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Comparative in vitro study of nonoxynol-9: Effects on human, bull and boar spermatozoa
Contraception
45:229-237,
COMPARATIVE
EFFECTS
Arambagi&
1992
IN
VITRO
ON HUMAN,
B. Milan’);
STUDY
BULL
Subotin
OF
AND BOAR
NONOXYNOL-9: BPERMATOSOA
M. Lazar2);
Quality Beograd,
Stani
1)
"ICN-GALENIKA-Institute*@, Biology Laboratory, 11.000 Yugoslavia
2)
Reproduction and Embryotechnology l'Agroekonomik-PKB1*, 11.213 Padinska Skela,
3)
Institute of Biology and Medicine, University of Visegradska 26., Yugoslavia
Mirsanda3)
Control Center, 29. Novembra ill., Center, Yugoslavia
Human Genetics, Belgrade, 11.000
School of Beograd,
Abstract
Spermicidal activity of different dilutions of nonoxynol-9 (nonyl-phenoxy-polyethoxy-ethanol) on human, bull and boar spermatozoa was studied using the modified Sander-Cramer method. The results obtained showed that differences in spermatozoa1 resistance among the mentioned species to nonoxynol-9 were statistically very significant (P << 0.0005): bull > man > boar. The results obtained were compared to available literature results.
Address for correspondence: Milan B. Aramba%iE "ICN-GALENIKA-Institute" Quality Control Center Biological Laboratory YU-11.000 Beograd 29. Novembra ill., Yugoslavia
Submitted for publication Accepted for publication
Copyright
June 11, 1991 January 28, 1992
63 1992 Butterworth-Heinemann
Contraception
230 Introduction
(nonyl-phenoxy-polyethoxy-ethanol, Nonoxynol-9 C33H60is an anhydrous liquid mixture of mononyl0101 Mw=616.8), with ethers of polyethylene glycols strong phenyl As such, nonoxynol-9 is an active spermicidal activity. concentrations (2-12.5%) ingredient contained in different contraceptives (local anticonceptives) in the in vaginal or film (Delfen Foam, form of foam, cream, gel, pessary Delfen Cream, Emko Foam, Orthocreme, Gynol II, Orthoforms, The mechanism of nonoxynol-9 C-Film, Double-Check [1,2]). on damaging the membrane structure of activity is based which results in lesions at the acrosome and spermatozoa, There are also some changes in equatorial segment levels. chromatin and mitochondria [3-51. The nuclear membrane, irreversible these changes is a rapid consequence of devitalization of spermatozoa (within a few seconds), which finally leads to their immobilization. The study of the effect of nonoxynol-9 on spermatozoa and from man obtained both from different animal species showed that the level of resistance to nonoxynol-9 was To be precise, species-related. the most resistant were rabbit spermatozoa (spermicidal concentration 1 mg/ml), and human resistant were spermatozoa (spermicidal the least concentration 0.125 mg/ml) [3]. The purpose of this paper was to compare spermicidal effects of nonoxynol-9 on human and animal (bull and boar) the most order to: a) choose suitable spermatozoa in and b) work out a potential method for biological material, routine control of both native and frozen sperm of human or animal origin. Material
and Methods
The spermicidal effect of nonoxynol-9 solution on the bulls (Simmental and Holstein spermatozoa of man, breeding and breeding boar (Landrace strain) was studied strains) Sander-Cramer method 161, which is using the modified lo-second agitation of a mixture of 0.2 ml of sperm gentle, and 1 ml of a nonoxynol-9 dilution in physiological saline water, A drop of the in redistilled (0.9% NaCl w/v)mixture is then placed on the microscope slide, covered, and observed within 10 seconds under magnification, 400X; total time for the test is 20 seconds. The dilution is increased By motility, to the level when spermatozoa remain motile. movements of the spermatozoa or we mean, either progressive The previous highest dilution which tail movements only. immobilized all spermatozoa is taken as the end-point of the The mixture of 0.2 ml of sperm of a certain origin study. and observed in and 1 ml of physiological saline, prepared the same manner, serves as the control.
231
Contraception
Human sperm was obtained from volunteering donors who abstained sexually for 4-5 days, that of bull was obtained using artificial vagina, and boar sperm was collected using The donors as well as the breeding bull manual stimulation. and boar were clinically healthy, and spermicidal activity of various nonoxynol-9 dilutions was tested on 24 bull, 12 human and 8 boar sperm samples. The sperm samples were incubated at 37OC, and the study was carried out immediately or within one hour of collecting the sperm, at the latest. Prior to the test, ejaculum characteristics were determined: volume, concentration, percentage of motile spermatozoa1 spermatozoa and pH. Different nonoxynol-9 dilutions were made from the stock solution (8% nonoxynol-9 in redistilled water, w/v) by adding physiological saline. The concentration of the stock solution (8% nonoxynol-9) was chosen because that is the concentration of the substance in Emko foam. The pH values of various nonoxynol-9 dilutions (l:lOO, 1:200, etc.) is 6.30 and osmotic pressure is in the range of 308.152 (1:900 mOsmol/l dilution) to 309.305 mOsmol/l (1:100 and dilution). The pH osmotic pressure values for physiological saline are 6.30 and 308.008 mosmol/l, respectively. Statistical analysis: the comparison of the mean values for the highest dilution that immobilize all spermatozoa and those for the lowest dilutions at which the spermatozoa are motile was done using one-way analysis of variance according to Plohinskij algorithm [7] and Scheffee's method [8]. Results Table I presents the mean values + S.D. (standard deviations) for characteristic ejaculum parameters for 3 mammal species: man, bull and boar, the mean values + S.D. for the highest nonoxynol-9 dilutions that immobilize all spermatozoa of a species (A) and the mean values + S.D. for the lowest nonoxynol-9 dilutions at which spermatozoa of a certain species are motile (B). Variation coefficient values are given for each presented data (C.V.) (%). Based on the mean values for the highest nonoxynol-9 dilutions that immobilize all spermatozoa of a certain species, most resistant is the the sperm of bull (1:137.50+64.69 ml), the next in the resistance scale is human sperm (1:541.67+122.17 ml), while the least resistant is the sperm of boar (1:912.50+145.77 ml). The comparison of the mean values for these dilutions showed that the differences in the resistance to nonoxynol-9 between bull and human sperm (P << 0.0005; F exp. = 130.963 >> FO 0005 (degrees of freedom (d.f.) 2; 41) z 9.25); bull and-boar sperm (P << 0.0005; F exp. = 361.147 >> F0.0005 (d.f.) 2; 41) Z 9.25); human and boar sperm (P << 0.0005; F exp. =
18.11
10.84
1:577.27+235.97 1:966.67+103.28 1:200.00+51.08 40.88 10.68 25.54 C.V. (%) ________-______---__~~~~~~~~~~~~~~~~~~~~~~~~~ ____________________~-~~~~~~~-~~~__~~~__ 8 12 24 Number of samples _________________-__~~~~~~~~~~~~~~~~~~~~~~_~~~~~~---~___-~~~~__-~~~_--_~~~_~~~_______
1:137.50+64.69 1:541.67+122.17 1:912.50+145.77 15.98 C.V. (%) 47.05 22.55 ________-________-__-~~~~~~~~~~~~ _______--_____--____~~~~~~--~~~~~~~~~~~~~~~~~~~~~~~~ (B) THE LOWEST NONOXYNOL-9 DILUTION AT WHICH SPERMATOZOA ABE MOTILE
7.32kO.16 7.10+0.11 6.71+0.06 PH 0.89 2.19 1.55 C.V. (%) _______________________^________________~~~~~~~~~~~~~~~~~~__~~~~~~~~~~__~~~~~~~~~~~_~ (A) THE HIGHEST NONOXYNOL-9 DILUTION THAT IMMOBILIZES ALL SPERMATOZA
6.08
78.75k4.79
72.00+13.04
83.9329.10
% of motile spermatozoa C.V. (%)
170.00+13.54 7.98
92.5Ok24.50 26.49
1123.482176.06 15.67
;o;c.($O9/1)
--__________________~--~~~~~~~~~~__~~~~~~~~~~_~~~~~~~-~~~~----~~~~~~~~___~~~~~-~~~~_~ MAN BOAR BULL ________-_____----__~~~~-~~~~~~~~~~~~-~~~-~~~~~~~~~~-~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 3.6721.60 190.00+56.57 6.59k2.22 . . I? 33.69 43.60 29.77 p:*
Comparative presentation of average ejaculum characteristic values + S.D. for human, boar and bull sperm, average values + S.D. for the highest nonoxynol-9 dilution that immobilizes all spermatozoa (A) and average values + S.D. for the lowest nonoxynol-9 dilution at which spermatozoa1 motility is observed (B); variation coefficient values are given for each presented data (C.V.) (%)
Table I.
233
Contraception
66.149 >> FO.0005 very significant.
(d.f.) 2; 41) = 9.25) were
statistically
Similar results were obtained by the comparison of the lowest nonoxynol-9 dilutions at which spermatozoa1 motility human: was (bull: 1:200.00+51.08 ml; observed ml). The 1:577.27+235.97 ml; and boar: 1:966.67+103.28 differences in the resistance to nonoxynol-9 between bull and human sperm (P << 0.0005; F exp. = 60.877 >> FO.0005 (d.f.) 2; 41) = 9.25); bull and boar sperm (P << 0.0005; F exp. = 33.381 >> FO,OOO5 (d.f.) 2; 41) Z 9.25) were also statistically very significant. Discussion The results obtained show that spermatozoa1 resistance to nonoxynol-9 depends on the origin of spermatozoa. The most resistant are bull spermatozoa, then human, and the The differences between least resistant are those of boar. are very the species statistically significant (P << 0.0005). The highest nonoxynol-9 dilutions that immobilize all spermatozoa of a given species have the following concentration (in that is, as %): bull mg/ml), man 1:137.50+64.69 ml = 0.582kO.352 mg/ml = 0.00582%; 1:541.67+122.17 ml = 0.148+ 0.035 mg/ml = 0.0148%; and boar: 1:912.5+145.77 ml = 0.088+0.014 mg/ml = 0.0088%. The value for nonoxynol-9 spermicidal concentration human for spermatozoa (0.148+0.035 mg/ml) is close to that obtained by Diao et al. [3] (0.125 mg/ml = O-0125%), and that obtained by Chijioke et al. [9] (0.120 mg/ml = 0.0120%). These concentration values are much lower than that which, in Schill and Wolff's [5] experiments, caused irreversible changes in human spermatozoa (0.05% or 0.5 mg/ml). The lowest nonoxynol-9 dilutions at which spermatozoa1 is observed motility of a species have the following values (in mglml, that is, %): concentration bull: 1:200.00+51.08 ml = 0.400+0.109 mg/ml = 0.0400%; man: 1:577.27+235.97 ml = 0.139+0.068 mg/ml = 0.0139%; and boar: 1:966.67+103.28 ml = 0.083+0.0099 mg/ml = 0.0083%. The results obtained are representative since the ejaculum values for average volumes, spermatozoa1 concentrations and percentage of motile spermatozoa were within the corresponding average value limits found in the However, the average human and bull ejaculum literature. values for this study were somewhat higher than those found in the literature [lO,ll], while the ejaculum values for boar were somewhat lower than those in the literature [12]. The variation coefficients (C.V.) seen in Table I show that the result validity and the size of the difference between experimental and tabular values for Fischer's coefficient (F) points to the significance of the differences in the
234
spermatozoa1 species.
Contraception
resistance
to
nonoxynol-9
among
various
Based on both literature and our data on nonoxynol-9 spermicidal concentrations, we can state that spermatozoa1 resistance to nonoxynol-9 declines in the following order: rabbit (1.0 mg/ml [3]) > bull (0.582kO.352 mg/ml) > dog (0.25 mg/ml [3]) > man (0.148+0.035; or 0.125 mg/ml [3]; or 0.120 mg/ml [9]) > monkey (0.125 mg/ml [3]) > boar It should be noted that spermatozoa1 (0.088+0.014 mg/ml). resistance of monkey is similar to that of man. For the sake of showing the differences in spermatozoa1 resistance to nonoxynol-9 among rabbit, bull, dog, man and boar, we have tried to compare normal values for ejaculate of spermatozoa and osmotic characteristics: pH, number pressure, which are comparatively presented in Table II. The quoted pH values of the ejaculates and the number of spermatozoa in rabbit and dog, as well as osmotic pressure values for all 5 species, are taken from the literature [11,13]. The values presented in Table II show fairly wide ejaculate osmotic tolerance of the various species (290-392 which correlates with the fact that mammal mOsmol/l), are subjected to different osmotic pressures spermatozoa while passing through the female genital tract [14] and, therefore, osmotic tolerance is necessary. Osmotic pressure values for 0.9% NaCl solution and various nonoxynol-9 dilutions range from 308.008 and 309.305 mOsmol/l, which is within the range of the quoted literature data. Thus, the spermatozoa1 resistance differences of the mentioned species to nonoxynol-9 could not be explained by different osmotic resistance. A similar case is seen for ejaculate pH values. The optimal pH value for mammalian spermatozoa is about neutral observed in [I31 I with a tolerance which is particularly The pH value for 0.9% Nacl solution and various rabbits. nonoxynol-9 dilutions is 6.30, i.e., somewhat lower than minimal normal values presented in Table II. However, Table II shows that there is some tendency towards decrease of spermatozoa1 resistance of certain species to nonoxynol-9 with increase of ejaculate pH value for the same species. is quite different with spermatozoa1 The situation Table II shows that there concentration in the ejaculate. is a drastic difference in the average number of spermatozoa in the ejaculate of the mentioned species, which is, except boar, directly dependent on spermatozoa1 resistance of these species to nonoxynol-9. Based on this, it is possible to assume that the spermatozoa1 resistance of certain species probably on nonoxynol-9 depends the spermatozoa1 to concentration in the ejaculate of that species - the higher
6.67 - 6.76 [13] 7.10 - 7.50
DOG
MAN
92.5
300 [ll]
1123
296 - 312 [13]
312 - 323 [13]
290 - 392 [13]
290 - 339 [13] 170 7.00 - 7.20 BOAR ____________________~~~~-~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~-~-~~~~~~~-~~~~~-~-~--
6.60 - 6.80
BULL
____________________~~~~~~~~~~~~~~~~~~~~-~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ Concentration Source of Osmotic pressure of spermatozoa ejaculates pH values of of ejaculates ejaculates (mean values) (in order of resistance) (mOsmol/l) (X109/1) -----------------------~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 296 - 317 [13] 200000 [ll] 6.59 - 8.86 [13] RABBIT
Comparative presentation of normal pH and osmotic pressure values of the ejaculate, as well as of mean spermatozoa1 concentration values for rabbit, bull, dog, man and boar ejaculates; in the quoted paper [13], osmotic pressure values of the ejaculates were presented as freezing-point depression (OC), so conversion to mOsmol/l was made
Table II.
236
Contraception
the spermatozoa1 concentration in the ejaculate, the higher the resistance to nonoxynol-9. Based on the differences in concentrations in spermatozoa1 the ejaculate of the the spermatozoa1 resistance differences mentioned species, of those species to nonoxynol-9 could probably be explained. The results obtained in this paper as well as quoted based on the average ejaculum literature data show that, resistance and availability, bull sperm volume, nonoxynol-9 is the most suitable biological material to be used for biological control of contraceptives. Acknowledgement authors would like to thank Prof.Dr. Milan The Jevremovie and Prof.Dr. Vasilije MiljkoviG for reading the manuscript and making valuable comments on it, as well as Zlatica MilutinoviG for translating the paper into Mrs. English. References 1.
Non-prescription Bounds W. 1986;236:521-23.
contraceptives.
2.
The Extra Pharmacopoeia. 29th Martindale, Press, JEF, ed. London: The Pharmaceutical
3.
Diao XH, Chen Q, Wailer DP, Kaminski J, Zaneveld UD. spermicidal Comparison of the activity and acute toxicity of nonoxynol-9 and agent 741 [alkylphenoxy polyethoxy ethanol (lo)]. Contraception 1986;33:1-5.
4.
JJ. Wilborn WN, Hahn DW, McGuire Scanning electron microscopy of human spermatozoa after incubation with the spermicide nonoxynol-9. Fertil Steril 1983;39:71719.
5.
Schill WB, spermatozoa nonoxynol-9 nonoxynol-9.
6.
Sander FV, Cramer SD. the spermicidal action Fertil 1941;6:134-37.
7.
Plohinskij Moskovskogo
8.
Basic statistical nonPetz B. methods for SveuEili.&na naklada "Liber", Zagreb: mathematicians. [Serbocroatian]. 1981;299.
Wolff HH. Ultrastructure the presence of the in and a vaginal contraceptive Andrologia 1981;13:42-49.
Pharm
J
ed. Reynolds 1989;1245.
of human spermicide containing
A practical method for testing of chemical contraceptives. Hum
Algoritmy biometrii. NA. Un-ta, 1967;48-49.
Moskva:
Izd-vo
237
Contraception 9.
Comparison of the Chijioke PC, Pearson RM, Zaman S. chlorhexidine and nonoxynol-9 potency of D-propranolol, Contraception 1986;34:207in the Sander-Cramer test. 11.
10.
Jondet M, Millet D. Die heterologe (donogene) Besamung Fachtagung fur Fortpflanzung bei Menschen. 16. Inter. Austria. In: Miljkovib. F. und Besamung, Wels, animals. Beograd: Artificial fertilization of Privredno-finansijski vodig, 1979;266 [Serbocroatian].
11.
Paufler SK, Bader H, Bonfert A, Foot RM, Salamon S, Kunstliche Besamung und Vaserling HF. Eitransplantation bei Tier und Mensch. Hanover, Verlag Mil jkovi6 F. Artificial Schaper MH, 1974. In: Beograd: Privrednofertilization of animals. finansijski vodib, 1979;9 [Serbocroatian].
12.
Lipu J. Bogdan AT, Bogdan I, Probleme der Fortpflanzungbiologie und ker Kiinstliche Besamung bei Schweine-produktion. 7-te Industriellbetribener Internationale Kongress fiir Fortpflanzung und Besamung, Munchen, 1972, Buch 2, s. 1250.
13.
Mann T. The biochemistry reproductive tract. London:
14.
The effects of temperature, Mahi CA, Yanagimachi R. ion osmolarity and concentration on the hydrogen reaction of golden activation and acrosome hamster spermatozoa. J Reprod Fert 1973;35:55-66.
of semen and of the Methuen, 1964;18,86,88.
male
45:229-237,
COMPARATIVE
EFFECTS
Arambagi&
1992
IN
VITRO
ON HUMAN,
B. Milan’);
STUDY
BULL
Subotin
OF
AND BOAR
NONOXYNOL-9: BPERMATOSOA
M. Lazar2);
Quality Beograd,
Stani
1)
"ICN-GALENIKA-Institute*@, Biology Laboratory, 11.000 Yugoslavia
2)
Reproduction and Embryotechnology l'Agroekonomik-PKB1*, 11.213 Padinska Skela,
3)
Institute of Biology and Medicine, University of Visegradska 26., Yugoslavia
Mirsanda3)
Control Center, 29. Novembra ill., Center, Yugoslavia
Human Genetics, Belgrade, 11.000
School of Beograd,
Abstract
Spermicidal activity of different dilutions of nonoxynol-9 (nonyl-phenoxy-polyethoxy-ethanol) on human, bull and boar spermatozoa was studied using the modified Sander-Cramer method. The results obtained showed that differences in spermatozoa1 resistance among the mentioned species to nonoxynol-9 were statistically very significant (P << 0.0005): bull > man > boar. The results obtained were compared to available literature results.
Address for correspondence: Milan B. Aramba%iE "ICN-GALENIKA-Institute" Quality Control Center Biological Laboratory YU-11.000 Beograd 29. Novembra ill., Yugoslavia
Submitted for publication Accepted for publication
Copyright
June 11, 1991 January 28, 1992
63 1992 Butterworth-Heinemann
Contraception
230 Introduction
(nonyl-phenoxy-polyethoxy-ethanol, Nonoxynol-9 C33H60is an anhydrous liquid mixture of mononyl0101 Mw=616.8), with ethers of polyethylene glycols strong phenyl As such, nonoxynol-9 is an active spermicidal activity. concentrations (2-12.5%) ingredient contained in different contraceptives (local anticonceptives) in the in vaginal or film (Delfen Foam, form of foam, cream, gel, pessary Delfen Cream, Emko Foam, Orthocreme, Gynol II, Orthoforms, The mechanism of nonoxynol-9 C-Film, Double-Check [1,2]). on damaging the membrane structure of activity is based which results in lesions at the acrosome and spermatozoa, There are also some changes in equatorial segment levels. chromatin and mitochondria [3-51. The nuclear membrane, irreversible these changes is a rapid consequence of devitalization of spermatozoa (within a few seconds), which finally leads to their immobilization. The study of the effect of nonoxynol-9 on spermatozoa and from man obtained both from different animal species showed that the level of resistance to nonoxynol-9 was To be precise, species-related. the most resistant were rabbit spermatozoa (spermicidal concentration 1 mg/ml), and human resistant were spermatozoa (spermicidal the least concentration 0.125 mg/ml) [3]. The purpose of this paper was to compare spermicidal effects of nonoxynol-9 on human and animal (bull and boar) the most order to: a) choose suitable spermatozoa in and b) work out a potential method for biological material, routine control of both native and frozen sperm of human or animal origin. Material
and Methods
The spermicidal effect of nonoxynol-9 solution on the bulls (Simmental and Holstein spermatozoa of man, breeding and breeding boar (Landrace strain) was studied strains) Sander-Cramer method 161, which is using the modified lo-second agitation of a mixture of 0.2 ml of sperm gentle, and 1 ml of a nonoxynol-9 dilution in physiological saline water, A drop of the in redistilled (0.9% NaCl w/v)mixture is then placed on the microscope slide, covered, and observed within 10 seconds under magnification, 400X; total time for the test is 20 seconds. The dilution is increased By motility, to the level when spermatozoa remain motile. movements of the spermatozoa or we mean, either progressive The previous highest dilution which tail movements only. immobilized all spermatozoa is taken as the end-point of the The mixture of 0.2 ml of sperm of a certain origin study. and observed in and 1 ml of physiological saline, prepared the same manner, serves as the control.
231
Contraception
Human sperm was obtained from volunteering donors who abstained sexually for 4-5 days, that of bull was obtained using artificial vagina, and boar sperm was collected using The donors as well as the breeding bull manual stimulation. and boar were clinically healthy, and spermicidal activity of various nonoxynol-9 dilutions was tested on 24 bull, 12 human and 8 boar sperm samples. The sperm samples were incubated at 37OC, and the study was carried out immediately or within one hour of collecting the sperm, at the latest. Prior to the test, ejaculum characteristics were determined: volume, concentration, percentage of motile spermatozoa1 spermatozoa and pH. Different nonoxynol-9 dilutions were made from the stock solution (8% nonoxynol-9 in redistilled water, w/v) by adding physiological saline. The concentration of the stock solution (8% nonoxynol-9) was chosen because that is the concentration of the substance in Emko foam. The pH values of various nonoxynol-9 dilutions (l:lOO, 1:200, etc.) is 6.30 and osmotic pressure is in the range of 308.152 (1:900 mOsmol/l dilution) to 309.305 mOsmol/l (1:100 and dilution). The pH osmotic pressure values for physiological saline are 6.30 and 308.008 mosmol/l, respectively. Statistical analysis: the comparison of the mean values for the highest dilution that immobilize all spermatozoa and those for the lowest dilutions at which the spermatozoa are motile was done using one-way analysis of variance according to Plohinskij algorithm [7] and Scheffee's method [8]. Results Table I presents the mean values + S.D. (standard deviations) for characteristic ejaculum parameters for 3 mammal species: man, bull and boar, the mean values + S.D. for the highest nonoxynol-9 dilutions that immobilize all spermatozoa of a species (A) and the mean values + S.D. for the lowest nonoxynol-9 dilutions at which spermatozoa of a certain species are motile (B). Variation coefficient values are given for each presented data (C.V.) (%). Based on the mean values for the highest nonoxynol-9 dilutions that immobilize all spermatozoa of a certain species, most resistant is the the sperm of bull (1:137.50+64.69 ml), the next in the resistance scale is human sperm (1:541.67+122.17 ml), while the least resistant is the sperm of boar (1:912.50+145.77 ml). The comparison of the mean values for these dilutions showed that the differences in the resistance to nonoxynol-9 between bull and human sperm (P << 0.0005; F exp. = 130.963 >> FO 0005 (degrees of freedom (d.f.) 2; 41) z 9.25); bull and-boar sperm (P << 0.0005; F exp. = 361.147 >> F0.0005 (d.f.) 2; 41) Z 9.25); human and boar sperm (P << 0.0005; F exp. =
18.11
10.84
1:577.27+235.97 1:966.67+103.28 1:200.00+51.08 40.88 10.68 25.54 C.V. (%) ________-______---__~~~~~~~~~~~~~~~~~~~~~~~~~ ____________________~-~~~~~~~-~~~__~~~__ 8 12 24 Number of samples _________________-__~~~~~~~~~~~~~~~~~~~~~~_~~~~~~---~___-~~~~__-~~~_--_~~~_~~~_______
1:137.50+64.69 1:541.67+122.17 1:912.50+145.77 15.98 C.V. (%) 47.05 22.55 ________-________-__-~~~~~~~~~~~~ _______--_____--____~~~~~~--~~~~~~~~~~~~~~~~~~~~~~~~ (B) THE LOWEST NONOXYNOL-9 DILUTION AT WHICH SPERMATOZOA ABE MOTILE
7.32kO.16 7.10+0.11 6.71+0.06 PH 0.89 2.19 1.55 C.V. (%) _______________________^________________~~~~~~~~~~~~~~~~~~__~~~~~~~~~~__~~~~~~~~~~~_~ (A) THE HIGHEST NONOXYNOL-9 DILUTION THAT IMMOBILIZES ALL SPERMATOZA
6.08
78.75k4.79
72.00+13.04
83.9329.10
% of motile spermatozoa C.V. (%)
170.00+13.54 7.98
92.5Ok24.50 26.49
1123.482176.06 15.67
;o;c.($O9/1)
--__________________~--~~~~~~~~~~__~~~~~~~~~~_~~~~~~~-~~~~----~~~~~~~~___~~~~~-~~~~_~ MAN BOAR BULL ________-_____----__~~~~-~~~~~~~~~~~~-~~~-~~~~~~~~~~-~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 3.6721.60 190.00+56.57 6.59k2.22 . . I? 33.69 43.60 29.77 p:*
Comparative presentation of average ejaculum characteristic values + S.D. for human, boar and bull sperm, average values + S.D. for the highest nonoxynol-9 dilution that immobilizes all spermatozoa (A) and average values + S.D. for the lowest nonoxynol-9 dilution at which spermatozoa1 motility is observed (B); variation coefficient values are given for each presented data (C.V.) (%)
Table I.
233
Contraception
66.149 >> FO.0005 very significant.
(d.f.) 2; 41) = 9.25) were
statistically
Similar results were obtained by the comparison of the lowest nonoxynol-9 dilutions at which spermatozoa1 motility human: was (bull: 1:200.00+51.08 ml; observed ml). The 1:577.27+235.97 ml; and boar: 1:966.67+103.28 differences in the resistance to nonoxynol-9 between bull and human sperm (P << 0.0005; F exp. = 60.877 >> FO.0005 (d.f.) 2; 41) = 9.25); bull and boar sperm (P << 0.0005; F exp. = 33.381 >> FO,OOO5 (d.f.) 2; 41) Z 9.25) were also statistically very significant. Discussion The results obtained show that spermatozoa1 resistance to nonoxynol-9 depends on the origin of spermatozoa. The most resistant are bull spermatozoa, then human, and the The differences between least resistant are those of boar. are very the species statistically significant (P << 0.0005). The highest nonoxynol-9 dilutions that immobilize all spermatozoa of a given species have the following concentration (in that is, as %): bull mg/ml), man 1:137.50+64.69 ml = 0.582kO.352 mg/ml = 0.00582%; 1:541.67+122.17 ml = 0.148+ 0.035 mg/ml = 0.0148%; and boar: 1:912.5+145.77 ml = 0.088+0.014 mg/ml = 0.0088%. The value for nonoxynol-9 spermicidal concentration human for spermatozoa (0.148+0.035 mg/ml) is close to that obtained by Diao et al. [3] (0.125 mg/ml = O-0125%), and that obtained by Chijioke et al. [9] (0.120 mg/ml = 0.0120%). These concentration values are much lower than that which, in Schill and Wolff's [5] experiments, caused irreversible changes in human spermatozoa (0.05% or 0.5 mg/ml). The lowest nonoxynol-9 dilutions at which spermatozoa1 is observed motility of a species have the following values (in mglml, that is, %): concentration bull: 1:200.00+51.08 ml = 0.400+0.109 mg/ml = 0.0400%; man: 1:577.27+235.97 ml = 0.139+0.068 mg/ml = 0.0139%; and boar: 1:966.67+103.28 ml = 0.083+0.0099 mg/ml = 0.0083%. The results obtained are representative since the ejaculum values for average volumes, spermatozoa1 concentrations and percentage of motile spermatozoa were within the corresponding average value limits found in the However, the average human and bull ejaculum literature. values for this study were somewhat higher than those found in the literature [lO,ll], while the ejaculum values for boar were somewhat lower than those in the literature [12]. The variation coefficients (C.V.) seen in Table I show that the result validity and the size of the difference between experimental and tabular values for Fischer's coefficient (F) points to the significance of the differences in the
234
spermatozoa1 species.
Contraception
resistance
to
nonoxynol-9
among
various
Based on both literature and our data on nonoxynol-9 spermicidal concentrations, we can state that spermatozoa1 resistance to nonoxynol-9 declines in the following order: rabbit (1.0 mg/ml [3]) > bull (0.582kO.352 mg/ml) > dog (0.25 mg/ml [3]) > man (0.148+0.035; or 0.125 mg/ml [3]; or 0.120 mg/ml [9]) > monkey (0.125 mg/ml [3]) > boar It should be noted that spermatozoa1 (0.088+0.014 mg/ml). resistance of monkey is similar to that of man. For the sake of showing the differences in spermatozoa1 resistance to nonoxynol-9 among rabbit, bull, dog, man and boar, we have tried to compare normal values for ejaculate of spermatozoa and osmotic characteristics: pH, number pressure, which are comparatively presented in Table II. The quoted pH values of the ejaculates and the number of spermatozoa in rabbit and dog, as well as osmotic pressure values for all 5 species, are taken from the literature [11,13]. The values presented in Table II show fairly wide ejaculate osmotic tolerance of the various species (290-392 which correlates with the fact that mammal mOsmol/l), are subjected to different osmotic pressures spermatozoa while passing through the female genital tract [14] and, therefore, osmotic tolerance is necessary. Osmotic pressure values for 0.9% NaCl solution and various nonoxynol-9 dilutions range from 308.008 and 309.305 mOsmol/l, which is within the range of the quoted literature data. Thus, the spermatozoa1 resistance differences of the mentioned species to nonoxynol-9 could not be explained by different osmotic resistance. A similar case is seen for ejaculate pH values. The optimal pH value for mammalian spermatozoa is about neutral observed in [I31 I with a tolerance which is particularly The pH value for 0.9% Nacl solution and various rabbits. nonoxynol-9 dilutions is 6.30, i.e., somewhat lower than minimal normal values presented in Table II. However, Table II shows that there is some tendency towards decrease of spermatozoa1 resistance of certain species to nonoxynol-9 with increase of ejaculate pH value for the same species. is quite different with spermatozoa1 The situation Table II shows that there concentration in the ejaculate. is a drastic difference in the average number of spermatozoa in the ejaculate of the mentioned species, which is, except boar, directly dependent on spermatozoa1 resistance of these species to nonoxynol-9. Based on this, it is possible to assume that the spermatozoa1 resistance of certain species probably on nonoxynol-9 depends the spermatozoa1 to concentration in the ejaculate of that species - the higher
6.67 - 6.76 [13] 7.10 - 7.50
DOG
MAN
92.5
300 [ll]
1123
296 - 312 [13]
312 - 323 [13]
290 - 392 [13]
290 - 339 [13] 170 7.00 - 7.20 BOAR ____________________~~~~-~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~-~-~~~~~~~-~~~~~-~-~--
6.60 - 6.80
BULL
____________________~~~~~~~~~~~~~~~~~~~~-~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ Concentration Source of Osmotic pressure of spermatozoa ejaculates pH values of of ejaculates ejaculates (mean values) (in order of resistance) (mOsmol/l) (X109/1) -----------------------~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 296 - 317 [13] 200000 [ll] 6.59 - 8.86 [13] RABBIT
Comparative presentation of normal pH and osmotic pressure values of the ejaculate, as well as of mean spermatozoa1 concentration values for rabbit, bull, dog, man and boar ejaculates; in the quoted paper [13], osmotic pressure values of the ejaculates were presented as freezing-point depression (OC), so conversion to mOsmol/l was made
Table II.
236
Contraception
the spermatozoa1 concentration in the ejaculate, the higher the resistance to nonoxynol-9. Based on the differences in concentrations in spermatozoa1 the ejaculate of the the spermatozoa1 resistance differences mentioned species, of those species to nonoxynol-9 could probably be explained. The results obtained in this paper as well as quoted based on the average ejaculum literature data show that, resistance and availability, bull sperm volume, nonoxynol-9 is the most suitable biological material to be used for biological control of contraceptives. Acknowledgement authors would like to thank Prof.Dr. Milan The Jevremovie and Prof.Dr. Vasilije MiljkoviG for reading the manuscript and making valuable comments on it, as well as Zlatica MilutinoviG for translating the paper into Mrs. English. References 1.
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2.
The Extra Pharmacopoeia. 29th Martindale, Press, JEF, ed. London: The Pharmaceutical
3.
Diao XH, Chen Q, Wailer DP, Kaminski J, Zaneveld UD. spermicidal Comparison of the activity and acute toxicity of nonoxynol-9 and agent 741 [alkylphenoxy polyethoxy ethanol (lo)]. Contraception 1986;33:1-5.
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JJ. Wilborn WN, Hahn DW, McGuire Scanning electron microscopy of human spermatozoa after incubation with the spermicide nonoxynol-9. Fertil Steril 1983;39:71719.
5.
Schill WB, spermatozoa nonoxynol-9 nonoxynol-9.
6.
Sander FV, Cramer SD. the spermicidal action Fertil 1941;6:134-37.
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Plohinskij Moskovskogo
8.
Basic statistical nonPetz B. methods for SveuEili.&na naklada "Liber", Zagreb: mathematicians. [Serbocroatian]. 1981;299.
Wolff HH. Ultrastructure the presence of the in and a vaginal contraceptive Andrologia 1981;13:42-49.
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Algoritmy biometrii. NA. Un-ta, 1967;48-49.
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Jondet M, Millet D. Die heterologe (donogene) Besamung Fachtagung fur Fortpflanzung bei Menschen. 16. Inter. Austria. In: Miljkovib. F. und Besamung, Wels, animals. Beograd: Artificial fertilization of Privredno-finansijski vodig, 1979;266 [Serbocroatian].
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Paufler SK, Bader H, Bonfert A, Foot RM, Salamon S, Kunstliche Besamung und Vaserling HF. Eitransplantation bei Tier und Mensch. Hanover, Verlag Mil jkovi6 F. Artificial Schaper MH, 1974. In: Beograd: Privrednofertilization of animals. finansijski vodib, 1979;9 [Serbocroatian].
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Lipu J. Bogdan AT, Bogdan I, Probleme der Fortpflanzungbiologie und ker Kiinstliche Besamung bei Schweine-produktion. 7-te Industriellbetribener Internationale Kongress fiir Fortpflanzung und Besamung, Munchen, 1972, Buch 2, s. 1250.
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Mann T. The biochemistry reproductive tract. London:
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The effects of temperature, Mahi CA, Yanagimachi R. ion osmolarity and concentration on the hydrogen reaction of golden activation and acrosome hamster spermatozoa. J Reprod Fert 1973;35:55-66.
of semen and of the Methuen, 1964;18,86,88.
male