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Effect of thawing temperature on the loss of acrosin and hyaluronidase enzymes from bovine spermatozoa
EFFECT AND
OF
THAWING
TEMPERATURE
HYALURONIDASE
ENZYMES
Imam ~bulatorische
El
ON
THE
FROM
LOSS
BOVINE
OF
ACROSIN
SPERMATOZOA
Danasouri
und Geburtshilflische
Veterin~rklinik
Justus-Liebig-Universit~t 6300 Giessen,
FRG
Received for publication: &me 29, Accepted:
Apti
1987
7, 1988
ABSTRACT Bovine semen frozen in 0.5ml straws was used to determine the loss of the acrosamal enzymes acre in and hyaluronidase (from spermatozoa after thawing at 5 C for 50 set, 37 Q for 15 set and at 70 C for 7 sec. The highest acrosin activity remaining in the spermatozoa and the loweat hyaluronidase activity in the media were observed after thawing at 70 C (P<8.01). Indications of high acrosomal damage were observed after thawing at 5 C (PiO.01). INTRODUCTION Rapid thawing rates of semen packaged in plastic straws hqve been shown to increase significantly the percentage of unstained(live} spermatozoa (If, percentage of motile spermatozoa (Z-5) and postthaw acrosomal integrity (4-6). However, all the investigations of acrosomal integrity were based on the morphological changes of acrosomes. Correlations between morphological changes of acrosomes and acrosin or hyaluronidase activity loss from the spermatozoa were not proven (7,8).
Present address:
REI Lab. GYN/OB Dept., Medical Center, Stanford University, Stanford, CA 94305.
Acknowledgments: The auther wishes to acknowledge the support and the help of Prof. Dr. W. Gehring, Ambulatorische und Geburtshilflische Veteringrklinik, Giessen; Dr. F. Miller-Schl~sser, Zentralbesa~ungsstation, Giessen, for suppling the semen;and Dr. K.J. Becker, Institute fur Biochmie und Endokrinologie, Giessen, for technical assistance.
JUNE 1988 VOL. 29 NO. 6
1343
THERIOGENOLOGY
The acrosomal enzymes acrosin and hyaluronidase are necessary for the penetration of ova by spermatozoa (9,lO). Release orloss of the acrosomal enzymes into the medium should provides a sensitive indicator for the acrosomal damage sustained by the spermatozoa during the different processes of semen handling (7,11,12). The investigations of Pace et al. (13) showed higher acrosin activity remaining in the spermatozoa after thawinq at 37'C than after thawing at 10°C or in-ice water: The objective of this study was to determine the effect of rapid thawing on the loss of the acrosomal enzymes,acrosin,and hyaluronidase bovine spermatozoa,.
from
MATERIALS AND METHODS
One ejaculate from each of five mature Schwarzbunt and five.Rotbunt bulls at the Zentralbesamungsstation., 8iessen,.were6diluted in Q-is-egg yolk- glycerin and packed in 0.5-ml straws (30 X IO sperm/ straw) and frozen in liquid nitrogen. Thawingoprocedure was done by total immersion of thg straws in a water bath at 5 C for 50 set, at 37 C for 25 set and at 70 C for 7 sec. Ten straws from each ejaculate were used for every thawing temperature. Samples were centrifuged immediatly after thawing at 600 g for 10 mni. Acrosin Assay The sperm pellet was resuspended twice in salige and centrifuged and extracted with 0.25 ml of 2% (V/V) acetic acid at 4 C. Enzyme activity was determined using hydrolysis of N-benzoyl-L-arginine ethyl ester (BAEE) according to the method q$ Schleuning and Fritz (14). The enzyme activity was calculated in mU/ 10 sperm. Hyaluronidase
Assay
The supernatant was subsequently centrifuged at 3000 g for 20 min and taken for calorimetric measurment of the total hyaluronidase activity using the method described by Linker (15). RESULTS
AND
DISCUSSION
The mean activities from all ejaculates for the acrosin and hyaluronidase enzymes after thawing at different thawing temperatures are presented in-the table below. Tablel.The
mean activities
Thawing Aemperature ( C)
___.
.-
5 37 70 .-_-_-_-
of acrosin and hyalur~nidase Total hyaluronidase activity
-
24.58 10.6 1.48
enzymes after thawing
Acrosin.acajivity in mU in 10 sperm
._.---- .l.l. __.l.-..__.__I__.
Thawing at 37'C resulted in a higher &P ~0.01) posthaw acrosin activity of the spermatozoa than after thawing at 5 C. Furthermore, increased thawing
1344
JUNE 1988 VOL. 29 NO. 6
temperature to 70°C resulted in an increase in the extractable acrosin activity (P
1. Aamadal, J. and Andersen, K. Fast Zuchyhyg. 3:22-24
of
bull semen frozen in straws.
I. Photogrammetrische Untersuchungen nach unterschiedlichen Auftaut~peraturen. (1986).
2. El Danasouri, ersperma Giessen,
thawing
(1968). zur Motilit&t von RfndVet. Med. Diss.
3. Ennen, B-D., Berndtson, W.F., Wortimer, R.G. and Pickett. E.W. Effect of processing procedures on motil?ty of bovine spermatozoa frozen fn 0.25-ml straws. J. Anim. Sci, -43:651-656 (1976). 4, Gilbert, G.R. and Almquist, 3.0. Effect of processing procedures on postthaw acrosomalretention and motility of bovine spermatozoa packaged in 0.3-mf straws at room temperature. J. At-tint. ScS, 46:225-231 (1978). als Fru~htbarkeitparameter 5. Papa, F.D. Motifit'iitund Ak~somenintegr~~~t tiefgefrorenen Bullenspermas in AbhZngigkeit von verschiedenen Auftauverfahren unter berticksichtigung von Kglteschok-, Thermoresistenz und Sephadex-Filtertest. Vet. Med. Diss. Wannover, (1982), 6. De Abreu, R-M., Berndtson, W.E., Smith, R.L. and Pickett, B.W. Postthaw warming on viability of bovine spermatozoa thawed at different rates in French straws. J. Dairy S&i. -62:1449-1454 (1979). bei Bu~lenspe~~~n unter besonderer Beriick7. Wendt, If. Akros~nbest~~ung sichtigung des efnzelnen Spermiums. Vet. Med. Diss. Miinchen, (1974). 8. Molzmann, A., Strahl, H., Jahn, 3. and Bamberg, E. Correlation between spermatological results and hyaluronidase activity in bull semen tuchthyg. u:li!'L-126 (1978). 9. Srivastava, P.M., Adams, C.E. and Hartee, E-F. Enzymatic action of acrosomaf preparation on the rabbit ovum in vitro. J, Reprod. Fertil, -10:61-67 (1965). 10. Stambaugh, R. and Buckley, Y. Zona pellucida dSssolution rabbit sperm head. Science -* 161*585-586 (1968).
enzymes of the
II. Church, K.E. and Graves, C.N. Loss of acrosin from bovine spermatozoa following cold shock:protective effect of seminal plasma. Cryobiology x:341-346 (1976). auf die GOT und 12. Klenner, A. Auswr'rkung der Tie~~efrjerkonservierung Akrosinaktivjtlt sowie die Akrosommorphologie von Eberspermien. Vet, Med. Diss. Mdnchen, (1975)"
JUNE 1988 VOL. 29 NO. 6
1345
TI-IERIOGENOLOGY
Elliott, F.I., Graham, E.F. and Coulter, G.H. 13. Pace, M.M., Sulivan, J.J., Effect of thawing temperature, number of spermatozoa and spermatozoa1 quality on fertility of bovine spermatozoa packaged in 0.5 ml French straws. J. Anim. Sci. -53:693-701 (1981). 14. Scleuning, W.D. and Fritz, H. Sperm acrosin. In: Colowjck, S.P. and N.O. Kaplan (eds). Methods in Enzymology. Vol. XLVTcademic Press, New York, 1974, pp. 330-342.j 15. Linker, A. Hyaluronidase. In: Bergmeyer, H.U. (ed.!.Methods of Enzymatic New York, Academic Press, 1974, PP. 944 Analysis. Weinheim Verlag xemie, 948.
1346
JUNE 1988 VOL. 29 NO. 6
OF
THAWING
TEMPERATURE
HYALURONIDASE
ENZYMES
Imam ~bulatorische
El
ON
THE
FROM
LOSS
BOVINE
OF
ACROSIN
SPERMATOZOA
Danasouri
und Geburtshilflische
Veterin~rklinik
Justus-Liebig-Universit~t 6300 Giessen,
FRG
Received for publication: &me 29, Accepted:
Apti
1987
7, 1988
ABSTRACT Bovine semen frozen in 0.5ml straws was used to determine the loss of the acrosamal enzymes acre in and hyaluronidase (from spermatozoa after thawing at 5 C for 50 set, 37 Q for 15 set and at 70 C for 7 sec. The highest acrosin activity remaining in the spermatozoa and the loweat hyaluronidase activity in the media were observed after thawing at 70 C (P<8.01). Indications of high acrosomal damage were observed after thawing at 5 C (PiO.01). INTRODUCTION Rapid thawing rates of semen packaged in plastic straws hqve been shown to increase significantly the percentage of unstained(live} spermatozoa (If, percentage of motile spermatozoa (Z-5) and postthaw acrosomal integrity (4-6). However, all the investigations of acrosomal integrity were based on the morphological changes of acrosomes. Correlations between morphological changes of acrosomes and acrosin or hyaluronidase activity loss from the spermatozoa were not proven (7,8).
Present address:
REI Lab. GYN/OB Dept., Medical Center, Stanford University, Stanford, CA 94305.
Acknowledgments: The auther wishes to acknowledge the support and the help of Prof. Dr. W. Gehring, Ambulatorische und Geburtshilflische Veteringrklinik, Giessen; Dr. F. Miller-Schl~sser, Zentralbesa~ungsstation, Giessen, for suppling the semen;and Dr. K.J. Becker, Institute fur Biochmie und Endokrinologie, Giessen, for technical assistance.
JUNE 1988 VOL. 29 NO. 6
1343
THERIOGENOLOGY
The acrosomal enzymes acrosin and hyaluronidase are necessary for the penetration of ova by spermatozoa (9,lO). Release orloss of the acrosomal enzymes into the medium should provides a sensitive indicator for the acrosomal damage sustained by the spermatozoa during the different processes of semen handling (7,11,12). The investigations of Pace et al. (13) showed higher acrosin activity remaining in the spermatozoa after thawinq at 37'C than after thawing at 10°C or in-ice water: The objective of this study was to determine the effect of rapid thawing on the loss of the acrosomal enzymes,acrosin,and hyaluronidase bovine spermatozoa,.
from
MATERIALS AND METHODS
One ejaculate from each of five mature Schwarzbunt and five.Rotbunt bulls at the Zentralbesamungsstation., 8iessen,.were6diluted in Q-is-egg yolk- glycerin and packed in 0.5-ml straws (30 X IO sperm/ straw) and frozen in liquid nitrogen. Thawingoprocedure was done by total immersion of thg straws in a water bath at 5 C for 50 set, at 37 C for 25 set and at 70 C for 7 sec. Ten straws from each ejaculate were used for every thawing temperature. Samples were centrifuged immediatly after thawing at 600 g for 10 mni. Acrosin Assay The sperm pellet was resuspended twice in salige and centrifuged and extracted with 0.25 ml of 2% (V/V) acetic acid at 4 C. Enzyme activity was determined using hydrolysis of N-benzoyl-L-arginine ethyl ester (BAEE) according to the method q$ Schleuning and Fritz (14). The enzyme activity was calculated in mU/ 10 sperm. Hyaluronidase
Assay
The supernatant was subsequently centrifuged at 3000 g for 20 min and taken for calorimetric measurment of the total hyaluronidase activity using the method described by Linker (15). RESULTS
AND
DISCUSSION
The mean activities from all ejaculates for the acrosin and hyaluronidase enzymes after thawing at different thawing temperatures are presented in-the table below. Tablel.The
mean activities
Thawing Aemperature ( C)
___.
.-
5 37 70 .-_-_-_-
of acrosin and hyalur~nidase Total hyaluronidase activity
-
24.58 10.6 1.48
enzymes after thawing
Acrosin.acajivity in mU in 10 sperm
._.---- .l.l. __.l.-..__.__I__.
Thawing at 37'C resulted in a higher &P ~0.01) posthaw acrosin activity of the spermatozoa than after thawing at 5 C. Furthermore, increased thawing
1344
JUNE 1988 VOL. 29 NO. 6
temperature to 70°C resulted in an increase in the extractable acrosin activity (P
1. Aamadal, J. and Andersen, K. Fast Zuchyhyg. 3:22-24
of
bull semen frozen in straws.
I. Photogrammetrische Untersuchungen nach unterschiedlichen Auftaut~peraturen. (1986).
2. El Danasouri, ersperma Giessen,
thawing
(1968). zur Motilit&t von RfndVet. Med. Diss.
3. Ennen, B-D., Berndtson, W.F., Wortimer, R.G. and Pickett. E.W. Effect of processing procedures on motil?ty of bovine spermatozoa frozen fn 0.25-ml straws. J. Anim. Sci, -43:651-656 (1976). 4, Gilbert, G.R. and Almquist, 3.0. Effect of processing procedures on postthaw acrosomalretention and motility of bovine spermatozoa packaged in 0.3-mf straws at room temperature. J. At-tint. ScS, 46:225-231 (1978). als Fru~htbarkeitparameter 5. Papa, F.D. Motifit'iitund Ak~somenintegr~~~t tiefgefrorenen Bullenspermas in AbhZngigkeit von verschiedenen Auftauverfahren unter berticksichtigung von Kglteschok-, Thermoresistenz und Sephadex-Filtertest. Vet. Med. Diss. Wannover, (1982), 6. De Abreu, R-M., Berndtson, W.E., Smith, R.L. and Pickett, B.W. Postthaw warming on viability of bovine spermatozoa thawed at different rates in French straws. J. Dairy S&i. -62:1449-1454 (1979). bei Bu~lenspe~~~n unter besonderer Beriick7. Wendt, If. Akros~nbest~~ung sichtigung des efnzelnen Spermiums. Vet. Med. Diss. Miinchen, (1974). 8. Molzmann, A., Strahl, H., Jahn, 3. and Bamberg, E. Correlation between spermatological results and hyaluronidase activity in bull semen tuchthyg. u:li!'L-126 (1978). 9. Srivastava, P.M., Adams, C.E. and Hartee, E-F. Enzymatic action of acrosomaf preparation on the rabbit ovum in vitro. J, Reprod. Fertil, -10:61-67 (1965). 10. Stambaugh, R. and Buckley, Y. Zona pellucida dSssolution rabbit sperm head. Science -* 161*585-586 (1968).
enzymes of the
II. Church, K.E. and Graves, C.N. Loss of acrosin from bovine spermatozoa following cold shock:protective effect of seminal plasma. Cryobiology x:341-346 (1976). auf die GOT und 12. Klenner, A. Auswr'rkung der Tie~~efrjerkonservierung Akrosinaktivjtlt sowie die Akrosommorphologie von Eberspermien. Vet, Med. Diss. Mdnchen, (1975)"
JUNE 1988 VOL. 29 NO. 6
1345
TI-IERIOGENOLOGY
Elliott, F.I., Graham, E.F. and Coulter, G.H. 13. Pace, M.M., Sulivan, J.J., Effect of thawing temperature, number of spermatozoa and spermatozoa1 quality on fertility of bovine spermatozoa packaged in 0.5 ml French straws. J. Anim. Sci. -53:693-701 (1981). 14. Scleuning, W.D. and Fritz, H. Sperm acrosin. In: Colowjck, S.P. and N.O. Kaplan (eds). Methods in Enzymology. Vol. XLVTcademic Press, New York, 1974, pp. 330-342.j 15. Linker, A. Hyaluronidase. In: Bergmeyer, H.U. (ed.!.Methods of Enzymatic New York, Academic Press, 1974, PP. 944 Analysis. Weinheim Verlag xemie, 948.
1346
JUNE 1988 VOL. 29 NO. 6