Role of oestrogens in male erectile function 2

SHORT-TERM STUDY GROUPS NUMBER OF RATS DURATION LONG-TERM STUDY GROUPS NUMBER OF RATS DURATION Table 1: Experimental Groups of Rats Individual treatment groups n=10 administered two dos

Chapter 3 MONITORING FOOD INTAKE AND BODY WEIGHT CHANGES IN OESTROGEN TREATED ANIMAL MODELS

3 MONITORING FOOD INTAKE AND BODY WEIGHT CHANGES

IN OESTROGEN TREATED ANIMAL MODELS

3.1 Study Groups

All the animals used in this investigation were supplied and maintained by the Laboratory Animal Centre and the Animal Holding Unit of the National University of Singapore, Singapore With due compliance of the International guiding principles for animal research, the experimental protocol was approved by the ethics committee at this centre Cage-side examinations were conducted once daily and the animals were individually examined for abnormal behaviour or physical changes once weekly throughout the study

3.1.1 Rat Model

Taking into consideration the possibility of species difference in response to administered steroids (Biegel et al., 1998), which includes oestradiol, the time-tested and conventional rat strain, Sprague-Dawley (SD) male rats were used in this investigation Rat is also an established whole animal / in vivo model for studies estimating sexual behaviour (Chapter 4) and cavernous pressure response to erectile nerve stimulation (Chapter 5) The animals on arrival were thoroughly assessed for general physical fitness and sixty healthy and sexually mature adult male rats were randomly divided into six experimental groups (involving two control and four treatment groups, n=10 each) The animals were housed in pairs under ambient temperature (approximately 22°C) and reversed dark - light cycle (08.00am−20.00pm with the individual dark and light phases of 12 hours) All the experimental animals had ready access to food and water The standard rat diet (Glen Forrest Stock Feeders, Western Australia) was provided as 11mm diameter pellets and the balanced formula included wheat, millmix, lupins, soya meal, fish meal, vegetable oil, tallow, salt, added vitamin and mineral premix Essential dietary constituents of the rat feed are listed in Appendix 1

3.1.2 Rabbit Model

Being docile, rabbits as experimental animals have several advantages over other species with regard to basic studies Furthermore, in this area of impotence research, the good structural and functional correlation identified between the rabbit corpus cavernosum (CC) and the human counterpart (Azadzoi et al., 1988; Bischoff, 2001) prompted the choice of rabbits to investigate the physiopharmacological aspects of the human erectile disorder in this animal model (Chapter 6)

Twenty-four genetically pure-bred strain of New Zealand White (NZW) healthy male rabbits were procured for the study At the initiation of the experimental procedure, the animals were sexually mature young adults (six months old) They were assigned at random to four experimental groups (n=6, each) and housed in individual steel cages (18’x16’x13’) under a stable air-conditioned atmosphere with adequate daily care and ready access to standard rabbit diet (pellets) and water The pellets were procured in bulk quantities from Glen Forrest Stock Feeders of Western Australia They were well balanced to meet the daily nutritional requirement of the animals The constituents of the standard dietary supply included barley, oats, millmix, lupins, soya meal, lucerne, limestone, methionine, salt and added vitamin and mineral premix (Appendix 2)

3.2 Administration of Oestrogen and Phytoestrogen

Individual rat groups were treated with 0.01mg or 0.1mg of oestradiol valerate (Progynova, Schering) prepared as a suspension in sterile water (in fixed volume of 0.3ml) and administered through oral gavage daily for one week (n=20, short-term study) and 12 weeks (n=20, long-term study) respectively The valerate salt of oestradiol has optimal pharmacokinetic parameters for systemic effects / toxicity (Kuhnz and Putz, 1989) and the chosen doses were reported to produce significant modulations in body

weight and hormonal parameters in rats (Brewster et al., 1997) The control groups

(n=10) for each duration of treatment received the same volume of vehicle (sterile water)

through gavage The treatment groups are described in Table 1

SHORT-TERM STUDY GROUPS NUMBER OF RATS DURATION

LONG-TERM STUDY GROUPS NUMBER OF RATS DURATION

Table 1: Experimental Groups of Rats

Individual treatment groups (n=10) administered two doses of oestradiol valerate for 1

week or 12 weeks and their respective untreated, time-matched control animals

Rabbits from the treatment groups (n=6, each) received 0.1mg of oestradiol valerate (this

dose produced significant changes during investigation in the rat model) or

phytoestrogen, soybean isoflavone daidzein (Sigma) 0.01mg or 0.1mg as suspensions in

sterile water (fixed volume of 1ml) through oral gavage daily for 12 weeks While

soybean is the richest known source for phytoestrogen isoflavones daidzein and genistein

(Nicholls et al, 2002), the choice of the specific isoflavone was based on earlier reports

from other centres of better bioavailability (Xu et al., 1994) and functional efficacy

(Picherit et al., 2000) of daidzein over genistein Control rabbits of the study consumed

the same volume of sterile water by gavage daily The different treatment groups are

listed in Table 2

STUDY GROUPS NUMBER OF RABBITS DURATION

Table 2: Experimental Groups of Rabbits

Individual treatment groups (n=6) administered oestradiol valerate or two doses of

phytoestrogen isoflavone daidzein for 12 weeks and the untreated, time-matched control

animals

3.3 Estimation of Body Weight and Food Intake

Since oestrogen appears to have ubiquitous distribution and wide ranging physiological

functions in males, several mechanistic and biochemical endpoints such as body weight,

weight of peripheral androgen-dependent organs, serum hormone levels, sertoli cell

morphology and sperm analysis (Brewster et al., 1997; Cook et al., 1998) may be

evaluated to quantify the differential pharmacological effects of an administered

oestrogen on these variables Among these, reduction in body weight is favourably

modulated by oestrogens through central mechanisms (Anderson et al., 1988; Brewster et

al., 1997), peripheral pathways and metabolic parameters (Sanchis et al., 1997;

Loose-Mitchell and Stancel, 2001) The objective of comparative body weight estimation of

different experimental groups in the present investigation was to utilise this known

physiological effect for the efficacy of the pharmacological administration of oestrogens

in the rat and rabbit models

Using standard weighing scale (Shimadzu Corporation, Japan), rats of the short-term

study group were weighed on days 0 and 8 and those of the long-term study group and

rabbits at 0, 6 and 12 weeks Food consumption / animal / day were estimated at random

intervals throughout the treatment period, measured as the 24-hour difference in the weight of the pellets provided in the cage

3.3.1 Data Analysis

Statistical comparison was done using one way analysis of variance (ANOVA) from the Statistical Package for Social Sciences (SPSS) version 11.5 for windows All the results were expressed as mean±SEM (standard error of the mean) and the level of significance set at p<0.05

3.4 Animal Acclimatization

All experimental animals were allowed to acclimatise to experimental conditions for two weeks prior to oral administration of the test agents; this pre-investigative period was uneventful and the animals remained healthy and active Food and water intake was normal during this period and the baseline range of body weight for rats was 250-300g and that for rabbits was 2.5-3.0kg There was no resistance or untoward reaction to gavage feeding of the oestrogen suspensions when initiated and there was no animal mortality during the study period

3.4.1 Oestrogen Administered Rats

There was no significant change in body weight or average daily food consumption in the rats of the oestradiol treated groups (0.01mg and 0.1mg) of the short-term study compared to the control animals However, when the treatment was continued for 12 weeks (long-term study group), there was a time as well as dose-dependent decrease in body weight / weight gain which was statistically significant in the 0.1mg oestradiol treated rats at 12 weeks This also coincided with the gradual reduction in daily rat chow intake in this group The mean body weight and food intake at 1 week and 12 weeks are given in Table 3

SHORT-TERM STUDY GROUPS Body Weight (g, 1 wk) Food Intake / Rat / day (g)

Table 3: Body Weight and Food Intake in Rats

Reduced weight gain and food consumption in rat groups (n=10, each) at 1 week and 12

weeks The decrease in mean body weight for rats on higher oestradiol administration

was statistically significant (*p<0.05) in the 12 weeks treatment group compared to the

control animals

3.4.2 Oestrogen Administered Rabbits

There was a decrease in body weight gain and average food intake in rabbits of the oestradiol treated group (0.1mg) when compared to control animals Although this result failed to achieve statistical significance, this trend indicated the onset of oestrogen mediated homeostatic alteration of this parameter The average body weight and food consumption in this group at 12 weeks is given in Table 4

3.4.3 Phytoestrogen Administered Rabbits

There was no significant difference in average food intake or body weight gain in rabbits

of the phytoestrogen treated groups (0.01mg and 0.1mg) when compared with control animals However, there was an inter-group dose-dependency which indicated the possibility of onset of similar albeit milder homeostatic alterations / oestrogen-mimetic activity (Table 4)

STUDY GROUPS Body Weight (kg, 12 wks) Food Intake / Rabbit / day (g)

Oestradiol valerate (0.1mg) 3.2±0.1 133.4±1.1

Phytoestrogen Daidzein (0.01mg) 3.7±0.7 145.5±5.8

Phytoestrogen Daidzein (0.1mg) 3.3±0.1 141.1±2.5

Table 4: Body Weight and Food Intake in Rabbits

Effect of oestradiol valerate and phytoestrogen isoflavone daidzein on mean weight gain

and food consumption in the rabbit treatment groups (n=10, each) at 12 weeks

3.5 Discussion and Conclusion

The observations of the reduced body weight / weight gain in the rat and rabbit models with oestrogenic agents are consistent with reports from short term and long term treatment models (Sanchis et al., 1997; Kuhnz and Putz, 1989; Gibson et al., 1967; Beigel

et al., 1998; and Brewster et al., 1997) In rodents, both synthetic and natural oestrogens decreased mean growth rate (Hart, 1990); this being mediated through reduced food and water intake (Finkelstein, 1986; Hart, 1988) and decreased food efficiency (Cook et al., 1998) In suppressing appetite, oestrogens act centrally on the hypothalamus (Brewster et al., 1997) probably at the paraventricular or ventromedial nuclei (Nunez et al., 1980) both

of which are involved in regulation of food intake and satiation (Guyton and Hall, 2001) and/or peripherally decreasing leptin (Nedvidkova et al., 1997) and serum lipoprotein levels with decrease in LDL and increase in HDL cholesterols (Loose-Mitchell and Stancel, 2001) This may also be perceived as the functional antagonism by oestrogen of the androgenic anabolism and weight gain mediated through appetite stimulation (Wilson, 1996) There is a direct link between weight gain, obesity, leptin levels and heart disease (Rajapurohitam et al., 2003) and the oestrogenic cardioprotection may thus involve some of these multiple pathways With decrease in circulating oestrogen levels in

menopausal women, there is increased risk of obesity and alteration in body fat composition (Lovejoy, 2003) In this investigation, the end point was further influenced

by the duration and dose of oestrogenic treatment While conventional sympathomimetic anorexiants such as amphetamine and related agents produce only short term benefits limited by the development of tolerance (O’Brien, 2001) and their long term use is inherent with serious adverse and abuse potentials (Hoffman, 2001), our results indicate that an optimal and constant concentration of oestrogen will result in normal body weight regulation and maintenance However, the clinical usefulness of this beneficial effect in the males is an open question

Chapter 4 EFFECT OF OESTRADIOL ON PARAMETERS OF

SEXUAL BEHAVIOUR IN RATS

4 EFFECT OF OESTRADIOL ON PARAMETERS OF SEXUAL

BEHAVIOUR IN RATS 4.1 Choice of Animal Model / Objectives

Rats have been widely used for a variety of behavioural studies ranging from developmental learning experiments to those concerned with socio-sexual behaviour Their behavioural pattern is highly predictable and well-replicated in any laboratory setting They are economical, easily handled and very much adaptable They are uninhibited during studies and are therefore avidly used by researchers for evaluating behavioural changes (Agmo, 1997) Through animal studies of such spontaneous behaviour, it is possible to delineate changes in sexual function, especially those of motivation (desire/arousal/libido) from parameters of copulation (erectile capacity or performance) (Pfaus, 1996) Using these animal models, it is possible to obtain insights and information on certain components which cannot be studied in patients in a scientifically objective way due to ethical constraints (Barfield, 1993) Hence male rats were the subjects of this investigation which concerns with the delineation of erectile function/dysfunction in the presence of oestradiol induced hormonal changes

4.2 Materials and Methods

4.2.1 Animal Groups

Sixty healthy and sexually mature adult SD male rats were randomly divided into six experimental groups (involving two control and four treatment groups, n=10 each) All the animals were housed in a reversed light - dark cycle (20.00hrs−08.00hrs) with the initial acclimatisation phase of 3 weeks

4.2.2 Diet and Drug Administration

All the experimental animals had ready access to food and water Individual rat groups were treated with 0.01mg or 0.1mg of oestradiol valerate (Progynova, Schering) prepared

as a suspension in sterile water (0.3ml) and administered through oral gavage daily for one week (n=20, short-term study) and 12 weeks (n=20, long-term study) respectively (see Table 1)

4.2.3 Experimental Design

At the initiation of the experiment (0 week), all the 60 animals participating in the study were assessed sexually through copulation tests with proceptive female rats to record the baseline sexual activity in each group and to also render them sexually experienced Following administration of the drug / vehicle, these behavioural studies encompassing several parameters were once again carried out in the treated and control groups at 1 week for short-term study and at 6 and 12 weeks for the long-term study group

4.2.4 Induction of Female Receptivity

Thirty female SD rats were procured from the Laboratory Animal Centre and maintained

at the Animal Holding Unit for tests on the sexual behaviour At the initiation of the experiment, they were seven to eight weeks old and weighed between 175 - 200g Independently of whether intact or ovariectomised female rats are to be used, sexual behaviour is under the influence of ovarian hormones (Walker, 1990) In principle, it would be possible to use normally cycling females at the time of proestrus-oestrus In order to determine this phase, vaginal smear needs to be studied Furthermore, naturally cycling females conceive easily and cannot be used for subsequent investigations These complexities are the reasons for using ovariectomised and hormonally primed female rats

in this investigation Three weeks after their arrival and acclimatisation to the laboratory conditions, all the female rats were subjected to ovariectomy

4.2.4.1 Ovariectomy

The animal under halothane anaesthesia was laid on its ventral surface on the operating table A small 1 – 2cm midline dorsal skin incision was made approximately half way between the middle of the back (hump) and the base of the tail Through lateral muscle incisions, the ovaries with the surrounding fat were pulled out and excised along with the blood vessels, fallopian tubes and the uterine horns The incisions were then sutured and allowed to heal (Waynforth and Flecknell, 1992, Figure 6)

Figure 6: Principles of Rat Ovariectomy

(from Waynforth and Flecknell, 1992)

The animals were utilised for copulation tests after 3 weeks of convalescence and recovery

4.2.4.2 Priming of Female Rats

Oestrous behaviour was induced in ovariectomised female rats through sequential administration of oestrogen and progesterone Variable doses of these female sex hormones have been used by different researchers in their studies The dose of oestradiol

benzoate has ranged widely from 0.5µg to 100µg and that of progesterone from 100µg to 1mg (Winn, 1991; Murray et al., 1992 and Mani et al., 1994) In a preliminary titration at this centre (Srilatha, 1998), it was found that doses of 10µg/100g body weight of oestradiol benzoate (Sigma) and 0.5mg/100g body weight of progesterone (Biologici Italia Labs) produced optimal proceptive behaviour in the female rats and this dosage schedule was used in this investigation

Stock solution of oestradiol benzoate was made in absolute alcohol, refrigerated and subsequent dilutions prior to use were made in 50% alcohol The oestrogen was injected subcutaneously 48 hours before and the progesterone subcutaneously 4 hours before the initiation of copulation experiments The female rats with evidences for good proceptive induction in the form of hopping and darting movements, voluntary approaches to the male and lordosis were used in the study

4.2.5 Mating Experiments

Rats being nocturnal animals are best studied during the day by maintaining them under reversed lighting (Wilson CA, 1993) Copulation tests were carried out between 14.00 and 19.00hours in a separate room under dim red illumination with the animals in a rectangular perspex chamber (42X38X20cm) The males were allowed 10 minutes to acclimatise to the chamber conditions prior to introduction of the primed female Subsequent to placing the receptive female, the parameters of male sexual behaviour (McGill, 1962) were recorded and testing of each pair was concluded after a complete copulatory series leading to ejaculation and the beginning of the next series with mounting

4.2.5.1 Parameters of Sexual Receptivity in Female Rats

Oestrous / hormonally primed female rats usually initiate mating behaviour through scent-marking and orientation towards the male rats This will be followed by an initial period of mutual anogenital investigation and abrupt run away by the female causing the male to pursue the female After a brief chase, the female rat will stop and assume a pre-lordosis or presenting posture which becomes complete lordosis when the male rat mounts the female (Figure 7)

Figure 7: Female Lordosis / Receptivity

4.2.5.2 Parameters of Sexual Receptivity in Male Rats (Agmo 1997; Figures 8-11)

a Mount Latency: (ML): This is the time interval between the introduction of female rat

to the first mount by the male rat

b Intromission Latency: (IL): The interval from the time of introduction of female to the

first intromission Intromissions are distinguished behaviourally from mounts by the presence of deep thrusts and rapid springing dismounts

c Ejaculation Latency: (EL): This is the time interval calculated from first intromission

to ejaculation The ejaculation patterns were characterised by longer, deeper thrusts, slow dismounts and a period of rest following ejaculation

d Post-Ejaculation Interval or Refractory Period (PEI / PERP): The time interval of

refractoriness between the first ejaculation and the subsequent mounting of the female rat

by the male rat

e Mount Frequency: (MF): The number of mounts without intromission from the time of

introduction of female and the ejaculation by the male rat

f Intromission Frequency: (IF): The number of intromissions counted between the

introduction of female and the ejaculation by the male rat

Mount latency, frequency and post-ejaculatory mounting interval are indicators of sexual desire (central) and arousal (central and peripheral) states Intromission latency, frequency and ejaculatory latency are indicators of erectile capacity and sexual performance

4.2.5.3 Short-Term Treatment Groups

After recording the baseline rate of copulation, the male rats of these groups were treated daily with oestradiol valerate 0.01mg / 0.1mg / vehicle perorally through a gavage tube for a total duration of 1 week The copulation tests were repeated on the 8th day with proceptive female rats and the responses compared

4.2.5.4 Long-Term Treatment Groups

After recording the baseline sexuality parameters, the male rats of these groups were treated daily with oestradiol valerate 0.01mg / 0.1mg / vehicle perorally through a gavage tube for a total duration of 12 weeks Mating experiments in the presence of hormonally primed and receptive female rats were conducted at the end of 6 weeks and 12 weeks and the responses compared

4.2.6 Data Analysis

Statistical studies were done using one way analysis of variance (ANOVA) and the non parametric tests for K independent samples from the Statistical Package for Social Sciences (SPSS) version 11.5 for windows All the results were graphically expressed as mean±SEM (standard error of the mean) and the level of significance set at p<0.05

Figure 8: Mounting by the Male Rat

Figure 9: Intromission by the Male Rat

Figure 10: Ejaculation by the Male Rat

Figure 11: Post-Ejaculatory Interval / Refractory Period

4.3 Results

4.3.1 Short-Term Treatment Groups

Sexual behaviour was assessed before the initiation of drug therapy and then on the 8thpost treatment day Each pair was observed closely over the 30 minute period of interaction from the time of introduction of the female rat The results from the two treated groups were compared with those from age-matched controls (Table 5; Figures 12-17)

In control rats of this group, there were several brief mounts and shallow pelvic thrusts prior to actual insertion or intromission after which the males and females separated and groomed their genitals The average time taken to attempt mounting by the males of this group at 1 week was 0.4±0.1min These rats achieved successful intromission after 0.7±0.1min Each intromission lasted only a fraction of a second (0.25-0.5s) Ejaculation required 13.6±1.2 mounts and 16.4±1.3 intromissions Following ejaculation, the rats had

a brief period of sexual inactivity within the study duration of 30 minutes; this was followed by normal mounting of the female rat by the male rat when the observation was completed The slight difference in sexual behavioural parameters compared within the control group at 0 and 1 week was not statistically significant

Oestradiol (0.01mg) pretreatment slightly delayed both mounting and intromission (ML: 11±0.01s; IL: 4±0.02s) compared to the baseline values for this group obtained before initiating drug treatment (0.5±0.1 and 0.8±0.1min respectively) However, the animals had similar numbers of mounts and intromissions as the controls and ejaculated at 13.8±1.0min at the end of 1 week Despite continued medication, all 10 treated rats completed the entire copulatory pattern successfully

In the higher oestradiol treated rats, the mean ejaculation latencies at 0 and 1 week were 16.2±0.9min and 15.5±1.1min respectively The mount and intromission latencies increased by statistically insignificant fractions of 10.0±0.04s and 3±0.1s respectively from baseline values obtained prior to starting oral treatment (0.5±0.1min and 0.9±0.2min) when tested at the end of 1 week Between-group comparative results for all the behavioural parameters measured at the end of 1 week are given in Table 5

Short-Term Group ML (min) IL (min) MF IF EJL (min) PEI (min) Control 0.4 ±0.1 0.7±0.1 13.6±1.2 16.4±1.3 14.4±0.6 3.5±0.2

Oestradiol 0.01mg 0.6 ±0.1 0.8±0.1 14±1.1 17±1.3 13.8±1.0 4.3±0.3

Oestradiol 0.1mg 0.6 ±0.2 1.0±0.2 12.5±1.7 15.9±1.8 15.5±1.1 3.9±0.5

Table 5: Mating Parameters in Short-Term (1 week) Treatment Groups

Time taken for the first mount (ML), intromission (IL), ejaculation (EL) and subsequent mounting interval (PEI) and total numbers of mounts (MF) and intromissions (IF) in oestradiol treated rat groups and control animals (n=10, each) The difference in these parameters in the two treatment groups was not significant when compared to the values in control rats

MOUNT LATENCY

0 0.5 1

Figure 12: Mount Latency in Short-Term Treatment Group

Time taken for the first mount by the control and two doses of

oestradiol treated rats at 0 and 1 week The increase in latency at 1

week in the treatment groups was not statistically significant

(p>0.05)

INTROMISSION LATENCY

0 0.5 1 1.5

Figure 13: Intromission Latency in Short-Term

Treatment Group

Time taken for the first intromission by the control and two doses

of oestradiol treated rats at 0 and 1 week The difference in latency

at 1 week in the treatment groups was not statistically significant

(p>0.05)

MOUNT FREQUENCY

0 5 10 15

Figure 14: Mount Frequency in Short-Term Treatment Group

Total number of mounts by the male rats in three experimental groups compared at 0 and 1 week (n=10, each)

INTROMISSION FREQUENCY

0 5 10 15 20 25

Figure 15: Intromission Frequency in Short-Term

Treatment Group

Total numbers of intromissions by the male rats in three

experimental groups at 0 and 1 week (n=10, each)

EJACULATION LATENCY

0 5 10 15 20

Figure 16: Ejaculation Latency in Short-Term

Treatment Group

Time taken for the onset of ejaculation by the male rat of three

experimental groups compared at 0 and 1 week (n=10, each) The

difference was not statistically significant (p>0.05)

POST-EJACULATORY MOUNTING LATENCY

0 2 4 6

Figure 17: Post-Ejaculatory Latency in Short-Term

Treatment Group

Time taken for the first mount following ejaculation by the male

rat of three experimental groups at 0 and 1 week (n=10, each) The

difference was not statistically significant (p>0.05)

4.3.2 Long-Term Treatment Groups

Behavioural studies were carried out before the initiation of drug therapy and then at 6 weeks and 12 weeks of oestradiol administrations The study duration for mating experiments was a 30 minute period of interaction from the time of introduction of the female rat The results from the two treated groups were compared with those from age-matched and vehicle treated controls (Table 6; Figures 18-23)

Control rats of this group exhibited typical sexual behavioural pattern consisting of multiple mounts and intromissions The average time taken to attempt mounting by the males of this group at 12 weeks was 0.6±0.1min These rats achieved successful intromission after 1.0±0.1min Each intromission lasted only a fraction of a second (0.25-0.5s) In this group, ejaculation was completed after 14.8±1.2 mounts and 18.9±1.4 intromissions Following ejaculation the rats had a brief period of sexual inactivity within the study duration of 30 minutes The slight difference in sexual behavioural parameters compared within the control group at 0, 6 and 12 weeks was not statistically significant Oestradiol (0.01mg) pretreatment delayed both mounting and intromission (ML: 0.3±0.0min; IL: 1.3±0.3min) compared to the baseline values for this group However, the animals had similar numbers of mounts and intromissions as the controls and ejaculated at 15.8±0.9min when tested at the end of 12 weeks Despite continued medication, all 10 treated rats completed the 30 minute pattern successfully

In the higher oestradiol (0.1mg) treated rats of this group, there were statistically significant prolongations of intromission latency (1.4±0.3min) and post-ejaculatory interval (2.2±0.4min) when compared with the control group at 12 weeks Within the group, the mean ejaculation latencies at 0 and 12 weeks were 17.2±0.9min and 21.0±1.5min respectively At 12 weeks, mount latency increased by 0.4±0.01min, mount

and intromission frequencies reduced by counts of -2.5±0.2 and -4.3±0.1 when compared with their own baseline values Between-group comparative results for all the behavioural parameters measured at the end of 12 weeks are given in Table 6

Long-Term Group ML (min) IL (min) MF IF EJL (min) PEI (min) Control 0.6 ±0.1 1.0±0.1 14.8±1.2 18.9±1.4 13.9±0.8 3.5±0.2

Oestradiol 0.01mg 0.8 ±0.2 2.2±0.4 11.3±1.3 14.1±1.4 15.8±0.9 4.7±0.6

Oestradiol 0.1mg 1.1 ±0.1 2.4±0.5 11.3±1.2 14.4±1.1 21.0±1.5 5.8±0.7

Table 6: Mating Parameters in Long-Term Treatment Groups

Time taken for the first mount (ML), intromission (IL), ejaculation (EL) and subsequent mounting interval (PEI) and the total numbers of mounts (MF) and intromissions (IF) in oestradiol treated rat groups compared with the parameters in untreated control animals (n=10, each) The prolongations of intromission and post-ejaculatory mounting latencies in the higher oestradiol treated group were statistically significant (*p<0.05)

MOUNT LATENCY

0 1 2

) Control E2:0.01mg E2:0.1mg

Figure 18: Mount Latency in Long-Term Treatment Group

Time taken for the first mount by control and two doses of oestradiol treated rats at 0, 6 and 12 weeks (n=10, each) The increase in latency at 12 weeks in the oestradiol treatment groups was not significant

INTROMISSION LATENCY

0 1 2 3

Figure 19: Intromission Latency in Long-Term

Treatment Group

Time taken for the first intromission by control and two doses of oestradiol treated rats at 0, 6 and 12 weeks (n=10, each) The increase in latency at 12 weeks in the higher oestradiol treated group (0.1mg) was statistically significant (*p<0.05) compared to the mean value in control rats

MOUNT FREQUENCY

0 5 10 15 20

Figure 20: Mount Frequency in Long-Term Treatment Group

Total number of mounts by the male rats in three experimental groups at 0, 6 and 12 weeks (n=10, each) The decrease in counts

at 6 and 12 weeks in the oestradiol treated rats was not statistically significant

INTROMISSION FREQUENCY

0 5 10 15 20 25

Figure 21: Intromission Frequency in Long-Term

Treatment Group

Total number of intromissions by the male rats in three

experimental groups at 0, 6 and 12 weeks (n=10, each) The

decrease in counts at 6 and 12 weeks in the oestradiol treated rats

was not statistically significant