Materials and methods

Animals

Adult Wistar rats of proven fertility kept under standardized animal house conditions (temperature: 28-31&degreeC, photoperiod: approximately 12 hours with natural light per day, relative humidity: 50-55%) were used. They had free access to pelleted food (Master feed Ltd., Colombo, Sri Lanka) and tap water. Each rat was used only once and except at the time of experimental procedures the animals were handled only during cage cleaning.

Drugs

SNP and ether (Fluka Chemica, Buchs, Switzerland), oestradiol benzoate and progesterone (Sigma Chemical Company, St. Louis, MO, USA) were used. SNP was dissolved in 0.1 mL of isotonic saline (0.9% NaCl, w/v) to obtain the required dose immediately prior to testing.

Effect on sexual behaviour

Sexual ability was examined in a noncompetitive copulation test. Thirty-five male rats were randomly assigned into four groups and intraperitoneally (i.p.) treated with SNP in the following manner: (1) SNP, 20 μg/kg, n = 10, (2) SNP, 30 μg/kg, n = 7 (3) SNP 60 μg/kg, n = 3 and (4) vehicle control receiving saline, 0.1 mL/kg, n = 12. The treated rats were placed individually in cages for 30 min following administration of vehicle or SNP. Following a 10 min adaptation period a female that had been brought into oestrous (oestradiol benzonate 12 μg in olive oil injected intramuscularly 8 hours prior to pairing) was placed in the cage. 8 Ratnasooriya WD, Dharmasiri MG, Wadsworth RM. Reduction in libido and fertility of male rats by administration of the nitric oxide (NO) synthase inhibitor N-nitro-L-arginine methyl ester. Int J Androl 2000; 23: 187-91. The precoital behaviour was observed for 15 min or until ejaculation. During this period the mount latency (the time from the introduction of the receptive female to the first mount); intromission latency (the time from the introduction of the receptive female to the first intromission) and ejaculation latency (the time from the introduction of the receptive female to ejaculation), number of mounts and number of intromissions displayed by the rats were recorded. A mount was defined as the male's pelvis intentionally coming into contact with the female's haunch with accompanying hip movements from the male. An intromission was identified when the male mounted and achieved insertion of the penis into the female as marked by a deeper than normal thrust usually followed by abrupt movement away from the female, urgent front leg movements and grooming. Ejaculation was marked by a more profound thrust than that of regular intromission, and was followed not by movement away from the female, but by sudden limpness and immobility until female moves away. After ejaculation and immobility, the male engaged in a long period of grooming (5 – 10 min) before settling into rest. When there is no intromission and ejaculation during the observation period their latencies were taken as 15 min. Using these data the following parameters were computed,% mounted,% intromitted,% ejaculated, copulatory efficiency [(number of intromissions/number of mounts) x 100], and intercopulatory interval (average time between intromissions).

These rats were also observed for overt signs of toxicity (salivation, lachrymation, ptosis, teeth exposure, wilting, convulsions, stupor, tremor, rapid rotational movement of head, rolling, jumping, postural changes), stress (erection of fur and exopthalmia), aversive behaviours (biting and scratching behaviour, licking at tail or paw, or vocalization), and nonsexual behaviour (such as cleaning of face, self grooming, climbing in the cage, rearings).

Partner preference test

Twelve male rats were randomly assigned into two equal groups (n = 6). One group was i.p. treated with 30 μg/mL of SNP and the other with 0.1 mL of saline. These rats were then individually caged. Sixty minutes later, one oestrous (receptive) and one dioestrous (nonreceptive) female were introduced to the cage of each of the treated males. The cumulative time spent by a male in physical contact with each of the females was recorded for 15min. Partnerpreference index was calculated by subtracting the time spent with dioestrous female from the time spent with the oestrous female. A positive score indicates preference for the sexually receptive female while a negative score indicates the preference for the nonreceptive female. 9 Edwards DA, Griffis KT, Tardivel C. Olfactory bulb removal: effects on sexual behaviour and partnerpreference in male rats. Physiol Behav 1990; 48: 447-50.

Effect on fertility

Twenty-four male rats were randomly assigned into two equal groups. One group was injected with 30 μg/kg SNP and the other with 0.1 mL saline i.p. at 16.00 h. Each male was then individually paired overnight with a pro-oestrous female at 16.30-17.00 h. Vaginal smears of the paired females were taken on the following morning (at 8.00–8.30 h) and examined microscopically (x 100). If the spermatozoa were present, their numbers and gross morphology were noted and this was taken as the onset of pregnancy. If spermatozoa were absent then daily vaginal smearing was undertaken for at least 14 days to determine the appearance of pregnancy or pseudopregnancy. At 14 days after mating, the females were subjected to uterotomy under ether anaesthesia to determine the number, viability and the size of fetuses and the number of resorption sites. The gross appearance of corpora lutea was also recorded. The following reproductive indices were computed: index of libido [(number mated/number paired) x 100], quantal pregnancy [(number pregnant/number mated) x 100], fertility index [(number pregnant/number paired) x 100], implantation index [(total number of implantation/number mated) x 100], preimplantation loss [(number of corpora lutea – number of implants)/number of corpora lutea] x 100, and postimplantation loss [(number of implants – number of viable implants)/number of implants] x 100.

Effect on yawning and penile erections

Twelve male rats were randomly assigned into two equal groups. One group was treated i.p. with 30 μg/kg of SNP and the other with 0.1 mL of saline. Shortly after treatment, these rats were placed individually in plexiglass cages and the number of yawns and penile erections (characterized by penile body elongation and its dorsiflexion, reddening and engorgement of glans and its flaring) were recorded for 30 min. 10 De Mateos-Verchere JG, Leprince J, Tonson MC, Vaudry H, Costetin J. The octadecaneuropeptide ODN inhibits apomorphine-induced yawning in rats. Eur J Pharmacol 1998; 357: 121-26.

Effect on muscle strength and co-ordination

Twelve male rats were randomly divided into two equal groups. The first group was i.p. administered with 30 μg/kg of SNP and the other with 0.1 mL saline. Thirty minutes following treatment, the rats were subjected to bar holding test for 1min to determine muscle strength, and the latency to fall was recorded. 11 Plaznic A, Stefanski R, Palejko W, Kotawski W. The role of accumbens GABA-B receptors in the regulation of rat behaviour. Neurosci Res Commun 1993; 12: 23-30. Immediately after this the rats were subjected to Bridge test to evaluate muscle coordination, and the latency to slide off was recorded.

Evaluation of sedative activity

Sedative activity of SNP was evaluated using the rat hole-board technique. 12 File SE, Wardill A. Validity of head-dipping as a measure of exploration in modified hole-board. Psychopharmacology 1975; 44: 53-57. Randomly selected male rats were i.p. treated with 30 μg/kg of SNP (n = 6) or 0.1 mL saline. Thirty minutes later, these rats were individually placed on the centre of the rat hole-board and give 7.5 min trial period. The number of head dips, rears, locomotory activity and the number of faecal boluses produced were recorded. The time per head dip was then computed.

Evaluation of analgesic activity

Twelve rats were randomly assigned into two equal groups. One group was treated i.p. with 1 mL of physiological saline and the other group with 30 μg/kg of SNP. Following 30 min analgesic activity, they were evaluated using tail flick and hot plate tests, with a cut-off time of 5 and 15 min respectively to prevent tissue damage. 13 Langerman L, Zakouski MI, Piskoun B, Grant GJ. Hot plate versus tail flick evaluation of acute tolerance to continuous morphine infusion in the rat model. J Pharmacol Toxicol Methods 1995; 34: 23-28.

Statistical analyses

Data are given as mean &plusminus SEM. Statistical comparisons were made using G-test and Mann–Whitney U-test as appropriate. 14 Frank H, Althoen SC. Statistics; concepts and applications, Low price edition. Cambridge: Cambridge University Press, 1995. P < 0.05 was accepted as indicating significance.

Results

None of the rats receiving the highest dose of SNP survived: these rats exhibited signs of stress and developed severe tremors and convulsions within 15 min of treatment and, died by 30 min. In contrast, no overt signs of toxicity, stress or aversive behaviours were evident in rats treated with mid and low doses of SNP.

Table 1 summarizes the data obtained with the sexual behaviour study. The results of the control animal are agreeable with those of our previous studies,,, Both low and mid doses of SNP significantly (P < 0.05–0.01) altered all the parameters of male sexual behaviour investigated except the intromission ratio. None of the rats that mounted exhibited aberrant mounting behaviours. Further, SNP-induced effects on sexual behaviour appear to be dose-related.

In the partner preference test the mid dose of SNP did not impair the partner preference index (control versus treatment: 77.5&plusminus12.5 versus 42.2&plusminus6.8 s).

In the fertility study, with the mid dose of SNP, all the rats that ejaculated had apparently normal numbers of sperm, with no gross detectable morphological defects. Further, none of the fertility parameters investigated was significantly (P > 0.05) impaired (data not shown).

The mid dose of SNP provoked significant (P < 0.05) increase in yawning (control versus treatment 0.0&plusminus0.0 versus 2.3&plusminus0.1) and spontaneous erections (0.0&plusminus0.0 versus 2.5&plusminus1.5). These erections were usually accompanied with genital grooming but not with spontaneous ejaculations. Further, micturition was never observed during the test period.

In the bar holding test, mid dose of SNP did not significantly (P > 0.05) increase the latency to fall (control versus treatment: 1.0&plusminus0.0 versus 1.0&plusminus0.0).

However, the same dose of SNP caused a significant (P < 0.05) reduction (by 35%) on the latency to slide off in the Bridge test (control versus treatment: 45.1&plusminus0.8 versus 29.3&plusminus8.9 s).
In the rat hole-board experiment (Table 2) the mid dose of SNP markedly and significantly (P < 0.05–0.01) impaired locomotory activity (by 36%), number of rearings (by 38%) and the number of head dips (by 59%). In contrast, time per head and the number of faecal boluses produced remained significantly (P > 0.05) unaltered.

The mid dose of SNP did not significantly (P > 0.05) alter the reaction times in both tail flick (control versus treatment: 2.1&plusminus0.1 versus 2.0&plusminus0.5 s) and hot plate test (control versus treatment: 10.6&plusminus1.5 versus 12.1&plusminus2.0 s).

Discussion
This study examined the impact of SNP (given i.p.) on male reproductive competence using gonadally intact rats and noncompetitive copulation tests. We have chosen the i.p. administration rather than intrapenile administration. In conscious rats, intrapenile injection is stressogenic and often causes cavernous fibrosis. Stress impairs male rat sexual behaviour. 15 Retana-Marquez S, Bonilla-Jaime H, Vazquez-Palacios G, Martinez-Garcia R, Velazquez-Moctezuma. Changes in masculine sexual behaviour, corticostorone and testosterone in response to acute and chronic stress in male rats. Horm Behav 2003; 44: 327-37. Rats are one of the most widely used animals in reproductive studies, and copulation is the most natural and frequently examined behaviour of the laboratory tests of sexual functions. 16 Yonezawa A, Watanabe C, Ando R, Furuta S, Sakurda S, Yoshimara H, Iwanaga T, Kimura Y. Characterization of p-chloroamphitamine-induced penile erection and ejaculation in anaesthetized rats. Life Sci 2000; 67: 3031-39. 17 Sachs BD. Contextual approaches to the physiology and classification of erectile function, erectile dysfunction and sexual arousal. Neurosci Behav Rev 2002; 24: 541-60. Furthermore, it has been proposed that there are obvious homologies between human copulatory behaviour and that of rat including the mechanism of penile erection. 18 Pfaus JG. Homologies of animal and human sexual behaviours. Horm Behav 1996; 30: 187-200. The results show that the highest dose of SNP tested was toxic and caused rapid death possibly from cyanide toxicity: SNP releases cyanide in vivo, and cyanide toxicity is a true complication of its clinical use. 19 Arnold WP, Longnecker DE, Epstein RM. Phototdegradation of sodium nitroprusside: biological activity and cyanide release. Anaesthesiology 1984; 61: 254-60. On the other hand, mid and low doses of SNP caused a marked overall disruption of sexual competence in a dose-related fashion. Interestingly, however, SNP had apparently no effect on ejaculation in terms of vaginal sperm counts in paired females or immediate fertility in terms of number of uterine implants, quantal pregnancy, fertility index and implantation index which may be attributable to SNP's rapid and short-lasting pharmacological action (NO donor). 2 Anonymous: British National Formulary 2000. London: The British Medical Association and the Royal Pharmaceutical Society of Great Britain, 2000. The onset of this impairment in sexual competence was quite rapid (with in 1 hour), suggesting a centrally mediated action. SNP is a NO donor, and a threshold level of NO in brain is claimed to be essential for the expression of normal male sexual function.2 Anonymous: British National Formulary 2000. London: The British Medical Association and the Royal Pharmaceutical Society of Great Britain, 2000.5 Ratnasooriya WD, Dharmasiri MG. L-arginine, the substrate of nitric-oxide synthase inhibits fertility of male rats. Asian J Androl 2001; 3: 97-103.
>8 Ratnasooriya WD, Dharmasiri MG, Wadsworth RM. Reduction in libido and fertility of male rats by administration of the nitric oxide (NO) synthase inhibitor N-nitro-L-arginine methyl ester. Int J Androl 2000; 23: 187-91.Luckily, this SNP-induced impairment of sexual behaviour was short-lasting and fully reversible (within 6.5–8.0 hours), as in the mating study all the SNP-treated rats ejaculated and exhibited normal libido in terms of index of libido and fertility: in rats, natural mating usually occurs between 22.00 and 24.00 h of the day of pro-oestrous.20 Ratnasooriya WD, Wadsworth RM. Effect of mating on sperm distribution in the reproductive tract of the male rat. Gamete Res 1987, 17: 261-66.
With the low and mid doses of SNP, there were no overt signs of general toxicity, stress or aversive behaviours. Further, SNP-treated rats showed no signs of fatigue or hyperactivity: fatigue plays a role in sexual activity and hyperactive rats are usually hyposexual.21 Kohlert JG, Bloch GJ. Hyperactivity in hyposexual male rats. Physiol Behav 1996; 59: 171-78. Thus, the inhibition of sexual behaviour patterns could be attributed to a selective action of SNP, possibly an elevated NO level and/or SNP-specific side effect. However, further experiments are required to resolve this point.
In the partner preference test, SNP-treated rats displayed a positive score as the control rats, indicating that SNP treatment has not suppressed sexual motivation.9 Edwards DA, Griffis KT, Tardivel C. Olfactory bulb removal: effects on sexual behaviour and partnerpreference in male rats. Physiol Behav 1990; 48: 447-50.
