Effect of photoperiod on the diurnal rhythm of plasma testosterone, dihydrotestosterone and androstenedione in mature male chickens

Camp. Biochem.

Physiol.

Vol. 8lA, No.

3, pp. 715-779,

1987

Printed in Great Britain

0

0300-9629/87 $3.00 + 0.00 1987 PergamonJournalsLtd

EFFECT OF PHOTOPERIOD ON THE DIURNAL RHYTHM OF PLASMA TESTOSTERONE, DIHYDROTESTOSTERONE AND ANDROSTENEDIONE IN MATURE MALE CHICKENS SUZANNE E. BACHMAN,JEANNETTEM. BACHMANand MAGDI M. MASHALY* Department of Poultry Science, The Pennsylvania State University, University Park, PA 16802, USA (Received 14 October 1986) Abstract-l. The effects of different photoperiods on the concentrations of plasma androgens, testes weight and on the diurnal rhythm of plasma testosterone (T), dihydrotestosterone (DHT) and androstenedione (A) in mature, single comb White Leghorn male chickens were studied. 2. Birds were exposed to either 14 hr of light (lights on at 0600-2000 hr) or to 24 hr of light per day. 3. Blood samples were collected from birds in both groups at 3-hr intervals and plasma levels of T, DHT and A were measured using radioimmunoassays. Following blood collection, birds were weighed, killed and testis weights were recorded. 4. Under 14 hr of light, there was a diurnal rhythm of T and DHT with hormone concentrations peaking at the end of the dark period. There was no obvious rhythm for A. Exposure to 24 hr of light abolished the diurnal rhythm found under 14 hr of light. 5. There was an increase not only in testis weights but also in body weight and hormone concentrations under 24 hr of light. 6. It was concluded that .ohotoDeriod davs an important role in controlling the concentration and . rhythm of androgens in mature male chickens. 1

I~

INTRODUCITON

The reproductive cycle of several species of birds and mammals may be influenced by changes in photoperiod. Light stimulates the release of FSH and LH which exert their effects on the testis to stimulate spexmatogenesis and androgen production. Rhythms of androgens have been demonstrated in rams (Lincoln et al., 1977), rhesus monkeys (Plant, 1981) and man (Miyatake er al., 1980) with the highest concentration of testosterone found during the dark period. In a majority of temperate zone birds, reproductive functioning is dependent on seasonal changes in photoperiod length (van Tienhoven and Planck, 1973). Exposure to long photoperiods induces gondal growth and increases concentrations of gonadotrophins and testosterone in the Japanese quail (Nicholls et al., 1973; Follett, 1976) and the ring dove (Balthazart et al., 1981) while short photoperiods result in gonadal regression and decreases concentrations of gonadotrophins and testosterone. Although the domestic fowl is not a seasonal breeder, diurnal rhythms of spermatogenesis (Riley, 1940; McCartney, 1942), ejaculate volume (Lake and Wood-Gush, 1956) and testicular temperature (Langford and Howarth, 1972) have been demonstrated in chickens. The time of greatest spermatogenic activity varies between reports, however, it is suggested to occur during the dark period. This time period coincides with the time of increased testosterone levels (Schanbacher et al., 1974). *To whom correspondence should be addressed at: Department of Poultry Science, 202 Henning Building, University Park, PA 16802, USA.

Variations in reported testosterone levels in the domestic fowl may be due to differences in age or breed (Sturkie, 1976); however, since testosterone exhibits a diurnal rhythm (Schanbacher et al., 1974), the time of blood sampling or the length of the photoperiod may affect the hormone concentration. Knowledge of the pattern of androgen fluctuations is important in the determination of optimum sampling time for androgen analysis. The objectives of this study were to investigate the diurnal rhythm of androgens under different light regimes and the effect of photoperiod length on serum androgen concentrations and testis weight. MATERIALS AND METHODS Single comb White Leghorn male chickens at 31 weeks of age were randomly divided into two groups of 10 birds each. Birds were housed in individual cages and exposed to either 14 hr of light (0600-2000 hr) and 10 hr of darkness (2000+%0 hr) or 24 hr of light. Prior to the beginning of the experiment, birds were exposed to 14 hr of light. Food and water were available od libitum. Birds were maintained under the different photoperiods (14 or 24 hr of light) for a 4-week adaptation period prior to blood collection. Two days before blood collection, a polyethylene cannula (Intramedic PE-50) containing heparin (100 units/ml) was inserted into the left brachial vein of each chicken. The cannula was secured to the blood vessel using surgical silk, occluded with wax and taped to the wing. Serial blood samples (4 ml) were obtained from the birds at 3-hr intervals (0900, 1200, 1500, 1800, 2100, 0300 and 0600 hr). Blood was immediately centrifuged at 17OOgand plasma removed and frozen until assayed. Red blood cells were resuspended in sterile physiological saline and reinjected into the bird at each time interval to maintain blood volume

775

176

SUZANNE E. BACHMAN etal.

and hematocrit. After the last blood sample was obtained, birds were weighed, sacrificed and testis weights were recorded. Plasma levels of testosterone Q, dihydrotestosterone (DHT), and androstenedione (A) were analyzed by radioimmunoassay techniques as described by Mashaly and Click (1979). Testosterone and DHT were assayed using antibody supplied by Dr Gordon Niswender of Colorado State University. The antibody crossreacts 100% with T and 69% with DHT. Testosterone and DHT were separated by column chromatography. Androstenedione antibody was purchased from Miles-Yeda Ltd. The cross reactivity of the antibody with A and T is 100 and 2.85%, respectively. All samples for each hormone were measured in one assay. One way analysis of variance was used to determine statistical significance due to differences in the time of blood sampling. Student’s t-test was used to determine significance due to differences in light exposure. RESULTS In the 14-hr group, individual values for each bird were averaged because the hormone profiles were similar. The diurnal rhythms of T and DHT in the 14-hr group are shown in Fig. 1. In this group significant peaks of plasma T (2245 +_780 pg/ml) and plasma DHT (105 + 18 pg/ml) were observed at 0600 hr (end of the dark period). The highest concentration of plasma A (2503 + 193 pg/ml) was also

14 hours

z

0 IO

0 05

d 2100

2400

0300

0600

0900

1200

I600

Fig. 1. Concentrations (ng/ml) of T, DHT, and A in plasma of 35-week-old single comb White Leghorn male chickens exposed to 14 hr of light (0600-2000 hr). Values are means f SEM. Dark bars indicate the dark period, *Denotes values which are significantly different (P > 0.05) from values at other time periods for each hormone.

6!-

Time

Fig. 2. Concentration

1500

Time

(ng/ml) of testosterone in plasma of 35-week-old individual single comb White Leghorn male chickens exposed to 24 hr of light.

777

Rhythm of androgens in chickens

observed

at 0600 hr, but it was not significantly

Table 1. Hormone concentrations

(pg/ml) in 35-week-old single comb White Leghorn male chickens exposed to 14 hr of light

(P < 0.05)different from the other time periods. Furthermore, unlike T and DHT which reached a single peak, A exhibited additional peaks during the light period. Average concentrations of T and A were higher during the dark period whereas the average DHT concentration was higher during the light period (Table 1); however, differences were not significant (P > 0.05). Birds subjected to 24 hr of light did not exhibit the synchronized rhythm of hormone concentrations found in the 14-hr group but rather has a freerunning rhythm. Concentrations of T (Fig. 2) and DHT (Fig. 3) exhibited several peaks although none were significant (P > 0.05). Most birds had one of A (Fig. 4); however, only the peak at 1200 hr in the second group of birds was significant (P < 0.05). Comparison of average hormone concentrations between the 14-hr and 24-hr groups reveals a dramatic increase in hormone concentration under 24 hr of light. Exposure to 24 hr of light resulted in threefold. 46-fold, and four-fold increases in T, DHT, and A concentration, respectively (Table 2). Body weight and testis weight were also significantly higher (Table 2). These birds tended to be more aggressive and possessed combs which were larger and brighter in color.

Hormone

Light period

Dark period

Testosterone DHT Androstenedione

669 k 140 88 * 39 2062+ 117

850 +- 238 59 + 9 2102 + 134

Values are means + SEM. There were no significant differences (P > 0.05) between the light and dark periods for any of the hornones.

during the dark period with a peak value at the end of the dark period. It is known that at the onset of darkness there is an increase in LH concentration (Scanes et al., 1978) and melatonin concentration Table 2. Comparison of various parameters (means j, SEM) between 35-week-old single comb White Leghorn male chickens exposed to 14 or 24 hr of light Light treatment 14hr

24 hr

Testis wt (g) (P < 0.01)

6.91 & 0.80

13.06% 1.69

Body wt (g) (P < 0.01)

1997 + 52

2282 +- 66

0.3468 +_0.400

0.5714 * 0.800

2.080 + 0.088

8.357 + 1.328

0.763 + 140

I.983 k 0.299

0.053 + 0.006

2.883 + 0.288

Parameter

Relative testis wt (g/loo g body wt) (P < 0.001) Androstenedione (P < 0.001)

DISCUSSION

(ng/ml)

Testosterone (q/ml) (P < 0.001)

The peak in plasma T observed in the 14-hr group is consistent with results reported by Schanbacher et al. (1974). They also found an increase in serum T

Dihydrotestosterone

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(P
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778

SUZANNE

E.

BACHMAN et al.

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Fig. 4. Concentration (ng/ml) of androstenedione in plasma of 35week-old individual single comb White Leghorn male chickens exposed to 24 hr of light.

(Wilson and Cogburn, 1983). However, the melatonin concentration declines before the end of the dark period (Wilson and Cogburn, 1983). Therefore, the T peak observed at the end of the dark period could be explained by the increase in LH and the decline in melatonin. The T rhythm demonstrated in the 14-hr group is paralleled by a similar rhythm of DHT. This suggests that the increased concentration of T accompanied by a corresponding increase in conversion to DHT. The concentration of A, a precursor for T, also increased although non-significantly at this time to compensate for the increased concentration of T. The melatonin rhythm disappears after 2 weeks of exposure to constant light (Ralph et at., 1975) thereby resulting in physiological pinealectomy. It has been suggested that the pineal is necessary for the rhythmic release of LH @canes et al., 1980). Therefore, under constant light there might be a lack of the normal rhythm of LH which could explain the random pulsatile releases of androgens observed in the 24-hr group in the present study. It has been shown that pineal extracts have an inhibitory effect on the synthesis of LH releasing hormone (Harrison et af., 1974). Therefore, the higher concentrations of androgens found in the 24-hr group could be explained by the reduced in-

hibition of LH releasing hormone resulting in higher LH concentrations. Balemans (1972) found that melatonin has an antigonadal effect in the mature Leghorn resulting in decreased testis weight and comb growth. The increase in testis weight and comb growth exhibited by the 24-hr group could be due to the inhibition of melatonin in this group. Increased testis weight may also be associated with increased spermatogenic activity stimulated by extended photoperiod as shown in chicken-pheasant hybrids (Purohit and Basrur, 1977). In conclusion, exposure to 14 hr of light results in an entrained endogenous circadian rhythm of T and DHT. Extending the photoperiod to 24 hr stimulates testis growth and increases hormone concentrations but results in a free-running pattern of hormone release. These results indicate that photoperiod plays an important role in controlling the rhythm of androgens in male chickens. REFERENCES

Balemans M. (1972) Age dependent effects of 5-methoxytryptophol and melatonin on testes and comb growth of the White Leghorn (Gallua domesficus). J. Neural Transm. 33, 179-194.

Balthazart J., Reboulleau C. and Cheng M. (1981) Diurnal variations of plasma FSH, LH and testosterone in male

Rhythm of androgens in chickens ring doves kept under different photoperiods. Gen. camp. Endocr. 44, 202-206. Follett B. (1976) Plasma FSH during photoperiodically induced sexual maturation in male Japanese quail. J. Endocr. 69, 117-126. Harrison P., Organek C. and Cogbum L. (1974) Northeastern Regional Report (NE-61). Lake P. and Wood-Gush D. (1956) Diurnal rhythm in semen yields and mating behavior in the domestic cock. Nature, Lund. 178, 853. Langford B. and Howarth B. (1972) Diurnal rhythm of testicular temperature and spermatogenic activity in the domestic fowl. Pot&. Sci. 51, 1828 (Abstract). Lincoln G., Peet M. and Cunningham R. (1977) Seasonal and circadian changes in the episodic release of FSH, LH and testosterone in rams exposed to artifical photoperiods. J. Endow. 72, 337-349. Mashaly M. and Glick B. (1979) Comparison of androgen levels in normal males and in males made sexually inactive by embryonic exposure to testosterone proprionate. Gen. camp. Endocr. 38, 105-110. McCartney E. (1942) Diurnal rhythm of mitotic activity in the seminiferous tubules of the domestic fowl. PO& Sci. 21, 130-135. Miyatake A., Morimoto Y., Oishi T. and Hanasaki N. (1980) Circadian rhythm of serum testosterone and its relation to sleep: comparison with variation in serum LH, PRL and cortical in normal men. J. clin. Endocr. Metab. 51, 1365-1371. Nicholls T., Scanes C. and Follett B. (1973) Plasma and pituitary LH in Japanese quail during photoperiodically induced gonadal growth and regression. Gen. camp. Endocr. 21, 84-98.

Plant T. (1981) Time courses of ~n~ntrations

C.B.P. I7/3A-4

of circu-

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lating gonadotrophin, PRL, testosterone and cortisol in adult male rhesus monkeys throughout the 24 hour light-dark cycle. Biol. Retwod. 25. 24252. Purohit V. and Basrur P. (i977) E&t of extended photoperiod on the spermatogenic activity of chicken-pheasant hybrids. Br. P&t. Sci. 18, 651655. Ralph C., Binkley S., Macbride S. and Kiein D. (1975) Regulation of pineal rhythms in chickens: effects of blinding, constant light, constant dark and superior ganglionectomy. ~nducr~no~ogy 97, 1375.1378. Riley G. (1940) Diurnal variations of spermatogenic activity in domestic fowl. Pouft. Sri. 19, 360 (Abstract). Scanes C., Chadwick A., Sharp. J. and Bolton N. (1978) Diurnal variations in serum LH, GH and PRL concentrations in intact and pinealectomized chickens. Gen. camp. Endocr. 34, 45-49.

Scanes C., Harvey S., Chadwich A., Gales L. and Newcomer W. (1980) Diurnal variations in Serum LH, GH and PRL concentrations in intact pinealectomized chickens. Gen. camp. Endocr. 41, 266-269. Schanbacher B., Gomes W. and Van Demark N. (1974) Diurnal rhythm in serum testosterone levels and thymidine uptake by testes in the domestic fowl. J. Ann. Sci. 38, 1245-1248.

Sturkie P. (1976) Reproduction in the male, fertilization and early embryonic development. In Auiun Physiology, pp. 332-347. Springer, New York. van Tienhoven A. and Planck R. (1973) The effect of light on avian reproductive activity. In Endocrinology II, Part 1, in Handbook of Physiology, pp. 79-107. Section of Endocrinology, Waverly Press, Baltimore, MD. Wilson S. and Cogbum L. (1983) Daily melatonin rhythm in young chickens and its abolition in pinealectomy. Pot&. 5%. 62, 1524 (Abstract).