FULL PAPER Anatomy

Seasonal Changes of Testicular Weight, Sperm Production, Serum , and in Vitro Testosterone Release in Korean Ring-Necked Pheasants (Phasianus colchicus karpowi)

In Shik KIM1) and Hong Hyun YANG1)

1)Department of Anatomy, College of Veterinary Medicine, Chonbuk National University, Chonju 561–756, Republic of Korea

(Received 17 July 2000/Accepted 26 September 2000)

ABSTRACT. To study the biology of reproduction of male Korean ring-necked pheasants kept under natural conditions of temperature and photoperiod, testicular weight, serum testosterone concentrations, testosterone release from the luteinizing (LH)-stimulated tes- tis in vitro and sperm production were measured. Significant changes associated with seasonal cycles were found. Testis weight decreased dramatically in August, remained low until February, rapidly increased from March to high levels to June, and decreased sub- sequently. Serum testosterone concentrations remained little from August until February, but increased sharply in March to reach the highest levels in April. Thereafter, the concentrations decreased significantly from June. The testosterone release was low from August to February, increased abruptly in March to reach the highest levels in May, and showed rapid decrease thereafter. Sperm production decreased to nondetectable levels from August to February, increased markedly in March, reached a peak in May, and sharply decreased thereafter. Thus, the pheasants breed from late March to late June. These results indicate that the Korean ring-necked pheasant, under natural conditions, exhibits characteristics of a seasonal cycle in reproduction. KEY WORDS: Korean ring-necked pheasant, male seasonal breeder. J. Vet. Med. Sci. 63(2): 151–156, 2001

Many avian species exhibit annual reproductive cycles producing steroid in response to and cyclic variations in reproductive hormones that are stimulation during the sexual cycle have been demonstrated associated with structural changes in the testis and the in various vertebrate classes [17], testicular responsiveness changing patterns of spermatogenic cells [8, 9] to ovine LH (oLH) has also been studied during the annual LH is the primary steroidogenic hormone acting on the cycle. testicular cells; therefore androgen production is controlled by the cyclic hypophyseal activity [2, 28]. However, in sev- MATERIALS AND METHODS eral seasonal breeders [1, 8, 9] it has also been shown that a different seasonal responsiveness of the gonads to gonado- Experimental animals: Adult male Korean ring-necked tropin stimulation plays a role in determining steroid pro- pheasants (1.2–1.4 kg) hatched in June 1997 were used. files. In the present study, an ovine gonadotropin prepara- They were the third generation of this species reared in cap- tion was used to stimulate the pheasant testes, because ovine tivity at Bio-Safety Institute, Chonju, Republic of Korea. LH has been shown to possess a high specificity in androgen The pheasants were maintained in a large outdoor pen (15 × stimulation, similar to that shown by bird LH [11]. 15 × 2 m) under natural conditions of temperature and pho- Androgens are known to play an important role in male toperiod. They were fed with assorted chicken feed (Sam avian reproduction and many studies have described sea- Yang Co., Seoul) every morning and provided water ad libi- sonal variations in plasma testosterone levels in birds such tum during the experimental periods. They were sacrificed as penguins [30], mallards [7], gray partridges [9], ostriches at monthly intervals from October 1998 until September [5], geese [6], Indian rose-ringed parakeets [14], and wild 1999. The Chonju area (35° 49’N, 127° 09’E) is in the songbirds [29]. In many birds, it has been reported that tes- southwestern part of the Korean peninsula. In Chonju, sum- tosterone levels changed in parallel with spermatogenic mer days have 14 hr of light and the light phase is 10 hr in activity in the testis [8]. However, little is known of the winter. The Chonju area has typical temperate zone weather reproductive endocrinology of the pheasant. Sakai and Ishii and the rainfall is similar to that seen in Monsoon areas. [25] reported on the annual reproductive cycles of Japanese Thus, it has warm springs (9–17°C), hot summers (23– common pheasants, but to our knowledge, there are no stud- 28°C), cool falls (8–21°C) and cold winters (2–4°C), and ies concerning the seasonal changes in the reproductive with heavy summer rainfall. activity of male Korean ring-necked pheasants. Sample collection: A blood sample was collected on the Therefore, the present study has analyzed the seasonal third Monday of each month from a wing vein of 6 ran- changes in testicular weight, serum testosterone levels, and domly selected males between 1400 and 1500 hr for 1 year. sperm production in male Korean ring-necked pheasants to The serum was separated by centrifugation and stored at obtain basic information concerning their reproductive pro- –20°C for future determination of testosterone levels by cesses. Moreover, since different gonadal capabilities for RIA. Six males per month were killed by carbon dioxide 152 I. S. KIM AND H. H. YANG. asphyxiation after collection of the blood sample. Their tes- were stained with hematoxylin and eosin. The slides were tes were removed, and weighed. The left testis was placed observed and photographed using a Leica DMRBE micro- in liquid nitrogen for the subsequent estimation of total scope. sperm production while the right testis was placed in Krebs- Statistical analysis: Significant differences in the values Ringer bicarbonate buffer for determination of LH-stimu- (p<0.01) between the groups were determined by the Dun- lated testosterone secretory capacity in vitro. can’s multiple-range test after analysis of variance. Testosterone release from the LH-stimulated testis in- vitro: The right testes (n=6 pheasants per month) were RESULTS removed, cleaned of fat, decapsulated, and cut into frag- ments of similar size (200 mg). Testicular fragments from Testis histology: Most active spermatogenesis in the each animal were incubated in Krebs-Ringer bicarbonate Korean ring-necked pheasant occurred from March to June. buffer (pH 7.4, aerated for 10 min) containing 2% glucose During this time, large numbers of spermatozoa were and 1,000 ng per ml ovine LH (oLH-26, National Institutes detected within the lumen of seminiferous tubules (Figs. 1 of Health, Bethesda, MD, U.S.A.), at 37°C for 3 hr in a and 2), thereafter, spermatogenesis abruptly ceased. The shaking water bath at 90 oscillations per minute as described lumen of seminiferous tubules gradually narrowed in Sep- previously [21]. A similar dose of oLH stimulated maximal tember (Fig. 3) and nearly closed in December (Fig. 4). testosterone release in testicular incubations of the Japanese During this period, the interstitium was greatly expanded quail [3], the garden warbler [1] and the ostriches [18]. The and no spermatozoa were found in the lumen. The appear- incubation chamber was aerated continuously throughout ance of the seminiferous tubules and interstitium remained the incubation period. When the incubation was over, the similar through February. medium was transferred to plastic tubes. The tubes were Testis weight: The values for the testis weight are shown then centrifuged and the media were collected. Testoster- in Table 1. These data indicate that important changes are one in the incubation media was determined by RIA. associated with the annual seasonal cycle. Testis weights in Testosterone RIA: The amount of testosterone in the pheasants obtained during August-February were much serum and incubation medium was measured by radioim- lower than in those obtained at other months. Testis weight munoassay according to the method described previously increased markedly from March to maximum levels by [16, 23] with some modifications. Briefly, testosterone was April-May, and sharply decreased in July. extracted from serum (200 µl) and incubation medium (200 Serum testosterone: The serum testosterone profile is µl) with diethyl ether, evaporated in a freeze dryer, reconsti- shown in Table 2. The hormone remained little from tuted in RIA buffer, and then assayed without further purifi- August until February, but increased sharply in March to cation. Labeled testosterone ([1,2,6,7-3H] testosterone; 98 reach the highest levels in April. Thereafter, the concentra- Ci/mmol) was purchased from Amersham (UK) and test- tions decreased dramatically from June, and were lowest in osterone antiserum was donated by Dr. Yoon in Hanyang September. University (Seoul). Cross-reactivity of the antiserum to 5α- Testosterone release from the LH-stimulated testis in dihydrotestosterone was about 9% and that to the other vitro: Annual changes in LH-stimulated testosterone secre- androgenic steroids was negligible. The inter- and intraas- tory capacity in vitro are shown in Table 3. LH-stimulated say coefficient of variance was 10.6% and 9.4%, respec- (1,000 ng/ml) testosterone secretory capacity (calculated as tively. The lower limit of the assay sensitivity was 7 pg/ ng testosterone secreted/100 mg testis/ 3 hr) was low from tube. August-February, increased from March to maximum levels Estimation of sperm production: The left testis from each by April-May, and sharply decreased in June. The values of the pheasants (n=6 pheasants per month) was processed decreased lineally from June-September. for the determination of sperm production using the proce- Sperm production: Seasonal changes in the sperm pro- dure of Kirby et al. [13]. The weight of each thawed testis duction are shown in Table 4. Sperm production was not was recorded before and after decapsulation. After weigh- detectable from August to February, increased markedly in ing, the testicular parenchyma was homogenized in 10 vol- March, reached a peak by May, and sharply decreased in umes (w:v) of saline-triton-methiolate buffer (150 mM July. NaCl, 0.05% [v:v] Triton X-100, and 0.25 mM methiolate [Sigma Chemical Co., St. Louis, MO, U.S.A.]) in a semim- DISCUSSION icro blender. Homogenization was achieved by subjecting the tissue and buffer mixture to four or five 30 pulses. Elon- Most of birds are generally known as seasonal breeders gate spermatid nuclei that were resistant to homogenization [8]. Seasonal testicular cycles studied in the northern hemi- were counted on a hemocytometer. Each sample was sphere show that the reproductive activity coincides with counted in quadruplicate. The numbers of spermatid nuclei spring. The results of the present study indicate that the per testis were calculated. of the male Korean ring-necked pheas- Tissue preparation: Testes were fixed in 4% neutral buff- ant also experiences marked seasonal changes. Gonadal ered paraformaldehyde for 24–48 hr, embedded in paraplast, regression was observed in autumn-winter and the season and sectioned (5–6 µm) with a rotary microtome. Sections for maximal reproductive capacity was spring (March- TESTICULAR ACTIVITY IN KOREAN RING-NECKED PHEASANT 153

Figs. 1-4. Photographs of the testis in Korean ring-necked pheasants. The testes of pheasants were fixed, sectioned, and stained with hematoxylin and eosin and photographs were taken as described Materials and Methods. Testes were obtained in March (Fig. 1), May (Fig. 2), September (Fig. 3) and December (Fig. 4). All figures were of the same magnification (× 320). S, seminiferous tubule; I, interstitium; Arrows, Leydig cell.

May). Annual changes in gonads were coincident with the by histological studies [24] where involution of the seminif- changes in the serum testosterone levels, sperm production, erous tubules and absence of the spermatozoa are evident. and in the in vitro testicular response to gonadotropin stim- According to Ortavant et al. [22], in most of the species, ulation. spermatogenesis is not arrested but there is a significant The marked signs of gonadal regression experienced by decrease in the number of spermatozoa in winter. Korean ring-necked pheasants under natural conditions of Seasonal variations in the serum testosterone levels of the temperature and photoperiod during the winter season, char- Korean ring-necked pheasant were very similar to those of acterized by morphological changes have been confirmed Japanese common pheasants [25]. Indeed, in most respects 154 I. S. KIM AND H. H. YANG.

Table 1. Seasonal variations of testis weight in adult Korean ring- Table 3. Seasonal changes in the testosterone release from the LH- necked pheasants stimulated testis in vitro in adult Korean ring-necked pheasants Month Testicular weight (g) Month Testosterone production (ng/100 mg testis)

October 0.35 ± 0.02h) October 2.82 ± 0.37e) November 0.22 ± 0.01i) November 3.49 ± 0.69e) December 0.24 ± 0.01i) December 3.01 ± 0.26e) January 0.49 ± 0.01h) January 3.69 ± 0.19e) February 0.54 ± 0.02g) February 3.93 ± 0.44e) March 2.52 ± 0.12c) March 15.43 ± 2.78b) April 3.49 ± 0.2a) April 26.44 ± 4.18a) May 2.91 ± 0.16b) May 29.46 ± 3.99a) June 1.97 ± 0.05d) June 8.61 ± 0.58c) July 0.95 ± 0.03e) July 6.41 ± 0.45d) August 0.62 ± 0.02f) August 4.02 ± 0.19e) September 0.41 ± 0.02h) September 3.79 ± 0.27e) Six animals were used for each month. Values are expressed as Testicular fragments (200 mg) were incubated with ovine-LH. Six means ± SEM. animals were used for each month. Values are expressed as means ± a)-i) Means within a column with different superscripts are different at SEM. a)-e) Means within a column with different superscripts are p<0.01. different at p<0.01.

Table 2. Seasonal changes in the serum testosterone levels of adult Table 4. Seasonal variations in the sperm production of Korean Korean ring-necked pheasants ring-necked pheasants Month Serum testosterone (ng/dl) Month Sperm production (× 106) October 16.45 ± 1.32e) October ND November 12.84 ± 0.97e) November ND December 14.32 ± 1.12e) December ND January 15.56 ± 1.41e) January ND February 21.23 ± 1.89e) February ND ± b) March 155.16 ± 17.89b) March 971.8 67.8 ± a) April 214.67 ± 28.43a) April 1365.6 162.8 ± a) May 183.52 ± 20.79ab) May 1483.5 196.9 ± b) June 94.16 ± 7.52c) June 892.9 107.5 ± c) July 48.82 ± 2.51d) July 10.2 0.8 August ND August 22.47 ± 2.16e) September ND September 11.61 ± 0.89e) ND: not detected. Six animals were used for each month. Values are Six animals were used for each month. Values are expressed as expressed as means ± SEM. a)-c) Means within a column with means ± SEM. different superscripts are different at p<0.01. a)-e) Means within a column with different superscripts are different at p<0.01. the pattern of hormone secretion was not markedly dissimi- has been shown for other birds with a reproductive behavior lar to geese [6], mallards [7], songbirds [29], and gray par- strongly dependent on daylength [27], a seasonal cycle in tridges [9]. In female Korean ring-necked pheasants, egg reproduction with high correlation to the parameters here laying starts in April and ends in July, with a peak activity in studied exists in the Korean ring-necked pheasant. May-June [12]. In males, an increase in serum testosterone In several seasonal breeders [1, 8, 9] it has been shown occurred at the end of March, just prior to egg laying. Dur- that a different seasonal responsiveness of the gonads to ing the 3 to 4 months of the laying period, serum testoster- gonadotropin stimulation plays a role in determining steroid one levels were higher than those in other months. Similar profiles. In the present study, analysis of the testosterone relationship between egg laying and the blood testosterone production by testicular fragments indicates seasonal varia- level in males have been reported in Japanese common tions in their response to oLH stimulation. The serum test- pheasants [25]. osterone levels were closely related to the capacity of the Of importance are the factors that can regulate the circu- testicular tissue to respond to the gonadotropin stimulation mannual reproductive cycle of the male Korean ring-necked at the same time of the year. Therefore, these results indi- pheasant, such as temperature, photoperiod, rain, and the cate that, in Korean ring-necked pheasants, the seasonal tes- others. 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