerol. GlyceroI at high Ievels appeared to be the best cryopreservative under these conditions. REFERENCES 1. Allen, T. E., and Bobr, L. W. The fertiIity of fowl spermatozoain glycerol diluents after
2.
3.
4.
5.
intrauterine insemination. POUZt~ SCL 34, 1167-1169 ( 1955). Allen, T. E., and Grigg, G. W. Sperm tram+ port in the fowl. Au& J. A&. Rex 8, 788799 ( 1957). Burrows, W. H., and Quinn, J. P. The collection of spermatozoa from the domestic fowl and turkey. PO&~ Scl. 16, 19-24 (1937). Clark, C. E., and Shaffner, C. S. The fertilizing capacity of frozen chicken sperm and the inffuence of related in &TO processes. Poubry Sci. 39, 1213-1220 (1960). Harris, G. C., Jr., Thurston, R. J., and Cundall, J. Changes in the ultrastructure of the fowl spermatozoon due to rapid freeze-thaw. J. Reprod.
Fe&
34, 389-394
( 1972).
6. Harris, G. C., Jr., and Sweeney, M. J. Changes in the protein concentration of chicken seminal plasma after rapid freeze-thaw. Cryobiology 7, 209-215 ( 1971). 7. Ho, J. L. J,, and Meizel, S. Electrophoretic and biochemical characterization of trypsinlike activity in the spermatozoa of the domestic fowl. J. Reprod. Fwt. 23, 177179 ( 1970). 8. Howarth, B., Jr., and Digby, S. T. Evidence for the penetration of the vitelline membrane of the hen’s ovum by a trypsin-like acrosomal enzyme. J. Rep~od. Feti. 33, 12s 125 ( 1972).
9. Polge, C., Smith, A. V., and Parkes, A. S. Revival of spermatozoa after vitrification and dehydration at law temperatures. Nature (London)
164, 666 ( 1949).
10. Smith, A. V., and Polge, C. Survival of spermatozoa at low temperatures. Nature (London) 166, 668-669 (1950). 11. Steel, R. G. D., and Torrie, J. H. “Principles and Procedures of Statistics.” McGraw-Hill, New York, 1960. 12. Thurston, R. J., and Harris, G. C., Jr. Calcium45 uptake as a measurement sf freeze-thaw injury to chicken spermatozoa in the presence or absence of cryopreservntives. ~ryobiology 7, 100-106 ( 1970). 13. WestfaIl, F. D., and Harris, G. C., Jr. A protective digestion technique for evaluating freeze-induced damage to acrosomes of chicken spermatozoa.Cryobiology 11, 255259 ( 1974). 14. Zaneveld, L. J. D., Roberson, R. T., Kessler, M., and Williams, W. L. Inhibition of fertilization in U&O by pancreatic and seminal plasma trypsin inhibitors. J. Repod. Fe+t. 25, 387392 ( 1971),
12, 89-92
( 1975)
BRIEF COMMUNICATIONS The Ability of Cryopreservatives to Prevent Motility Loss and Freeze-Thaw Damage to the Acrosome of Chicken Spermatozoa1 F. D. WESTFALL
AND
G.
C.
HARRIS, JR.
Department of Animal Sciences, Univarsity of Arkansas, Fayetieuil~e,
Since the discovery that gIycero1 would preserve the motility of frozen chicken spermatozoa (9), much interest has been shown in the development of techniques for the preservation of semen from several species. Although glycerol was effective in protecting the motility of spermatozoa, the fertility of nonfrozen or frozen chicken semen treated with greater than 2% glycerol was greatly reduced (10). It was possible to attain a fertility level as high as 407% (4), if the glycerol was removed by dilution and centrifugation prior to insemination. Extensive membrane damage (5) and IOSS of proteins (6) from the chicken spermatozoon into the extraoellular media occurs after rapid freezing by direct immersion in liquid nitrogen ( - 198°C ) , Calcium45 uptake studies with chicken spermatozoa showed that a glycerol level of 12% was needed to provide some protection against the rigors of fast (direct immersion in liquid nitrogen) or slow (l”C/min) freezing (12). GIycerol at levels of 8 or IS% was unable to protect the membrane ultrastructure against damage during fast freezing by direct immersion in liquid nitrogen (5). However, partial protection of the
spermatozoon could be attained at lower gIycero1 concentrations with a sIow (1°C per min to -B”C)-fast (85°C per min to -196°C) freezing procedure. Two other cryopreservatives, dimethylsulfoxide (DMSO) and ethylene glycol, have received considerable attention as substitutes for glycerol. However, a deleterious response was obtained with DMSO when chicken semen was fast frozen (5, 12). Ethylene glycol was also Iess effective than glycerol for preserving the motility of frozen chicken spermatozoa (10). This cryopreservative at levels between 10 and 20% reduced the fertility of unfrozen spermatozoa. The acrosomes of chicken spermatozoa contain trypsin-like enzymes (7) which are essential for penetration of the egg (8, 14). A loss of these enzymes such as occurs as a result of freeze-thaw damage to the acrosome (13) would render the spermatozoon incapable of ovum penetration. Since the spermatozoa must also be motile to traverse the vagina and possibly to penetrate the ovum (2), spermatozoa which ‘are damaged either in the acrosome or in some structure which might cause loss of motility could not become involved in the fertilization process. In the present study three cryopreservatives (glycerol, DMSO, and ethylene
Received June 20, 1974. 1 PubIished with the approval of the Director of the Arkansas Agricultural. Experiment Station. 89 Copyright 0 1975 by Academic Press. Inc. All rights of reproduction in any form reserved.
Arkansas 72701
WESTFALL
90 TABLE EFFxxTs OF GLYCEROL CHICKEN
AND HARRIS, JR.
1
ON ho%L’x AND SPERMATOZOA~.~
drop.
Treatment
Nonfrozen
Frozen
0 2 4 8 12 16 24 0 2 4 8 12 16 24
SE
98s 97a 978 97” 9w 9% 8i-P
97Sb 99a 97”b 96”b 94&b” 9kw 96”b
Oh 33fg 43ef 54”e 53d* 63cd 32s
gp:de gfpfde
zk4
8@
87d” 8W 93abcd 93mbcd f2
1Each mean is the average of four replicate samples. 2Means with different superscripts are significantly different (P < 0.05) ; a-g indicates differences between means using the Duncan’s Multiple Range Test (see Materials and Methods).
gIyco1) were evaluated as to their ability to prevent motility loss and damage to the acrosome as measured by the membrane digestion technique. MATERIALS
Aliquots
(0.1 ml)
were removed and diluted 1:lO into Verona1 acetate buffer (pH 3.5). Small drops of this freshly diluted semen were placed on one end of the fixed gelatin membrane and smeared, Proteolytic digestion was evaluated as described in a previous paper (13). The data were anaIyzed by analysis of variance and the means were compared by Duncan’s MultipIe Range Test (Xl).
NUNFROZKN before and after freezing
AND METHODS
The effects of glycerol (0, 2, 4, 8, 12, 16, 24% ), dimethylsulfoxide ( DMSO > (0, 2, 4, 6, 8, 10, 12, 14% ), and ethylene glycol (0, 4, 8, 10, 12, 16, 24% > on proteolytic digestion and motility of chicken spermatozoa before and after freezing were examined. The semen was collected (2 f and diluted ( 1:lO) into monosodium glutamate solution before the cryapreservatives were added to give a final volume of 1.0 ml. The diluted semen was held at 5°C for 1 hr. Motility was estimated, before and after freezing in crushed dry ice for 1 hr by microscopic examination of a hanging
RESULTS
Glycerol at levels up to 16% (Table I) did not significantly affect motility before freezing, A further increase in glycerol level up to 25% significantly decreased the motility below that obtained with lower glycerol levels. Freezing significantly reduced motility at every level of glycerol. The motility of spermatozoa frozen in 16% glycerol was significantly higher than that obtained with glycerol levels of 0, 2, 4, or 24Gjo. The highest motility of spermatozoa after freezing was observed at the 16% glycerol level. No significant differences were noted between the motility obtained with the 16% glycerol level and either 8 or 12% levels. Motility of spermatozoa after freezing in a11 levels of glycerol was significantly higher than the (control) glutamate dihrent alone. No motility was noted for spermatozoa frozen in this diluent which did not contain any glycerol Glycerol had, no effect on proteolytic digestion of a membrane before freezing (Table 1 ), Freezing caused a significant reduction in proteolytic membrane digestion by spermatozoa frozen in the diluents containing 0, 2, 4, and 8% glycerol Membrane digestion by spermatozoa frozen in 12, 16, and 24% glycerol was not significantly affected by freezing. DMSO at levels of 8% or higher significantly depressed motihty of the spermatozoa before freezing (Table 2), After freezing, only the motility of the spermatozoa in the 8% level of DMSO was significantly
CRYOPRESERVATIVES
AND ACROSOMAL
higher than the motility of spermatozoa frozen without DMSO. DMSO had no effect on the number of spermatozoa capable of completely digesting the membrane before freezing (Table 2). Membrane digestion by spermatozoa frozen in 12 or 14% DMSO diluents was significantIy lower than at the 0, 2, or 4% DMSO IeveIs. Ethylene glycol (TabIe 3) at the 24% level significantly reduced the motiIity of unfrozen spermatozoa. After freezing, motility of spermatozoa in 46% ethylene glycol was significantly higher than the motility of spermatozoa frozen in the diIuent containing no ethylene glycol. However, motility was significantly reduced by freezing at all levels tested. Ethylene glycol had no effect on membrane digestion (Table 3) by unfrozen TABLE EFFECTS
OF
Nonfrozen
Frozen
SE
2
DMSO
ON FROZEN AND NONFROZEN CHICKKN SPF.I~M,\T~Z~.L~*~ Jlotile spermatozoa apermatoaoa cepshle of membrane t%) digestion (%)
0 2 4 6 8 10 12 14
94= 94= 908 82*b 64°C 580 47cd 38”e
97’L 95a’b 93=‘b0 91ab” 92*“0 94*b 9Zsbc
i’6d”’ 716’E 69 fg 64g *7
TABLE EFFECTS
91 3
OF ETHYLENE GLYCOL ON FROZEN NONFROZEN SP~RMATOZOA~J
Treatment
Level uf ethylene &ml (%“o)
Motile eparmatozoa (%)
APFD
Spermatoeos caprrble of membrane 0 d’“???“=
Nonfrozen
0 4 8 10 12 16 24
87s 87a 8P 83” 83a 77” 27b
1OtP 97ab loos 1OoB 998 9W 96abo
Rosen
0 4 8 10 12 16 24
oc 27b 33b 43b 43b 43b 22bc
90Cd 93b= 93b’: 87d 86d 7d 79
SE
zk.8
*2
1Each mean is the average of three replicate samples. 1 Means with different superscripts are significantly different (P < 0.05) ; a-g indicates differences between means using the Duncan’s Multiple Range Test (see Materials and Methods),
spermatozoa. After freezing, digestion by spermatozoa frozen in 16 or 24% Ievels of ethylene glycol was significantly lower than by spermatozoa frozen without added ethylene glycol.
@j&cd
szbcde &jnbcd szbcde slcdef
0 2 4 6 8 10 12 I4
DAMAGE
*4
1 Each mean is the average of five replicate samples. 2 Means with different superscripts are significantly different (P < 0.05) ; a-g indicates differences between means using the Duncan’s Multiple Range Test (see Materials and Methods).
DISCUSSION
Although freezing and thawing caused a significant reduction of the abiIity of chicken spermatozoa to completely digest a protein membrane, this reduction cannot fully explain the severe reduction in fertiIity caused by freezing these spermatozoa. It is more likely that either the loss of motility observed after freezing in all levels of these cryopreservatives or a general reduction in the stabihty of the spermatozoa is a more justifiable reason for decreased fertility of frozen spermatozoa. In view of the protective action of 16% glycerol on proteolytic digestion of the
WESTFALL
92
AND HARRIS, JR.
membrane and the preservation of motility also observed at this level, glycerol at high levels shouId be useful in preserving chicken spermatozoa, The observations that glycerol at 16% had no effect on motility or membrane digestion before freezing indicate that the Ioss of fertility which occurred (1) when unfrozen spermatozoa containing high glycero1 levels were inseminated into the vagina may be caused by an interaction between glycerol and the vagina which prevents the spermatozoa from moving up the female reproductive tract. This hypothesis is supported by the observation that unfrozen spermatozoa containing 15% glycero1 produce high fertility (1) when they are deposited in the uterus. Under the conditions employed, glycerol was the best cryopreservative tested for freeze-preservation of chicken spermatozoa. SUMMARY
Proteolytic membrane digestion and motility were used to determine the effects of cryopreservatives and freezing on acrosomal damage and survival of chicken spermatozoa. None of the cryopreservatives, glycerol, DMSO, or ethyIene glycol caused a decrease in proteolytic membrane digestion by the chicken spermatozoon before freezing. After rapid freezing, high levels (16 and 24% ) of glycerol prevented significant freeze-thaw reductions in proteolytic digestion. High levels of ethylene gIyco1 ( 16 and 24% ) and DMSO ( 12 and 14% ) sign%cantIy reduced the ability of frozen spermatozoa to compIeteIy digest the protein membrane. MotiIity after freezing was highest ( 63% ) in 16% glycerol. GlyceroI at high Ievels appeared to be the best cryopreservative under these conditions. REFERENCES 1. Allen, T. E., and Bobr, L. W. The fertiIity of fowl spermatozoain glycerol diluents after
2.
3.
4.
5.
intrauterine insemination. POUZt~ SCL 34, 1167-1169 ( 1955). Allen, T. E., and Grigg, G. W. Sperm tram+ port in the fowl. Au& J. A&. Rex 8, 788799 ( 1957). Burrows, W. H., and Quinn, J. P. The collection of spermatozoa from the domestic fowl and turkey. PO&~ Scl. 16, 19-24 (1937). Clark, C. E., and Shaffner, C. S. The fertilizing capacity of frozen chicken sperm and the inffuence of related in &TO processes. Poubry Sci. 39, 1213-1220 (1960). Harris, G. C., Jr., Thurston, R. J., and Cundall, J. Changes in the ultrastructure of the fowl spermatozoon due to rapid freeze-thaw. J. Reprod.
Fe&
34, 389-394
( 1972).
6. Harris, G. C., Jr., and Sweeney, M. J. Changes in the protein concentration of chicken seminal plasma after rapid freeze-thaw. Cryobiology 7, 209-215 ( 1971). 7. Ho, J. L. J,, and Meizel, S. Electrophoretic and biochemical characterization of trypsinlike activity in the spermatozoa of the domestic fowl. J. Reprod. Fwt. 23, 177179 ( 1970). 8. Howarth, B., Jr., and Digby, S. T. Evidence for the penetration of the vitelline membrane of the hen’s ovum by a trypsin-like acrosomal enzyme. J. Rep~od. Feti. 33, 12s 125 ( 1972).
9. Polge, C., Smith, A. V., and Parkes, A. S. Revival of spermatozoa after vitrification and dehydration at law temperatures. Nature (London)
164, 666 ( 1949).
10. Smith, A. V., and Polge, C. Survival of spermatozoa at low temperatures. Nature (London) 166, 668-669 (1950). 11. Steel, R. G. D., and Torrie, J. H. “Principles and Procedures of Statistics.” McGraw-Hill, New York, 1960. 12. Thurston, R. J., and Harris, G. C., Jr. Calcium45 uptake as a measurement sf freeze-thaw injury to chicken spermatozoa in the presence or absence of cryopreservntives. ~ryobiology 7, 100-106 ( 1970). 13. WestfaIl, F. D., and Harris, G. C., Jr. A protective digestion technique for evaluating freeze-induced damage to acrosomes of chicken spermatozoa.Cryobiology 11, 255259 ( 1974). 14. Zaneveld, L. J. D., Roberson, R. T., Kessler, M., and Williams, W. L. Inhibition of fertilization in U&O by pancreatic and seminal plasma trypsin inhibitors. J. Repod. Fe+t. 25, 387392 ( 1971),
