British Poultry Science
ISSN: 0007-1668 (Print) 1466-1799 (Online) Journal homepage: http://www.tandfonline.com/loi/cbps20
Sexual differentiation of copulatory behaviour in the male chick requires gonadal steroids N. Sayag , N. Snapir , E. Arnon , M. E. El Halawani , V. E. Grimm & B. Robinzon To cite this article: N. Sayag , N. Snapir , E. Arnon , M. E. El Halawani , V. E. Grimm & B. Robinzon (1991) Sexual differentiation of copulatory behaviour in the male chick requires gonadal steroids, British Poultry Science, 32:3, 607-614, DOI: 10.1080/00071669108417386 To link to this article: http://dx.doi.org/10.1080/00071669108417386
Published online: 08 Nov 2007.
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Citing articles: 7 View citing articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=cbps20 Download by: [Monash University Library]
Date: 11 November 2015, At: 10:21
British Poultry Science (1991) 32: 607-614
SEXUAL DIFFERENTIATION OF COPULATORY BEHAVIOUR IN THE MALE CHICK REQUIRES GONADAL STEROIDS
Downloaded by [Monash University Library] at 10:21 11 November 2015
N. SAYAG, N. SNAPIR, E. ARNON, M. E. EL HALAWANI, 1 V. E. GRIMM AND B. ROBINZON Department of Animal Science, Faculty of Agriculture, The Hebrew University of Jerusalem, POB 12, Rehovot 76100, Israel and 1Department of Animal Science, University of Minnesota, St. Paul, MN 55108, USA Received for publication 5th October 1989
Abstract 1. Embryonic injections of 0.3 mg/egg of tamoxifen (TAM), 0.2 mg/egg CI-628 (both antioestrogens), 0.5 mg/egg (ATD (aromatisation inhibitor), or antibodies to oestradiol (E), all suppressed male copulatory activity (MCA) in young male chicks. 2. Embryonic injections with either flutamide (F, androgen antagonist) or high dose of antibodies to testosterone (T) only slightly suppressed MCA. 3. TAM had no effect on embryonic plasma LH levels, 24 and 48 h after injection. 4. It seems that at the embryonic stage oestradiol is required for the normal differentiation of MCA.
INTRODUCTION
The differentiation of sexual behaviour in birds has been studied mainly in quails. Embryonic administration of testosterone (T) or oestradiol (E) caused demasculinisation of male sexual behaviour, but had no effect on female behaviour (Adkins, 1975; Whitsett et al., 1977) while antioestrogen administration resulted in masculinisation of female sexual behaviour and had no effect on male sexual activity (Adkins, 1976). Based on these observations, a general model for Aves has been suggested. According to this model, the male is behaviourally the neutral sex and thus normal masculine sexual behaviour can be manifested even if gonadal steroids are absent during the embryonic stage of life (Adkins, 1978). Furthermore, the male normally undergoes some demasculinisation owing to the embryonic aromatisation of endogenous T (Adkins-Regan, 1985) the high level of endogenous E in the female embryo neutralises its capacity to display masculine sexual behaviour (Adkins, 1976). Embryonic administration of exogenous T or E to the male chick demasculinised the sexual activity of the adult cockerel (Wilson and Glick, 1970) and the quail (Adkins, 1975). This was again confirmed in a study where the 607
Downloaded by [Monash University Library] at 10:21 11 November 2015
608
N. SAYAG ETAL.
masculine sexual response was measured in young chicks following activation with androgens (Sayag et al., 1991). In the male chick, embryonic injections of antioestrogen (tamoxifen; TAM), antiandrogen (flutamide; F), or aromatase inhibitor (ATD) caused demasculinisation as judged by the frequency of copulation at puberty (Sayag et al., 1989), in contrast to the model developed in the quail where ATD caused hypermasculinisation of the male (AdkinsRegan, 1985). Thus, in the male chicken embryo, an active process of masculinisation of copulatory behaviour may occur and E (produced by the gonads or aromatised from T in the brain) may play an important role in it. In the embryo TAM acts on the differentiation of chick testis (Weniger et al., 1983) and duck genital tubercle (Uchiyama and Mizuno, 1989) as an antioestrogen. However, it is possible that the steroid antagonists used previously (Sayag et al., 1989) had some agonistic effects, because high doses of TAM manifest oestrogen-agonistic effects in mammals (Patterson, 1981). In addition, it was established that interactions of oestrogen-receptor complexes with nuclear components of hypothalamic cells are a prerequisite for oestrogenic facilitation of some physiological and behavioural responses (Etgen, 1987). Because the antioestrogen CI-628 has been shown to translocate the cytosol receptors of oestrogen to hypothalamic cell nuclei (Roy and McEwen, 1979), there is a possibility that, at certain doses, the antioestrogen-receptor complex has slight oestrogenic properties. In young chicks TAM increases gonadal activity, presumably by reducing the negative feedback of gonadal steroids (Rozenboim et al, 1986; 1988; 1989; 1990). An increase in LH and gonadal steroid production in TAM- and ATD-treated embryos is also possible if a functional hypothalamo-pituitary-gonadal axis is assumed. In rats, high doses of F have been shown to increase serum LH (Furr et al., 1987). Both of the alternatives presented above suggest that the behavioural demasculinising effects observed in the male chicken following embryonic treatment with antisteroids and ATD (Sayag et al., 1989) could be caused by an increase, rather than a decrease, in oestrogenic activity. In the present study both of these possibilities were investigated by measuring masculine sexual behaviour of chicks following embryonic administration of chemical antagonists or specific antibodies to sex steroids. In addition, embryonic plasma LH was measured following injection of TAM or E.
MATERIAL AND METHODS
Fertile White Leghorn eggs were set in a rotating forced air incubator at 37*5°C and 60% relative humidity. On day 10 of incubation, a tiny hole was drilled in the small end of the shell, and one of the compounds (dissolved in 0-05 ml of vehicle) was injected into the albumen (Wilson and Glick, 1970). Hot paraffin wax was used to seal the injection hole. The injection had no effect on hatchability which was 85% for all the eggs in the incubator, regardless of the treatment. Following hatching, the sex of the chicks was identified after which they
Downloaded by [Monash University Library] at 10:21 11 November 2015
DIFFERENTIATION OF SEXUAL BEHAVIOUR
609
were placed on the floor of a heated room. During the whole experimental period, the birds were exposed to a 16:8 h light/dark cycle and food (commercial starter mash) and water were available ad libitum. Cockerels normally start displaying the full pattern of masculine sexual behaviour after 16 weeks of age (Guhl, 1961; Wood-Gush, 1971). Masculine sexual activity can, however, be activated at younger ages (Noble and Zitrin, 1942; Collias, 1950) and the method developed in a previous study (Sayag et al., 1991), in which typical male mating activity was successfully induced in male chicks by injection of androgen, was used here. On days 14 and 20, experimental chicks, of both sexes, were injected with 20 mg of testosterone oenanthate (TE; Sigma Chemical Co., USA), a long-acting androgen (Andrew, 1966) and for the behavioural test each bird was placed i n a l - 8 X l - 2 m enclosure together with 10 inact female chicks and observed for 5 min periods. The test was repeated 5 times on each bird, at 3-d intervals, from days 22 to 34 of age. Sexual activity of an intact male chick injected with TE was described as: "Crowing", a similar body posture to the "crowing" adult; Waltzing, in which the chick unfolds one wing and skips in little steps in an arc around the female; Mating attempts, seizing the neck feathers and mounting the female with one or two feet. As these three measures in chicks appear reliably to reflect.the masculine sexual motivation of the adult cockerel (Sayag et al., 1991), their frequencies were recorded in the present experiment. None of these behaviours were observed when male or female chicks of similar ages, inact or supplemented with the vehicle only, were tested under the same regimen (Sayag et al., 1991). Thus, control groups of these types were not included in the present study. Blood samples were collected from embryos on days 11 and 12 of development. The egg shell above the air space was removed (Ottinger and Bakst, 1981) and the internal content of the egg was carefully removed and placed in a small dish. Blood samples (0-2 to 0-5 ml/embryo) were immediately collected from the large extra-embryonic blood vessels with a 27G heparinised needle (Woods and Brazzill, 1981) and the sex of the embryo was determined by gonadal examination. In post-hatch chicks, blood was collected from the brachial vein at the end of the behavioural tests (35 days of age). All samples were centrifuged and plasma was stored at — 20°C pending assay. The samples were assayed individually and not in pools. Plasma was extracted with 5:2 petroleum ether (40°-60°) and benzene, in a ratio of 1:10, for the androgen assay which was determined in a single assay, using a commercial double antibody RIA kit (Boyle et al, 1984; Diagnostic Products Corporation, USA). The specific binding of the kit's antibody as defined by the supplier was: 100% testosterone, 34% 5a-DHT and 5/?-DHT, 4% 5/?-androstan-3/?,l7/?-diol, 3-3%11-hydroxytestosterone, 2-9% 5a-androstan3a,l7/?-diol, 2-7% 5a-androstan-3j?,17/?-diol, 2-1% and less than 1-0% with 31 other steroids. The minimal detectable dose for T was 2 pg/tube and the intraassay coefficient of variation was 2-4%. Plasma LH was determined in a single assay by the method of Burke et al. (1979). The minimal detectable dose was 0-1 ng/tube and the intra-assay coefficient of variation was 16-4%.
610
N. SAYAG ETAL.
Downloaded by [Monash University Library] at 10:21 11 November 2015
Experiment 1
Embryos were treated on day 10 of incubation, before the end of the critical period for the differentiation of sexual behaviour (Wilson and Glick, 1970). Each egg was injected with either 0-3 mg F, 0-3 mg TAM, 0-2 mg CI628, 0-6 mg ATD or vehicle (sesame oil). Flutamide (F), which is a pure antiandrogen (Poyet and Labrie, 1985) was kindly supplied by Schering Co., USA. The antioestrogen, TAM (Patterson, 1981) was obtained from Sigma Chemical Co., USA. CI-628 (a-4-Pyrrolidino-ethoxyl-phenyl-4-methoxy-a-nitrostilbene) was kindly supplied by Warner-Lambert Co., USA. CI-628 is also an antioestrogen which inhibits E uptake into brain cells in the rat (Roy and Wade, 1977). ATD (l,4,6-androstatriene-3,l7-dione), a potent aromatase inhibitor (Lieberburg et al., 1977), was obtained from Steraloids Inc., USA. All doses used in the present experiment were previously determined in our laboratory to be non-toxic, on the basis also of studies in quails (Adkins, 1976; Adkins-Regan et al., 1982; Adkins-Regan, 1985). After the masculine sexual response (induced by TE injections) was tested, plasma T concentrations were measured to ensure that the injections of TE produced similar blood T concentration in all the experimental birds. Experiment 2
On day 10 of incubation, embryos were injected with either antiserum to T, generated in rabbits by multiple injections of testosterone-7a-bovine serum albumin, or normal rabbit serum (NRS). Dialysis was performed to remove steroids from the antiserum and the NRS (1 ml serum in 5 1 saline+ 0-1% gelatin for 4 X 12 h). A dilution 1:25,000 of the antiserum bound about 50% of tritiated T and an estimate of the dose of antibodies necessary to neutralise the endogenous T in embryos on day 10 of incubation (Kalcheim et al., 1981) was made as follows: laboratory determinations showed that 1 pi\ of undiluted antiserum binds 200 pg T in an RIA system. The plasma T concentration in the male chick embryo reaches its peak at 300 pg/ml (Woods et al., 1975), and the total plasma volume on day 10 of incubation was estimated to be 1 ml. Based on these findings, three different doses of undiluted antiserum were used: low (L)—0-2 fA, medium (M)—0-6 fA and high (H)—1-7 fA, capable of inactivating 400, 1200 and 3400 pg of T respectively. The masculine sexual behaviour in response to TE was assayed in both females and males. Experiment 3
Embryos on day 10 of incubation were injected with either NRS or antiserum to E raised in rabbits by multiple injections of 17/?-oestradiol-6bovine serum albumin. Using an RIA system it was determined that 1 fA of antiserum binds approximately 100 pg of labelled E. The total plasma oestradiol in male and female chick embryos on day 10 of incubation was estimated to be 500 and 800 pg respectively (Woods and Brazzill, 1981). In the light of the results of experiment 2, only one dose of 28 fA/egg antiserum was
DIFFERENTIATION OF SEXUAL BEHAVIOUR
611
used capable of inactivating 2800 pg of E in in vitro conditions. The masculine sexual response of the male and female was observed.
Downloaded by [Monash University Library] at 10:21 11 November 2015
Experiment 4
On day 10 of incubation, embryos were injected with either 0-3 mg/egg of TAM, 0-5 mg/egg of oestradiol benzoate (EB; Sigma Che/nicals Co., USA) or oil. Blood samples were collected from half of the embryos at 24 h and from the rest at 48 h post injection. Plasma LH was determined. Statistical analysis of data for experiments 1, 2 and 4 was carried out using analysis of variance and Tukey's studentised range test, and for experiment 3 using the Mest (SAS User's Guide: Statistics, 1985).
RESULTS
Experiment 1
The mean values for the different characteristics of masculine sexual activity of the males are presented in Fig. 1. "Crowing" frequency was not affected by any of the embryonic injections. Both antioestrogens, TAM and CI628, reduced the manifestation of both waltzing and mating attempts (P
ISSN: 0007-1668 (Print) 1466-1799 (Online) Journal homepage: http://www.tandfonline.com/loi/cbps20
Sexual differentiation of copulatory behaviour in the male chick requires gonadal steroids N. Sayag , N. Snapir , E. Arnon , M. E. El Halawani , V. E. Grimm & B. Robinzon To cite this article: N. Sayag , N. Snapir , E. Arnon , M. E. El Halawani , V. E. Grimm & B. Robinzon (1991) Sexual differentiation of copulatory behaviour in the male chick requires gonadal steroids, British Poultry Science, 32:3, 607-614, DOI: 10.1080/00071669108417386 To link to this article: http://dx.doi.org/10.1080/00071669108417386
Published online: 08 Nov 2007.
Submit your article to this journal
Article views: 6
View related articles
Citing articles: 7 View citing articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=cbps20 Download by: [Monash University Library]
Date: 11 November 2015, At: 10:21
British Poultry Science (1991) 32: 607-614
SEXUAL DIFFERENTIATION OF COPULATORY BEHAVIOUR IN THE MALE CHICK REQUIRES GONADAL STEROIDS
Downloaded by [Monash University Library] at 10:21 11 November 2015
N. SAYAG, N. SNAPIR, E. ARNON, M. E. EL HALAWANI, 1 V. E. GRIMM AND B. ROBINZON Department of Animal Science, Faculty of Agriculture, The Hebrew University of Jerusalem, POB 12, Rehovot 76100, Israel and 1Department of Animal Science, University of Minnesota, St. Paul, MN 55108, USA Received for publication 5th October 1989
Abstract 1. Embryonic injections of 0.3 mg/egg of tamoxifen (TAM), 0.2 mg/egg CI-628 (both antioestrogens), 0.5 mg/egg (ATD (aromatisation inhibitor), or antibodies to oestradiol (E), all suppressed male copulatory activity (MCA) in young male chicks. 2. Embryonic injections with either flutamide (F, androgen antagonist) or high dose of antibodies to testosterone (T) only slightly suppressed MCA. 3. TAM had no effect on embryonic plasma LH levels, 24 and 48 h after injection. 4. It seems that at the embryonic stage oestradiol is required for the normal differentiation of MCA.
INTRODUCTION
The differentiation of sexual behaviour in birds has been studied mainly in quails. Embryonic administration of testosterone (T) or oestradiol (E) caused demasculinisation of male sexual behaviour, but had no effect on female behaviour (Adkins, 1975; Whitsett et al., 1977) while antioestrogen administration resulted in masculinisation of female sexual behaviour and had no effect on male sexual activity (Adkins, 1976). Based on these observations, a general model for Aves has been suggested. According to this model, the male is behaviourally the neutral sex and thus normal masculine sexual behaviour can be manifested even if gonadal steroids are absent during the embryonic stage of life (Adkins, 1978). Furthermore, the male normally undergoes some demasculinisation owing to the embryonic aromatisation of endogenous T (Adkins-Regan, 1985) the high level of endogenous E in the female embryo neutralises its capacity to display masculine sexual behaviour (Adkins, 1976). Embryonic administration of exogenous T or E to the male chick demasculinised the sexual activity of the adult cockerel (Wilson and Glick, 1970) and the quail (Adkins, 1975). This was again confirmed in a study where the 607
Downloaded by [Monash University Library] at 10:21 11 November 2015
608
N. SAYAG ETAL.
masculine sexual response was measured in young chicks following activation with androgens (Sayag et al., 1991). In the male chick, embryonic injections of antioestrogen (tamoxifen; TAM), antiandrogen (flutamide; F), or aromatase inhibitor (ATD) caused demasculinisation as judged by the frequency of copulation at puberty (Sayag et al., 1989), in contrast to the model developed in the quail where ATD caused hypermasculinisation of the male (AdkinsRegan, 1985). Thus, in the male chicken embryo, an active process of masculinisation of copulatory behaviour may occur and E (produced by the gonads or aromatised from T in the brain) may play an important role in it. In the embryo TAM acts on the differentiation of chick testis (Weniger et al., 1983) and duck genital tubercle (Uchiyama and Mizuno, 1989) as an antioestrogen. However, it is possible that the steroid antagonists used previously (Sayag et al., 1989) had some agonistic effects, because high doses of TAM manifest oestrogen-agonistic effects in mammals (Patterson, 1981). In addition, it was established that interactions of oestrogen-receptor complexes with nuclear components of hypothalamic cells are a prerequisite for oestrogenic facilitation of some physiological and behavioural responses (Etgen, 1987). Because the antioestrogen CI-628 has been shown to translocate the cytosol receptors of oestrogen to hypothalamic cell nuclei (Roy and McEwen, 1979), there is a possibility that, at certain doses, the antioestrogen-receptor complex has slight oestrogenic properties. In young chicks TAM increases gonadal activity, presumably by reducing the negative feedback of gonadal steroids (Rozenboim et al, 1986; 1988; 1989; 1990). An increase in LH and gonadal steroid production in TAM- and ATD-treated embryos is also possible if a functional hypothalamo-pituitary-gonadal axis is assumed. In rats, high doses of F have been shown to increase serum LH (Furr et al., 1987). Both of the alternatives presented above suggest that the behavioural demasculinising effects observed in the male chicken following embryonic treatment with antisteroids and ATD (Sayag et al., 1989) could be caused by an increase, rather than a decrease, in oestrogenic activity. In the present study both of these possibilities were investigated by measuring masculine sexual behaviour of chicks following embryonic administration of chemical antagonists or specific antibodies to sex steroids. In addition, embryonic plasma LH was measured following injection of TAM or E.
MATERIAL AND METHODS
Fertile White Leghorn eggs were set in a rotating forced air incubator at 37*5°C and 60% relative humidity. On day 10 of incubation, a tiny hole was drilled in the small end of the shell, and one of the compounds (dissolved in 0-05 ml of vehicle) was injected into the albumen (Wilson and Glick, 1970). Hot paraffin wax was used to seal the injection hole. The injection had no effect on hatchability which was 85% for all the eggs in the incubator, regardless of the treatment. Following hatching, the sex of the chicks was identified after which they
Downloaded by [Monash University Library] at 10:21 11 November 2015
DIFFERENTIATION OF SEXUAL BEHAVIOUR
609
were placed on the floor of a heated room. During the whole experimental period, the birds were exposed to a 16:8 h light/dark cycle and food (commercial starter mash) and water were available ad libitum. Cockerels normally start displaying the full pattern of masculine sexual behaviour after 16 weeks of age (Guhl, 1961; Wood-Gush, 1971). Masculine sexual activity can, however, be activated at younger ages (Noble and Zitrin, 1942; Collias, 1950) and the method developed in a previous study (Sayag et al., 1991), in which typical male mating activity was successfully induced in male chicks by injection of androgen, was used here. On days 14 and 20, experimental chicks, of both sexes, were injected with 20 mg of testosterone oenanthate (TE; Sigma Chemical Co., USA), a long-acting androgen (Andrew, 1966) and for the behavioural test each bird was placed i n a l - 8 X l - 2 m enclosure together with 10 inact female chicks and observed for 5 min periods. The test was repeated 5 times on each bird, at 3-d intervals, from days 22 to 34 of age. Sexual activity of an intact male chick injected with TE was described as: "Crowing", a similar body posture to the "crowing" adult; Waltzing, in which the chick unfolds one wing and skips in little steps in an arc around the female; Mating attempts, seizing the neck feathers and mounting the female with one or two feet. As these three measures in chicks appear reliably to reflect.the masculine sexual motivation of the adult cockerel (Sayag et al., 1991), their frequencies were recorded in the present experiment. None of these behaviours were observed when male or female chicks of similar ages, inact or supplemented with the vehicle only, were tested under the same regimen (Sayag et al., 1991). Thus, control groups of these types were not included in the present study. Blood samples were collected from embryos on days 11 and 12 of development. The egg shell above the air space was removed (Ottinger and Bakst, 1981) and the internal content of the egg was carefully removed and placed in a small dish. Blood samples (0-2 to 0-5 ml/embryo) were immediately collected from the large extra-embryonic blood vessels with a 27G heparinised needle (Woods and Brazzill, 1981) and the sex of the embryo was determined by gonadal examination. In post-hatch chicks, blood was collected from the brachial vein at the end of the behavioural tests (35 days of age). All samples were centrifuged and plasma was stored at — 20°C pending assay. The samples were assayed individually and not in pools. Plasma was extracted with 5:2 petroleum ether (40°-60°) and benzene, in a ratio of 1:10, for the androgen assay which was determined in a single assay, using a commercial double antibody RIA kit (Boyle et al, 1984; Diagnostic Products Corporation, USA). The specific binding of the kit's antibody as defined by the supplier was: 100% testosterone, 34% 5a-DHT and 5/?-DHT, 4% 5/?-androstan-3/?,l7/?-diol, 3-3%11-hydroxytestosterone, 2-9% 5a-androstan3a,l7/?-diol, 2-7% 5a-androstan-3j?,17/?-diol, 2-1% and less than 1-0% with 31 other steroids. The minimal detectable dose for T was 2 pg/tube and the intraassay coefficient of variation was 2-4%. Plasma LH was determined in a single assay by the method of Burke et al. (1979). The minimal detectable dose was 0-1 ng/tube and the intra-assay coefficient of variation was 16-4%.
610
N. SAYAG ETAL.
Downloaded by [Monash University Library] at 10:21 11 November 2015
Experiment 1
Embryos were treated on day 10 of incubation, before the end of the critical period for the differentiation of sexual behaviour (Wilson and Glick, 1970). Each egg was injected with either 0-3 mg F, 0-3 mg TAM, 0-2 mg CI628, 0-6 mg ATD or vehicle (sesame oil). Flutamide (F), which is a pure antiandrogen (Poyet and Labrie, 1985) was kindly supplied by Schering Co., USA. The antioestrogen, TAM (Patterson, 1981) was obtained from Sigma Chemical Co., USA. CI-628 (a-4-Pyrrolidino-ethoxyl-phenyl-4-methoxy-a-nitrostilbene) was kindly supplied by Warner-Lambert Co., USA. CI-628 is also an antioestrogen which inhibits E uptake into brain cells in the rat (Roy and Wade, 1977). ATD (l,4,6-androstatriene-3,l7-dione), a potent aromatase inhibitor (Lieberburg et al., 1977), was obtained from Steraloids Inc., USA. All doses used in the present experiment were previously determined in our laboratory to be non-toxic, on the basis also of studies in quails (Adkins, 1976; Adkins-Regan et al., 1982; Adkins-Regan, 1985). After the masculine sexual response (induced by TE injections) was tested, plasma T concentrations were measured to ensure that the injections of TE produced similar blood T concentration in all the experimental birds. Experiment 2
On day 10 of incubation, embryos were injected with either antiserum to T, generated in rabbits by multiple injections of testosterone-7a-bovine serum albumin, or normal rabbit serum (NRS). Dialysis was performed to remove steroids from the antiserum and the NRS (1 ml serum in 5 1 saline+ 0-1% gelatin for 4 X 12 h). A dilution 1:25,000 of the antiserum bound about 50% of tritiated T and an estimate of the dose of antibodies necessary to neutralise the endogenous T in embryos on day 10 of incubation (Kalcheim et al., 1981) was made as follows: laboratory determinations showed that 1 pi\ of undiluted antiserum binds 200 pg T in an RIA system. The plasma T concentration in the male chick embryo reaches its peak at 300 pg/ml (Woods et al., 1975), and the total plasma volume on day 10 of incubation was estimated to be 1 ml. Based on these findings, three different doses of undiluted antiserum were used: low (L)—0-2 fA, medium (M)—0-6 fA and high (H)—1-7 fA, capable of inactivating 400, 1200 and 3400 pg of T respectively. The masculine sexual behaviour in response to TE was assayed in both females and males. Experiment 3
Embryos on day 10 of incubation were injected with either NRS or antiserum to E raised in rabbits by multiple injections of 17/?-oestradiol-6bovine serum albumin. Using an RIA system it was determined that 1 fA of antiserum binds approximately 100 pg of labelled E. The total plasma oestradiol in male and female chick embryos on day 10 of incubation was estimated to be 500 and 800 pg respectively (Woods and Brazzill, 1981). In the light of the results of experiment 2, only one dose of 28 fA/egg antiserum was
DIFFERENTIATION OF SEXUAL BEHAVIOUR
611
used capable of inactivating 2800 pg of E in in vitro conditions. The masculine sexual response of the male and female was observed.
Downloaded by [Monash University Library] at 10:21 11 November 2015
Experiment 4
On day 10 of incubation, embryos were injected with either 0-3 mg/egg of TAM, 0-5 mg/egg of oestradiol benzoate (EB; Sigma Che/nicals Co., USA) or oil. Blood samples were collected from half of the embryos at 24 h and from the rest at 48 h post injection. Plasma LH was determined. Statistical analysis of data for experiments 1, 2 and 4 was carried out using analysis of variance and Tukey's studentised range test, and for experiment 3 using the Mest (SAS User's Guide: Statistics, 1985).
RESULTS
Experiment 1
The mean values for the different characteristics of masculine sexual activity of the males are presented in Fig. 1. "Crowing" frequency was not affected by any of the embryonic injections. Both antioestrogens, TAM and CI628, reduced the manifestation of both waltzing and mating attempts (P
