Journal of Ethnopharmacology, 28 (1990) 173Elsevier Scientific Publishers Ireland Ltd.
181
173
POST-TESTICULAR ANTIFERTILITY EFFECTS OF ABRUS PRECATORIUS SEED EXTRACT IN ALBINO RATS
BAKESH
SINHA*
Reproduction (India)
Physiology
Section, Department
of Zoology, Rajasthan
University,
Jaipur 302 004
(Accepted August 21, 1989)
Summary Oral administration of a 50% ethanol extract of Abrus precatorius seeds (250 mg/kg) in albino rats for 30 and 60 days induced an absolute infertility in males which was reversible. Suppression of sperm motility in the cauda epididymis was the most pronounced effect of the treatment. Such treatment may affect the oxidative/energy metabolism of the cauda epididymis. Histological and histocytometric observations in testis and parareproductive tissues appeared normal while the protein, sialic acid, acid phosphatase and succinic dehydrogenase levels were significantly depleted.
Introduction In recent years, an increasing research emphasis is being placed on male contraceptives. Recently, attempts have been made to study the antifertility effects of plant products in both males and females (Kamboj, 1988). Seeds of Abrus precatorius Linn. (Hindi = Ghungchi, Ratti), belonging to the family Leguminosae, have been reported to induce abortion (Chopra et al., 1958; Desai and Sirsi, 1964) and to be toxic (Viswanathan and Joshi, 1983). Recently, Rao (1987) reported the antifertility effect of an ethanolic extract of A. precatorius seeds on male rats. In the present investigation, Abrus seed extract was studied in regard to antifertility strategy aimed at specific inhibition of the maturing sperm cell, either during post-meiotic spermiogenesis or during maturation in the epididymis.
*Present address: Department of Biological Sciences, College of Basic Sciences and Humanities, G.B. Pant University of Agriculture and Technology, Pantnagar 263 145, Dist. Nainital, UP., India. 037%8741/90/$3.50 Q 1990 Elsevier Scientific Publishers Ireland Ltd. Published and Printed in Ireland
174
Materials and methods General methods The seeds of A. precatorius were collected and identified by Dr. B.K. Sharma, Department of Botany, Rajasthan University, Jaipur. The seeds were dried, powdered and extracted with 5O”i, ethanol. The cooled extract was filtered through Whatman’s filter paper No. 1 and the residue extracted 3-4 times with lOO-ml amounts of double glass-distilled water. The filtrates were pooled and evaporated to dryness at room temperature to obtain a brown semi-solid mass. The yield was about 8- 10 g/kg of Abrus seeds. A total of 30 male albino rats (inbred colony) were selected and fed a common laboratory diet and water ad libitum. The rats were kept at a controlled temperature (26-30°C) with alternating 12-h light and dark periods. Veterinary supervision and hygenic conditions were maintained. Animals were divided randomly into a control and two experimental groups. The seed extract was administered orally at a dose of 250 mg extract/kg/day for a period of 30 and 60 days in the experimental groups, respectively. Rats were killed under ether anaesthesia within 24 h of the last dose. The testis, epididymis, seminal vesicles, ventral prostate and vas deferens were dissected free of fat and weighed. Fertility test Fertility rate in all groups was studied according to the World Health Organization MB-50 protocol (Task Force, 1983). The fertility of each male rat (control and treated groups) was assessed by mating with two proestrus/estrus females. The presence of spermatozoa in the vaginal smear or the formation of a vaginal plug on the following morning indicated successful mating. The females were separated and laparotomized under sterile conditions on day 8 and the number of implantation sites in the uterine horns recorded. Presence of implantation sites was taken as a criterion of successful insemination and the fertility test was considered to be positive. The fertility rate was 90% positive in the control group. After closing the surgical wound, the females were allowed to complete pregnancy to term and the number of pups delivered was recorded. Sperm count and sperm motility Epididymal spermatozoa were obtained quickly by a puncture of the cauda epididymis. The density of spermatozoa in the cauda epididymis, testis and motility of sperm in the epididymis were assessed using the method of Prasad et al. (1972). Sperm motility of caput, cauda epididymis and vas deferens relative to untreated rats was determined in vitro using a mixture of equal volumes of seed extract and Krebs-Ringer bicarbonate (KRB) buffer. Sperm motility was recorded at + 15, + 30, +45 and +60 min. Histology and quantitative morphology Histological sections of the testes, caput and cauda epididymis, vas deferens, seminal vesicle and ventral prostate were stained with haematoxylin and eosin, and
175
histocytometric measurements were carried out using an “ocular” eye piece and a micrometer scale (Gurr, 1962). 3ioche~ica~ studies Biochemical estimations were done for protein (Lowry et al., 1951), sialic acid (Warren, 1959), acid phosphatase (Fiske and Subbarow, 1925) and succinic dehydrogenase (Beatty et al., 1966). The levels of serum glutamic oxaloacetate (SGOT) and pyruvic transminase (SGPT) were determined by the method of Mohun and Cook (1957). Results Body and organ weights Body weights did not change significantly following seed extract treatment for 30 and 60 days, respectively (Table 1). The weights of testes, epididymis, seminal vesicles and ventral prostate did not alter signi~c~tly, although the weights of the seminal vesicles and prostrates appeared low in the 60”day treatment group in comparison to controls (Table 1). Sperm density Sperm density in the cauda epididymis did not change significantly after 30 days of treatment but it was significantly reduced after 60 days of treatment (Table 2). Sperm counts recovered significantly after 3 months of withdrawal of treatment. Sperm counts in the testes were also low after 60 days treatment and also recovered after the 3-month withdrawal. Sperm motility Sperm motility in the cauda epididymis was reduced significantly at both durations of treatment. Cessation of treatment for 3 months led to significant recovery in the sperm motility. The in vitro motility of spermatozoa from caput and cauda epididymis and vas deferens of untreated rats were observed in a time-related study using a medium containing equal volumes of KRB buffer and aqueous seed extract. It was found that the reduction of sperm motility from cauda epididymis was greater than from vas deferens at 45 min and 60 min (Table 3). Fertility rate The number of mated females with implantation sites was 4 out of 20 following seed extract treatment for 30 days. However, none of the females showed implantation sites after 60-day treatment. The withdrawal of extract brought about a recovery to 25Y;b and 75% mated females with implantation after 1 month and 3 months, respectively. biochemical changes The levels of protein, sialic acid, acid phosphatase and succinic dehydrogenase enzymes were all significantly reduced following extract treatment (Table 4). SGPT and SGOT in serum remained within the normal ranges.
176
5.51+0.52
7o.ort2.0
2oj20
Total sperm count, testes (million/ml)
Sperm motility, cauda epididymis (%)
Females with impl~tati~n sites vs. female mated (quantal) 4120
58.0f2.5*
3.84+0.60
48,0& 1.4
+ 60 days --
o/20
19.0& 1.5**
1.!2+_0.21**
22.01:1.1**
--
.-
-
4116
47.0+ 1.6**
3.20 + 0.90
42.0+ 1.O
_ -___ Withdrawal -~-_.. + 1 month
----~
12/16
68.Ok2.0
5.a4*0.71
59.oi3.0
..-..-
._._ . _.-+ 3 months ..--___ .----
.._..
WHEN FEMALE RATS WERE MATED WITH MALE RATS TREATED
._----^ Extract treatment -_.f 30 days
OF FERTILITY
Average implantation sites 12.Ok2.0 3.2+0.1 8.OkO.l per female (N) 0 4.0fO.l ~_ __________~_.. .-_ ___--.-. ___~ ______.-.... Significant relative to control: *P < 0.01, **P (: 0.001; N for males = 10 per treatment; all non-quanta1 values represent the mean i S.E.M.
55.6+2.1
_~ .-...- _____I_~-__ Total sperm count, cat.& epididymis (million/ml)
SPERM COUNT, MOTILITY AND INCIDENCE WITH ABRUS SEED EXTRACT ~I.~_____.__ ._~____ Parameter Control
TABLE 2
178
179
I:
180
Histological
changes
The histometric observations of the testis, i.e., tubular and Leydig cell diameters and epithelial cell height from caput and cauda epididymis, were not affected significantly after extract treatment. The histoarchitecture of testis, caput, cauda epididymis vas deferens, seminal vesicle and ventral prostate were also not changed. Discussion The results of the present investigation indicate that Abrus seed extract interferes with the motility and fertilizing ability of the sperm in the epididymis, without affecting the gonadal functions. Oral administrations of Abrus seed extract was effective in reducing the sperm motility in the cauda epididymis. The loss of fertility is attributed to alterations in sperm morphology as well as to changes in their metabolism (Chinoy and Sam, 1984). Suppression of oxidative/energy metabolism of sperm leads to deprivation of adenine nucleotide and impaired sperm function (Ford, 1982). ATP in the spermatozoa is probably related to their energy content and could influence their fertilizing potentiality (Comhaire et al., 1983). The decrease in the protein and sialic acid content of the caput and cauda epididymis may affect those glycoproteins secreted by the epididymis and coated on the sperm to stimulate its motility (Gupta et al., 1974). The extract may cause selective androgen deprivation to the reproductive organs, as the spermatozoa from cauda epididymis and vas deferens were nearly immotile and most were decapacitated, resulting in infertility in all the rats. Sperm density declined during long-term extract treatment, but the effects were reversible after extract withdrawal. These observations suggest that the effect might possibly be both on late spermatogenesis and in reducing the motility capacity of sperm in the epididymis (Phoolpramool and Srikaho, 1983) by acting as a spermatoxic agent on mature or maturing spermatozoa. Further studies to characterize the active principle(s) of A. precatorius seeds are in progress and shall be reported later. However, on the basis of the present study, it may be concluded that the active principle in the extract responsible for widespread changes in the testes and sex accessories might presumably be due to abrin. References Beatty, C.H., Basinger, G.M., Dolly, CC. and Bocek, R.M. (1966) Comparison of red and white voluntary skeletal muscles of several species of primates. j%urnal of Histochemistry and Cytochemistry 14, 590-600. Chinoy, N. J. and Sam, M.G. (1984) Induction of functional sterility in male rats by low dose Carica papaya seed extract treatment. Acta Europaea Fertilitatis 14, 425-432. Chopra, R.N., Chopra, I.C., Handa, K.L. and Kapur, I.D. (1958) Indigenous Drugs of India, 2nd Edn. U.N. Dhar and Sons Pvt. Ltd., Calcutta, p. 261. Comhaire, F., Vermeulen, L., Ghedira, K., Mas, J., Irvine, S. and Callipolitis, G. (1983) Adenosine triphosphate in human semen: A quantitative estimate of fertilizing potential. Fertility and Sterility 40.500-504.
181 Desai, R.V. and Sirsi, M. (1964) The effect of Abrus precatorius
on pregnancy of mice. Current Science
33, 585-587.
Fiske,
C.H.
Biological
and Subbarow, Chemistry
Y. (1925)
The
calorimetric
determination
of phosphorus.
Journal
of
66, 375-400.
Ford, W.C.L. (1982) The mode of action of 6-chloro-deoxysugars as antifertility agents in the male. In: S.L. Jeffcoate and M. Sandler (Eds.), Progress Towards a Male Contraceptive. John Wiley & Sons Ltd., London, pp. 159-184. Gupta, G., Rajalakshmi, M., Prasad, M.R.N. and Moudgal, N.R. (1974) Alteration of epididymal function and its relation to maturation of spermatozoa. Andrologia 6, 35-44. Gurr, E. (1962) Sraining Animal Tissues, Practical and Theoretical. Leonard Hill (Books) Ltd., London, p. 233. Kamboj, V.P. (1988) A review of Indian medicinal plants with interceptive activity. Indian Journal of Medical
Research
83, 336-355.
Lowry, O.H., Rosenberg, N.J., Farr, A.L. and Randell, R.J. (1951) Protein measurements with the Folin phenol reagent. Journal of Biological Chemistry 193, 265. Mohun, A.F. and Cook, I.J.Y. (1957) Simple method for measuring serum levels of the glutamic oxaloacetic and glutamic pyruvic transaminases in routine laboratories. Journal of Clinical Pathology 10,394-399. Phoolpromool, C. and Srikaho, A. (1983) Antifertility effect of sulfasalazine in the male rat. Contraception
28, 273-279.
Prasad, M.R.N., Chinoy, N.J. and Kadam, K.M. (1972) Changes in succinate dehydrogenase levels in the rate epididymis under normal and altered physiological conditions. Fertility and Srerility 23, 186-190.
Rao, M.V. (1987) Antifertility effect of alcoholic seed extract of Abrus precatorius Linn. in male albino rats. Acta Europaea Eertilitatis 18(3), 217-220. Task Force on Indigenous Plants for Fertility Regulation (1983) MB-SO: A Method for Examining the Effect of Plant Extracts Administered Orally on the Fertility of Male Rats (9914E, 31.8.83 APF/lP), World Health Organization Special Program of Research, Development and Research Training in Human Reproduction, Geneva, Switzerland. Viswanathan, N. and Joshi, B.S. (1983) Constituents of some Indian plants. Current Science 52, l-8. Warren, L. (1959) The thiobarbituric acid assay of sialic acid. Journal of Biological Chemistry 234, 1971-1975.
181
173
POST-TESTICULAR ANTIFERTILITY EFFECTS OF ABRUS PRECATORIUS SEED EXTRACT IN ALBINO RATS
BAKESH
SINHA*
Reproduction (India)
Physiology
Section, Department
of Zoology, Rajasthan
University,
Jaipur 302 004
(Accepted August 21, 1989)
Summary Oral administration of a 50% ethanol extract of Abrus precatorius seeds (250 mg/kg) in albino rats for 30 and 60 days induced an absolute infertility in males which was reversible. Suppression of sperm motility in the cauda epididymis was the most pronounced effect of the treatment. Such treatment may affect the oxidative/energy metabolism of the cauda epididymis. Histological and histocytometric observations in testis and parareproductive tissues appeared normal while the protein, sialic acid, acid phosphatase and succinic dehydrogenase levels were significantly depleted.
Introduction In recent years, an increasing research emphasis is being placed on male contraceptives. Recently, attempts have been made to study the antifertility effects of plant products in both males and females (Kamboj, 1988). Seeds of Abrus precatorius Linn. (Hindi = Ghungchi, Ratti), belonging to the family Leguminosae, have been reported to induce abortion (Chopra et al., 1958; Desai and Sirsi, 1964) and to be toxic (Viswanathan and Joshi, 1983). Recently, Rao (1987) reported the antifertility effect of an ethanolic extract of A. precatorius seeds on male rats. In the present investigation, Abrus seed extract was studied in regard to antifertility strategy aimed at specific inhibition of the maturing sperm cell, either during post-meiotic spermiogenesis or during maturation in the epididymis.
*Present address: Department of Biological Sciences, College of Basic Sciences and Humanities, G.B. Pant University of Agriculture and Technology, Pantnagar 263 145, Dist. Nainital, UP., India. 037%8741/90/$3.50 Q 1990 Elsevier Scientific Publishers Ireland Ltd. Published and Printed in Ireland
174
Materials and methods General methods The seeds of A. precatorius were collected and identified by Dr. B.K. Sharma, Department of Botany, Rajasthan University, Jaipur. The seeds were dried, powdered and extracted with 5O”i, ethanol. The cooled extract was filtered through Whatman’s filter paper No. 1 and the residue extracted 3-4 times with lOO-ml amounts of double glass-distilled water. The filtrates were pooled and evaporated to dryness at room temperature to obtain a brown semi-solid mass. The yield was about 8- 10 g/kg of Abrus seeds. A total of 30 male albino rats (inbred colony) were selected and fed a common laboratory diet and water ad libitum. The rats were kept at a controlled temperature (26-30°C) with alternating 12-h light and dark periods. Veterinary supervision and hygenic conditions were maintained. Animals were divided randomly into a control and two experimental groups. The seed extract was administered orally at a dose of 250 mg extract/kg/day for a period of 30 and 60 days in the experimental groups, respectively. Rats were killed under ether anaesthesia within 24 h of the last dose. The testis, epididymis, seminal vesicles, ventral prostate and vas deferens were dissected free of fat and weighed. Fertility test Fertility rate in all groups was studied according to the World Health Organization MB-50 protocol (Task Force, 1983). The fertility of each male rat (control and treated groups) was assessed by mating with two proestrus/estrus females. The presence of spermatozoa in the vaginal smear or the formation of a vaginal plug on the following morning indicated successful mating. The females were separated and laparotomized under sterile conditions on day 8 and the number of implantation sites in the uterine horns recorded. Presence of implantation sites was taken as a criterion of successful insemination and the fertility test was considered to be positive. The fertility rate was 90% positive in the control group. After closing the surgical wound, the females were allowed to complete pregnancy to term and the number of pups delivered was recorded. Sperm count and sperm motility Epididymal spermatozoa were obtained quickly by a puncture of the cauda epididymis. The density of spermatozoa in the cauda epididymis, testis and motility of sperm in the epididymis were assessed using the method of Prasad et al. (1972). Sperm motility of caput, cauda epididymis and vas deferens relative to untreated rats was determined in vitro using a mixture of equal volumes of seed extract and Krebs-Ringer bicarbonate (KRB) buffer. Sperm motility was recorded at + 15, + 30, +45 and +60 min. Histology and quantitative morphology Histological sections of the testes, caput and cauda epididymis, vas deferens, seminal vesicle and ventral prostate were stained with haematoxylin and eosin, and
175
histocytometric measurements were carried out using an “ocular” eye piece and a micrometer scale (Gurr, 1962). 3ioche~ica~ studies Biochemical estimations were done for protein (Lowry et al., 1951), sialic acid (Warren, 1959), acid phosphatase (Fiske and Subbarow, 1925) and succinic dehydrogenase (Beatty et al., 1966). The levels of serum glutamic oxaloacetate (SGOT) and pyruvic transminase (SGPT) were determined by the method of Mohun and Cook (1957). Results Body and organ weights Body weights did not change significantly following seed extract treatment for 30 and 60 days, respectively (Table 1). The weights of testes, epididymis, seminal vesicles and ventral prostate did not alter signi~c~tly, although the weights of the seminal vesicles and prostrates appeared low in the 60”day treatment group in comparison to controls (Table 1). Sperm density Sperm density in the cauda epididymis did not change significantly after 30 days of treatment but it was significantly reduced after 60 days of treatment (Table 2). Sperm counts recovered significantly after 3 months of withdrawal of treatment. Sperm counts in the testes were also low after 60 days treatment and also recovered after the 3-month withdrawal. Sperm motility Sperm motility in the cauda epididymis was reduced significantly at both durations of treatment. Cessation of treatment for 3 months led to significant recovery in the sperm motility. The in vitro motility of spermatozoa from caput and cauda epididymis and vas deferens of untreated rats were observed in a time-related study using a medium containing equal volumes of KRB buffer and aqueous seed extract. It was found that the reduction of sperm motility from cauda epididymis was greater than from vas deferens at 45 min and 60 min (Table 3). Fertility rate The number of mated females with implantation sites was 4 out of 20 following seed extract treatment for 30 days. However, none of the females showed implantation sites after 60-day treatment. The withdrawal of extract brought about a recovery to 25Y;b and 75% mated females with implantation after 1 month and 3 months, respectively. biochemical changes The levels of protein, sialic acid, acid phosphatase and succinic dehydrogenase enzymes were all significantly reduced following extract treatment (Table 4). SGPT and SGOT in serum remained within the normal ranges.
176
5.51+0.52
7o.ort2.0
2oj20
Total sperm count, testes (million/ml)
Sperm motility, cauda epididymis (%)
Females with impl~tati~n sites vs. female mated (quantal) 4120
58.0f2.5*
3.84+0.60
48,0& 1.4
+ 60 days --
o/20
19.0& 1.5**
1.!2+_0.21**
22.01:1.1**
--
.-
-
4116
47.0+ 1.6**
3.20 + 0.90
42.0+ 1.O
_ -___ Withdrawal -~-_.. + 1 month
----~
12/16
68.Ok2.0
5.a4*0.71
59.oi3.0
..-..-
._._ . _.-+ 3 months ..--___ .----
.._..
WHEN FEMALE RATS WERE MATED WITH MALE RATS TREATED
._----^ Extract treatment -_.f 30 days
OF FERTILITY
Average implantation sites 12.Ok2.0 3.2+0.1 8.OkO.l per female (N) 0 4.0fO.l ~_ __________~_.. .-_ ___--.-. ___~ ______.-.... Significant relative to control: *P < 0.01, **P (: 0.001; N for males = 10 per treatment; all non-quanta1 values represent the mean i S.E.M.
55.6+2.1
_~ .-...- _____I_~-__ Total sperm count, cat.& epididymis (million/ml)
SPERM COUNT, MOTILITY AND INCIDENCE WITH ABRUS SEED EXTRACT ~I.~_____.__ ._~____ Parameter Control
TABLE 2
178
179
I:
180
Histological
changes
The histometric observations of the testis, i.e., tubular and Leydig cell diameters and epithelial cell height from caput and cauda epididymis, were not affected significantly after extract treatment. The histoarchitecture of testis, caput, cauda epididymis vas deferens, seminal vesicle and ventral prostate were also not changed. Discussion The results of the present investigation indicate that Abrus seed extract interferes with the motility and fertilizing ability of the sperm in the epididymis, without affecting the gonadal functions. Oral administrations of Abrus seed extract was effective in reducing the sperm motility in the cauda epididymis. The loss of fertility is attributed to alterations in sperm morphology as well as to changes in their metabolism (Chinoy and Sam, 1984). Suppression of oxidative/energy metabolism of sperm leads to deprivation of adenine nucleotide and impaired sperm function (Ford, 1982). ATP in the spermatozoa is probably related to their energy content and could influence their fertilizing potentiality (Comhaire et al., 1983). The decrease in the protein and sialic acid content of the caput and cauda epididymis may affect those glycoproteins secreted by the epididymis and coated on the sperm to stimulate its motility (Gupta et al., 1974). The extract may cause selective androgen deprivation to the reproductive organs, as the spermatozoa from cauda epididymis and vas deferens were nearly immotile and most were decapacitated, resulting in infertility in all the rats. Sperm density declined during long-term extract treatment, but the effects were reversible after extract withdrawal. These observations suggest that the effect might possibly be both on late spermatogenesis and in reducing the motility capacity of sperm in the epididymis (Phoolpramool and Srikaho, 1983) by acting as a spermatoxic agent on mature or maturing spermatozoa. Further studies to characterize the active principle(s) of A. precatorius seeds are in progress and shall be reported later. However, on the basis of the present study, it may be concluded that the active principle in the extract responsible for widespread changes in the testes and sex accessories might presumably be due to abrin. References Beatty, C.H., Basinger, G.M., Dolly, CC. and Bocek, R.M. (1966) Comparison of red and white voluntary skeletal muscles of several species of primates. j%urnal of Histochemistry and Cytochemistry 14, 590-600. Chinoy, N. J. and Sam, M.G. (1984) Induction of functional sterility in male rats by low dose Carica papaya seed extract treatment. Acta Europaea Fertilitatis 14, 425-432. Chopra, R.N., Chopra, I.C., Handa, K.L. and Kapur, I.D. (1958) Indigenous Drugs of India, 2nd Edn. U.N. Dhar and Sons Pvt. Ltd., Calcutta, p. 261. Comhaire, F., Vermeulen, L., Ghedira, K., Mas, J., Irvine, S. and Callipolitis, G. (1983) Adenosine triphosphate in human semen: A quantitative estimate of fertilizing potential. Fertility and Sterility 40.500-504.
181 Desai, R.V. and Sirsi, M. (1964) The effect of Abrus precatorius
on pregnancy of mice. Current Science
33, 585-587.
Fiske,
C.H.
Biological
and Subbarow, Chemistry
Y. (1925)
The
calorimetric
determination
of phosphorus.
Journal
of
66, 375-400.
Ford, W.C.L. (1982) The mode of action of 6-chloro-deoxysugars as antifertility agents in the male. In: S.L. Jeffcoate and M. Sandler (Eds.), Progress Towards a Male Contraceptive. John Wiley & Sons Ltd., London, pp. 159-184. Gupta, G., Rajalakshmi, M., Prasad, M.R.N. and Moudgal, N.R. (1974) Alteration of epididymal function and its relation to maturation of spermatozoa. Andrologia 6, 35-44. Gurr, E. (1962) Sraining Animal Tissues, Practical and Theoretical. Leonard Hill (Books) Ltd., London, p. 233. Kamboj, V.P. (1988) A review of Indian medicinal plants with interceptive activity. Indian Journal of Medical
Research
83, 336-355.
Lowry, O.H., Rosenberg, N.J., Farr, A.L. and Randell, R.J. (1951) Protein measurements with the Folin phenol reagent. Journal of Biological Chemistry 193, 265. Mohun, A.F. and Cook, I.J.Y. (1957) Simple method for measuring serum levels of the glutamic oxaloacetic and glutamic pyruvic transaminases in routine laboratories. Journal of Clinical Pathology 10,394-399. Phoolpromool, C. and Srikaho, A. (1983) Antifertility effect of sulfasalazine in the male rat. Contraception
28, 273-279.
Prasad, M.R.N., Chinoy, N.J. and Kadam, K.M. (1972) Changes in succinate dehydrogenase levels in the rate epididymis under normal and altered physiological conditions. Fertility and Srerility 23, 186-190.
Rao, M.V. (1987) Antifertility effect of alcoholic seed extract of Abrus precatorius Linn. in male albino rats. Acta Europaea Eertilitatis 18(3), 217-220. Task Force on Indigenous Plants for Fertility Regulation (1983) MB-SO: A Method for Examining the Effect of Plant Extracts Administered Orally on the Fertility of Male Rats (9914E, 31.8.83 APF/lP), World Health Organization Special Program of Research, Development and Research Training in Human Reproduction, Geneva, Switzerland. Viswanathan, N. and Joshi, B.S. (1983) Constituents of some Indian plants. Current Science 52, l-8. Warren, L. (1959) The thiobarbituric acid assay of sialic acid. Journal of Biological Chemistry 234, 1971-1975.
