Developmental Profile of Plasma Androgens in Cockerels Genetically Selected for Mating Frequency1

Developmental Profile of Plasma Androgens in Cockerels Genetically Selected for Mating Frequency1 T. L. DE SANTO, H. P. VAN KREY, and P. B. SIEGEL Department of Poultry Science F. C. GWAZDAUSKAS

(Received for publication February 11, 1983) ABSTRACT Plasma levels of androstenedione (AE), testosterone (T), and dihydrotestosterone (DHT) were measured at 1, 56, 112, and 168 days of age by radioimmunoassay in lines of cockerels divergently selected for male mating frequency. Values for total androgen (total A = AE + DHT + T) were also computed. No significant differences in mean hormone values were found between lines at any age. Hormone patterns throughout development were also similar for both lines. Plasma AE and T increased between Days 1 and 56, stabilized through Day 112, and rose again prior to 168 days of age. In contrast, DHT levels were low throughout Day 112 and rose significantly by Day 168. Total A in the high mating line was low throughout Day 112 with significant increases occurring by Day 168. In the low mating line, total A was low on Day 1, increased significantly by Day 56, and it remained unchanged through Day 112. Peak values occurred by Day 168. Within line correlation analyses between AE, T, DHT, and total A revealed a more uniform hormonal state throughout development in the high mating line than in the low mating line. Because no differences were found between the mating lines in baseline levels of individual androgens or in concentration patterns of androgens up through the attainment of sexual maturity, it appears that neither posthatch baseline levels nor posthatch temporal androgen concentrations control male sexual behavior in the mating lines of birds. (Key words: mating frequency, plasma androgens) 1983 Poultry Science 62:2249-2254 INTRODUCTION A l t h o u g h a great deal of information has been published regarding male sexual behavior, c o n t r o l of t h e intensity of t h e behavior remains relatively vague. G r u n t and Y o u n g ( 1 9 5 2 ) d e m o n s t r a t e d t h a t e x o g e n o u s levels of testost e r o n e greater t h a n threshold were n o t a p r i m a r y factor regulating male guinea pig libido. Nevertheless, m a n y s u b s e q u e n t studies involving h o r m o n a l control of t h e intensity of male sexual activity have focused primarily o n t e s t o s t e r o n e w i t h relatively little regard given t o o t h e r androgenic and estrogenic c o m p o u n d s . Because o t h e r gonadal h o r m o n e s have b e e n implicated in t h e control of sexual behavior in a variety of animal classes including Aves (reviewed b y Adkins-Regan, 1 9 8 1 ) , it is con-

1 Data were presented in part at the 71st Annual Meeting of the Poultry Science Association, August 1982.

ceivable t h a t several gonadal steroids acting in concert m a y control sexual behavior and t h a t relative titers of a family of such steroids m a y be critical t o t h a t c o n t r o l . Developmental relationships m u s t also b e given consideration w h e n a t t e m p t i n g t o define h o r m o n a l c o n t r o l of male sexual behavior. Prehatch critical periods (i.e., periods during which t h e presence or absence of gonadal h o r m o n e s affect differentiation of neural tissue and, as a result, s u b s e q u e n t sexual behavior) are k n o w n t o exist in b o t h m a m m a l s (Gorski, 1 9 7 9 ) and birds (Adkins, 1 9 7 5 ) . F u r t h e r m o r e , similar p o s t h a t c h critical periods m a y also exist in domestic fowl. J o n e s ( 1 9 7 4 ) f o u n d t h a t testes a u t o t r a n s p l a n t a t i o n into t h e b o d y cavity of 4-week-old cockerels stimulated adult copulatory behavior. She speculated that transient declines in e n d o g e n o u s t e s t o s t e r o n e m a y have been a factor precipitating t h e response.

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Animal p o p u l a t i o n s selected for differences

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Department of Dairy Science, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061

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DE SANTO ET AL.

MATERIALS AND METHODS

Stock and Management. Birds utilized in this study were males obtained from within line matings of S2i generation high (HML) and low (LML) mating lines of chickens that had been selected divergently for cumulative number of completed matings (Siegel, 1972). When tested for cumulative mating frequency in eight 10-min trials, HML males from the S21 generation (parent stock) completed a mean of 23.45 matings, whereas the mean for the LML was .27 matings. Furthermore, the frequency of nonmaters for males of the S2i generation was 0% for the HML and 84% for the LML. For the initial phase of the experiment, newly hatched chicks were exsanguinated by cardiac puncture with a heparinized syringe and then laparotomized to verify the sex. Blood samples obtained from 18 males from each mating line were centrifuged at 4850 X g for 10 min; plasma fractions were recovered and stored at - 2 0 C. For the second part of the study, 20 HML and LML male chicks were utilized. Upon hatching, each bird was wingbanded, vaccinated against Marek's disease, and vent sexed. The males were raised in unisexual flocks in litter floor pens and received food and water ad libitum. A 16-hr light cycle (0600 to 2200 hr) was maintained for the duration of the experiment. Between 0800 and 0930 hr, when the birds were 56, 112, and 168 days of age, 3.0-ml blood samples were collected by cardiac puncture with a heparinized syringe and processed as described. Radioimmunoassays. One-milliliter plasma samples were extracted three times with 5.0 ml of iso-octane. Celite columns (68 X 6 mm) were used for separation of androgens (Abraham and Manlimos, 1977). The elution procedure employed was that of Saksena et al. (1977). The

dried eluate residues were redissolved in 2.0 ml of methylene chloride: methanol (9:1, v/v), and known aliquots were removed for assay and estimation of procedural losses. Androstenedione (AE) and dihydrotestosterone (DHT) were quantitated according to the methods of Abraham et al. (1975) and Abraham and Manlimos (1977). The AE and DHT antibodies were obtained from Optimox, Inc., Palos Verdes, CA. Testosterone (T) determinations were conducted according to the procedures of Smith and Hafs (1973) and Kattesh et al. (1979). Testosterone antibody was donated by J. Ireland, Michigan State University, East Lansing, MI. All assays were validated using a plasma pool obtained from S2i generation HML and LML adult males. The percentage of recovery, the between assay coefficient of variation, and the within assay coefficient of variation for each assay were as follows: AE, 69%, 4.0%, 6.6%; DHT, 50%, 13.6%, 8.3%; and T, 56%, 11.4%, 5.7%. Statistical Analyses. Means for each hormone were compared between lines at each age using analysis of variance. The age effect within a given hormone for each line was determined by analysis of variance and significant age differences were identified utilizing Duncan's multiple range test. Product moment correlations were calculated between the hormonal traits within each line. RESULTS AND DISCUSSION

Mean plasma concentrations of DHT, AE, T, and total A (DHT + AE + T) are presented in Table 1. No significant differences were found between the lines for any of the hormones tested at any age through sexual maturity. Levels of DHT remained relatively low in both of the mating lines through Day 112. By Day 168, mean plasma levels of DHT increased significantly. At this age, males from the selected lines exhibit full mating activity (Cook and Siegel, 1972). To help place the current DHT data into perspective, note that Mashaly and Glick (1979) found serum DHT levels for New Hampshire cockerels at 112 days of age (.059 ng/ml) comparable to those of this study. Values for sexually mature birds, however, were somewhat lower (.075 ng/ml) than those obtained in this study. Possible explanations for the discrepancy in hormone concentration would be differences in assay procedures and

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in libido should provide an excellent opportunity to examine possible relationships between hormonal levels and sexual behavior. Accordingly, this endocrinological study was conducted utilizing populations of chickens bidirectionally selected for mating frequency (Siegel, 1972). A profile of several selected testicular steroids was developed, their developmental patterns were assessed, and the relationships between the hormones in cockerels from hatch through the attainment of sexual maturity were analyzed.

.094 ± . 0 9 4 a (17)

.063 ± .010 a (10)

.074 + .008 a (12)

.146 ± .017 b (15)

.082 ± .014 a (16)

.058 ± .010 a (13)

.159 + .012 b (15)

LML

.072 ± .009 a ( 9) 3

HML

.967+ .129 c (20)

.483 ± .044 b (20)

.498 ± .089 b (20)

.091± .017^ (16)

HML

3

2

1

Differences between mating lines were not significant (P<.05).

Number of cockerels.

Includes only individuals for which a complete profile was obtained.

1.119±.138C (20)

.980 ± (20)

.518 + (20)

.578 ± . 0 8 5 b (20)

.461 ± .060 b (20) .908 ± .076 c (20)

.584 ± (20)

.455 ± . 0 9 7 a b (20)

.409 ± .074 b (20)

LML

.084 + (16)

, „

Testosterone

(ng/ml) .160 ± .084* (13)

,

HML

Plasm;a concentrations

.068 + .008 a (17)

LML

Androstenedione

a ' b Means (± SE) within columns with different superscripts are significant (P<.01).

168

112

56

1

(days)

Age

Dihydrotestosterone

TABLE 1. Plasma androgen levels in high (HML) and low mating line (LML) cockerels at v

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Total A in plasma in the HML did not differ significantly through 112 days of age (Table 1), but a significant increase did occur by Day 168. In the LML, total A was low on Day 1, increased significantly by Day 56, and remained unchanged through Day 112. Peak total A values occurred by Day 168. Although statistically significant differences were found between the mating lines for temporal patterns of total A, these differences may be misleading. A small sample size was available for the HML on Day 1; patterns of androgen concentration were actually quite similar between the lines. Correlations between plasma concentrations of the gonadal hormones at various ages posthatch are summarized in Table 2. The data show a higher incidence of significant correlations within the HML when compared to the LML. Although this may be suggestive of a relatively more orderly and interrelated hormonal profile in the HML males, no consistent patterns were evident. These data, therefore, imply that relative steroid titer ratios at

TABLE 2. Correlation coefficients (r) between plasma levels of dibydrotestosterone (DHT), androstenedione (AE), testosterone (T), and total androgen (total A) in high and low mating line cockerels at various ages posthatch High mating line r

n

r

Total A

T

AE

DHT

n

r

n

r

n

Low mating line DHT

(days) .12 -.18 .71** .22

AE

T

Total A

*P«.05. **P<.01.

Age

6 16 13 15

-.06 .03 .01 -.19

14 10 12 15

.23 .03 .40 .08

13 10 12 15

-.03 -.09 .46* .74**

14 20 20 20

.72* .07 .27 .05

11 10 12 15

-.04 .12 .89** .88**

11 10 12 15

.56*

6 16 13 15

.54 -.11 .82** .44

4 16 13 15

1 56 112 168

-.22 .86** .82** .70**

11 20 20 20

.79 .96** .91** .95**

4 16 13 14

1 56 112 168

-.18 .96** .98**

4 16 13 15

1 56 112 168

.41 -.16 70* *

07**

.41 .97** .90** .97**

11 10 12 15

1 56 112 168

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differences in breed or strain types. Also, Schanbacher et al. (1974) reported a definite daily rhythm in hormonal concentration in the domestic fowl. Our blood samples were all collected at a consistent time, but the time of day that blood samples were obtained by Mashaly and Glick (1979) is unknown. Developmental patterns for AE and T were similar for each of the lines (Table 1). Concentrations were low on Day 1, increased significantly by Day 56, and remained unchanged at Day 112. Further significant increases occurred in both lines by Day 168. Mashaly and Glick (1979) reported serum T levels in New Hampshire cockerels at 112 and 168 days of age that were comparable to the values obtained in the current study. Values reported by Tanabe et al. (1979) for newly hatched White Leghorn cockerels were, however, greater than those of this study. No report of AE levels in the domestic cockerel could be located in the literature for comparative purposes.

PLASMA ANDROGENS IN COCKERELS

been shown to occur in a variety of mammalian species following specific sexual behaviors (reviewed by Harding, 1981). For example, male guinea pigs exhibiting increased sexual activity showed greater plasma T levels following exposure to estrous females and after sexual encounters than did low activity males (Harding and Feder, 1976), and vigorously mating male rodents showed rapid increases in plasma T levels upon exposure to receptive females (Damassa et al, 1977; Batty, 1978). Also, behaviors preceding the consummatory act, rather than copulation itself, were correlated with increasing plasma T levels. Studies have also attempted to link mating behavior with pituitary and hypothalamic secretions. In rodents, it has been determined that plasma luteinizing hormone (LH) increases during the initial stages of sexual behavior (Kamel et al, 1977; Coquelin and Bronson, 1979). Furthermore, LH releasing hormone, when given in addition to gonadal hormones, was capable of facilitating male sexual behavior (Moss et al, 1975). Similar studies have not been conducted with avian males. In view of the aforementioned studies, as well as the interesting hypothesis of Leshner (1979), which takes into account the interrelationship of environmental stimuli, behavioral actions, and momentary hormonal fluctuations, an interesting possibility arises concerning the mating lines. It is conceivable that genetic selection has affected momentary hormonal responsiveness to behavioral and environmental stimuli, and because of limited initial hormonal surges at the onset of a mating sequence, copulatory behavior occurs less frequently in the LML males. This hormonal responsiveness hypothesis is currently under investigation in our laboratory.

ACKNOWLEDGMENTS

This study was supported in part by NSF Grant BNS 78-24493. In addition, acknowledgments are extended to the Hubbard Farms Charitable Foundation for their financial support. The authors also wish to thank D. Aalseth, G. Anderson, J. Bame, M. Lacy, and J. Shelton for their technical assistance; J. Ireland for the testosterone antibody; and M. Riley and L. Wirt for their assistance in the preparation of the manuscript.

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critical stages during development were not contributing to the differential mating patterns exhibited by adults from the mating lines. Because no differences were found between the mating lines with respect to baseline levels of individual androgens or in concentration patterns of androgens up through the attainment of sexual maturity, it appears that neither posthatch baseline levels nor posthatch temporal androgen concentrations control male sexual behavior in the mating lines of birds. These results are not in agreement with those of Jones (1974). She found that testes autotransplantation stimulated significant increases in copulatory behavior in HML males and, as a result, theorized that a transient relative gonadal steroid deficiency affected subsequent sexual behavior, i.e., a posthatch critical period. Although baseline concentrations or hormone ratios were not primary in the control of libido in the mating line cockerels, these factors should be investigated in embryos from the two lines. It is possible that prehatch sensitivity to hormones is greater than posthatch sensitivity. Furthermore, in view of the rapid rate of development of the avian embryo, it is conceivable that a difference of only several hours in the onset of steroidogenesis could have a profound effect on development of what has been termed the "behavioral substrate" (van Tienhoven, 1968). Alternate possibilities explaining line differences in sexual behavior exist. In chickens, T is reduced to both 5a- and 5(3-DHT, but predominately 5j3-reduction occurs in avian neural tissues (Nakamura and Tanabe, 1974; Massa et al, 1977; Balthazart and Hirschburg, 1979; Balthazart et al, 1979; Massa and Sharp, 1981). Although the data of Adkins-Regan (1981) suggest that 5]3-DHT does not stimulate copulatory behavior in male quail, stimulation of precocial copulatory behavior has been evoked in cockerels through the administration of 50-DHT (Balthazart and Hirschburg, 1979). The effect that 5/3-DHT exerts on male libido is unknown. The antibody employed for hormone analysis in this study was not specific for either form of DHT; the 5a-DHT antibody utilized crossreacted 30% with 5|3-DHT (Abraham, personal communication). Thus, line differences with respect to 5a- and 5/3 DHT ratios remain unknown. Another plausible explanation for the mating line differences concerns momentary hormonal fluctuations. This phenomenon has

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2254 REFERENCES

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