Changes in Plasma Concentrations of Luteinizing Hormone, Progesterone, and Testosterone in Turkey Hens during the Ovulatory Cycle

General and Comparative Endocrinology 106, 281–292 (1997) Article No. GC976884

Changes in Plasma Concentrations of Luteinizing Hormone, Progesterone, and Testosterone in Turkey Hens during the Ovulatory Cycle1 Jingying Yang, David W. Long, and Wayne L. Bacon The Ohio State University, Department of Animal Sciences, Ohio Agriculture Research and Development Center, Wooster, Ohio 44691 Accepted January 15, 1997

Changes in luteinizing hormone (LH), progesterone, and testosterone concentrations were determined in blood samples taken every 10 min for 26 hr during an ovulatory cycle in laying turkey hens. During the 26-hr sampling period, one peak of both LH and progesterone and numerous peaks of testosterone were detected. The concentration of LH in plasma increased from basal level (2.44–4.0 ng/ml) to maximum level (8.57–24.3 ng/ml) over 1 to 2 hr and then declined over 3 to 5 hr to a basal level. The duration of the descending portion of the peak was about double that of the ascending portion. The concentration of progesterone increased rapidly from a basal level of 1.18–1.65 ng/ml to a peak of 6.18–11.87 ng/ml and then maintained a plateau before rapidly declining to basal level. The concentration of testosterone increased from a basal level of 0.06–0.09 ng/ml to a peak level of 0.13–0.30 ng/ml. All maximum levels of testosterone preceded those of LH, and all maximum levels of LH preceded those of progesterone. The durations of the progesterone peaks were longer than those of the LH peaks. Progesterone concentrations returned to basal level after LH had returned to basal level, although the initial increase in progesterone concentration was earlier, later, or at the same time as LH. Peak durations of testosterone were variable. The preovulatory surges of LH and progesterone of five of nine sets of samples 1

Salaries and research support provided by State and Federal appropriated to the Ohio Agricultural Research and Development Center, The Ohio State University. Manuscript number 100-96. 0016-6480/97 $25.00 Copyright r 1997 by Academic Press All rights of reproduction in any form reserved.

started at the end of the scotophase and ended during the beginning portion of the photophase. In three of nine sets both the start and the end occurred during the scotophase and in one of nine sets during the photophase. It was concluded from this study that the patterns of secretion of LH, progesterone, and testosterone were similar in that the preovulatory surge was superimposed on a relatively stable basal level, while the temporal relationships of the ovulatory surges of these hormones were variable. The preovulatory surges were more tightly associated with ovulation rather than with photoperiod. Neither progesterone nor testosterone might be an initiator of the LH surge prior to ovulation. r 1997 Academic Press It is generally considered that luteinizing hormone (LH), progesterone, and testosterone are important hormones in controlling reproduction of hens. During the ovulatory cycle, the levels of LH, progesterone, and testosterone increase from a basal level to a peak level about 4 to 7 hr before ovulation in chicken hens (Kappauf and van Tienhovan, 1972; Furr et al., 1973; Lague et al., 1975; Etches and Cunningham, 1976; Proudman et al., 1984). In turkey hens, LH and progesterone increased to peak levels at 8 hr before ovulation (Mashaly et al., 1976; Proudman et al., 1984), and an androgen peak level was observed at 8 hr before ovulation (Sharp et al., 1981). In duck hens, LH and progesterone concentrations in plasma increased at 3 hr before ovulation (Tanabe et al., 1980). In Japanese

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quail hens, LH and testosterone concentrations in plasma reached a peak at 6 hr before ovulation; however, progesterone concentration increased to a peak level at 4 hr before ovulation (Doi et al., 1980). The temporal relationships between LH and progesterone have been determined. The increase of progesterone in plasma of chicken hens before ovulation preceded or occurred simultaneously with the increase of LH, but the increase of LH never preceded the increase of progesterone (Furr et al., 1973). The concentration of testosterone in chicken plasma increased to a peak level 10 to 6 hr prior to ovulation or 4 to 2 hr before the peaks of progesterone and LH (Johnson and van Tienhoven, 1980a). In the study by Etches and Cheng (1981), the highest and lowest values of LH in plasma in chicken hens were observed at 3 and 6 hr progesterone at 3 and 0 hr, and testosterone at 3 and 6 hr before oviposition, respectively. Taken together, all studies reported do not give high-resolution information concerning the relationships between LH, progesterone, and testosterone to time of ovulation, due to relatively long intervals between samples and different data analysis criteria. It is not possible to determine whether LH, progesterone, or testosterone increases first before ovulation in hens. Since clear determinations of the temporal relationships among these reproductive hormones have not been obtained, the present study was designed to determine at higher resolution the detailed changes in plasma concentrations of LH, progesterone, and testosterone during the ovulatory cycle in laying turkey hens.

MATERIALS AND METHODS Photosensitive turkey hens selected for increased egg production (E line) (Anthony et al., 1991) were used early after initiation of egg production. Five laying hens were housed individually in 60 3 60 3 80 cm cages with wood shavings as litter and maintained under a 14-hr light and 10-hr dark photoperiod. The birds were cannulated and serial samples were taken every 10 min for 26 hr (Chapman et al., 1994). Briefly, 1.0 ml blood was taken and the same volume of saline was injected through the cannula each time. Blood cells were collected and returned to the donor birds

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Yang, Long, and Bacon

after every three or four samples. Plasma samples were frozen at 220° until assay. The cannulas were maintained and the birds were reused 2 weeks later. A total of nine sets of blood samples associated with ovulation were obtained.

Hormone Assays LH The LH radioimmunoassay was as previously described in Bacon et al. (1994). Sample size was 50 µl plasma. The sensitivity of the assay was 0.025 ng/tube. The coefficients of variability of the intra- and interassays were 10.9 and 13.7%, respectively.

Progesterone Extraction. Progesterone was separated using reverse-phase chromatography on C18 solid-phase columns (Alltech, Deerfield, IL). Aliquotes of 50 µl of each plasma sample were used for assay. The samples were added to 12 3 75 mm disposable glass tubes with 1 ml sodium phosphate buffer (0.05 M, pH 7.6) and heated 1 hr at 65°. They were then applied to the individual C18 columns followed by an additional 1 ml sodium phosphate buffer used to rinse the tubes. The columns were preconditioned with 5 ml methanol followed by 5 ml distilled water. After applying the plasma samples, the columns were rinsed with 2 ml distilled water followed by 2 ml of 40% methanol. The progesterone fraction was eluted with 1 ml of 100% methanol. The eluates were dried under vacuum at about 60°. The columns were then reconditioned and reused for up to six samples. Radioimmunoassay (RIA). Dried samples were reconstituted with 1.25 ml RIA buffer (6.05 g/liter, 0.05 M Tris–HCl, 5.85 g/liter, 0.1 M NaCl, 1.0 g/liter, NaN3 and 1.0 g/liter gelatin; pH 8.0). After brief vortexing, the samples were sonicated 10 min in a water bath at room temperature. The samples were split into duplicates, 0.5 ml of each, for assay. [1,2,6,7-3H]Progesterone (0.01 ml of 50 µCi/ml, Amersham, Arlington Height, IL) was dried with air and then dissolved in 10 ml of assay buffer. Progesterone antiserum (0.02 ml of a 1:100 dilution, Arnel products. Co., New York, NY) was diluted with 20 ml of assay buffer. Cross-reactivity of

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the antiserum as determined by the supplier was: progesterone 100%, DOC-21 30%, 17-OH-progesterone 6%, testosterone 5%, androstenedione 4%, estradiol 4%, and others below 0.001%. Next, 0.1 ml of diluted [3H]progesterone and 0.1 ml of diluted antiserum were added and then the mixture was incubated overnight at 4°C. Next, 0.2 ml of dextran-coated charcoal (Dextran T-70, 0.0625 g, and charcoal (Norit A), 0.625 g, were dissolved in 100 ml assay buffer) was added and incubated 15 min in ice water. After 15 min centrifugation at 2500 rpm, the supernatant were decanted into scintillation vials and 5 ml Ecolite(1) (ICN Biomedical Co., Aurora, OH) was added. Counting was for 10 min. The progesterone standards used for each assay were 400, 200, 100, 50, 25, 12.5, 6.25, and 3.125 pg per 0.5 ml. The percent recovery was determined by mixing pooled plasma with tracer [3H]progesterone, and controls included high and low pooled plasmas and blanks (buffer). The sensitivity of the assay was 0.303 pg/ tube. The percentage of recovery was 56.7–64.7%. The intra- and interassay coefficients of variation were 9.5 and 6.7%, respectively. Data were corrected for recovery.

Testosterone Extraction and RIA were as described in Bacon et al. (1991). Aliquots of 150 µl of each plasma sample were used. The sensitivity of the assay was 0.035 pg/tube. The percent recovery was 70.5–80.5%. The coefficients of variability of the intra- and interassay were 8.7 and 9.3%, respectively. Data were corrected for recovery.

Data Analysis The hormone data were analyzed by Pulsar algorithm (Merriam and Wachter, 1982) to determine the incidence of peaks and peak durations of each hormone. The G values for LH and progesterone were G(1) 5 3.80, for testosterone G(1) 5 50, the others G(2) 5 2.60, G (3) 5 1.90, G(4) 5 1.50, G(5) 5 1.20 were the same for all three hormones. The assay SD calculated from a sample of the data was 10.90X for progesterone, 6.22X 1 4.36 for LH, and 2.09X 1 0.84 for testosterone, where X is the concentration of progesterone, LH, or testosterone in the individual sample.

Setting G(1) 5 50 for testosterone precluded detection of single point peaks. The mean values of basal level, peak amplitude, relative increase, and peak duration of the hormones were analyzed for differences between hormones by ANOVA. Differences (P , 0.05) between means were determined by t test. Each set was treated as independent due to the 2-week lag between blood collections.

RESULTS Similar patterns of LH, progesterone, and testosterone secretion were detected in the nine sets of samples, two each from four hens and one from one hen. One large preovulatory peak was detected for LH and progesterone in all nine sets, but for testosterone, one or two peaks were detected throughout the ovulatory cycle (Figs. 1A, 1B, 1C, and 1D). The concentration of LH in plasma increased over 1–2 hr from a basal level (2.44–4.0 ng/ml) to a peak level (8.57–24.3 ng/ml) and then declined over 3–5 hr to the basal level. The peak curve shapes showed that the ascending sides were steeper than the descending sides (Fig. 1, LH panels). The concentration of progesterone increased rapidly from a basal level ranging from 1.18 to 1.65 ng/ml to a peak level ranging from 6.18 to 11.87 ng/ml and then maintained a relatively stable plateau before rapidly declining to the basal level (Fig. 1, progesterone panels). Increase to peak levels of LH and progesterone concentrations occurred during the scotophase and decline to basal level occurred during the photophase in five of nine sets of samples; both increase and decline occurred during the scotophase in three of nine sets of samples and occurred during the photophase in one of nine sets of samples. Compared to the changes in LH and progesterone, testosterone concentrations were very low, and one or two peaks were detected during the 26 hr of sampling. Basal levels ranged from 0.06 to 0.09 ng/ml and peak levels ranged from 0.13 to 0.30 ng/ml (Fig. 1, testosterone panels). Compared with LH peaks, progesterone peaks were symmetrical, with a broad plateau, whereas LH peaks tended to be triangular, with the duration of the ascending limb about one-half the duration of the descending limb. Peak durations of progester-

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FIG. 1. Secretory patterns of luteinizing hormone (LH), progesterone, and testosterone in plasma taken every 10 min for 26 hr in laying turkey hens during the ovulatory cycle. For comparison, time 0 was arbitrarily set at the beginning of the scotophase. Bleedings were started 150 min before the scotophase. The hens were maintained on a 14-hr light: 10 h dark photoperiod. The heavy black bar at top indicates the scotophase. Solid circles and thin straight lines in each panel indicate the location and duration of the peak(s), open circles indicate the location and duration of the basal level(s). Data from four of the nine sample sets are presented. A, B, C, and D are from individual birds from sets 1, 3, 5, and 9, respectively.

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FIG. 1—Continued

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Yang, Long, and Bacon

FIG. 1—Continued

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Plasma Concentration Changes during the Ovulatory Cycle

FIG. 1—Continued

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Yang, Long, and Bacon

TABLE 1 The Basal Level, Peak Level, Peak Initiation, and Duration of Luteinizing Hormone (LH), Progesterone (P), and Testosterone (T) in Plasma in Turkey Hens during the Ovulatory Cycle

Set no.

Hormones

Basal level (ng/ml) (mean 6 SD)

1 (b1, t1) e

P T LH P T LH P T LH P T LH P T LH P T LH P T LH P T LH P T LH

1.60 6 0.01 0.08 6 0.01 2.44 6 0.03 1.18 6 0.03 0.08 6 0.02 3.27 6 0.78 1.65 6 0.19 0.08 6 0.02 2.47 6 0.32 1.61 6 0.10 0.08 6 0.01 3.38 6 0.13 1.26 6 0.01 0.06 6 0.00 4.00 6 0.14 1.69 6 0.10 0.07 6 0.01 2.50 6 0.07 1.53 6 0.12 0.06 6 0.01 2.76 6 0.11 1.49 6 0.14 0.09 6 0.00 3.25 6 0.13 1.37 6 0.42 0.07 6 0.01 3.20 6 0.06

2 (b2, t1)

3 (b3, t1)

4 (b4, t1)

5 (b1, t2)

6 (b2, t2)

7 (b3, t2)

8 (b4, t2)

9 (b5, t2)

Peak Amplitude (ng/ml)

Relative increase a

Position b (min)

Initiation c (min)

Duration d (min)

9.34 0.22 9.69 4.11 0.32 10.72 8.47 0.30 19.42 11.87 0.30 8.57 6.18 0.13 11.21 8.54 0.20 10.51 9.57 0.20 9.04 9.03 0.22 11.39 8.30 0.24 23.92

5.84 2.75 3.97 3.48 4.0 3.28 5.13 3.75 7.86 7.37 3.75 2.54 4.9 2.17 2.8 5.05 2.86 4.20 6.25 3.33 3.28 6.06 2.44 3.50 6.06 3.43 7.48

550 340 430 1270 1080 1200 220 140 220 520 290 330 870 530 660 760 410 630 790 590 640 860 630 710 580 400 420

310 260 (840) 300 960 270 (980) 960 410 40 (570) 40 150 130 210 490 500 (820) 540 450 170 470 440 440 (980) 530 460 150 590 290 170 330

490 280 (260) 630 — 90 (190) — 560 420 (170) 620 740 1040 330 690 290 (130) 450 680 600 460 640 480 (60) 400 660 760 350 600 460 400

a

Ratio of amplitude value to basal level value. Time 0 was arbitrarily set at the beginning of the 600 min (10 hr) scotophase. c Numbers in parenthesis are the second peak initiation time. d Numbers in parenthesis are the second peak duration time. e b, bird No., t, bleeding time. b

one(632.5 6 79.7 min) were also longer than those of LH (455 6 113.8 min) (P , 0.05). Testosterone peak durations were much more variable than those for LH or progesterone. Basal and maximum levels, peak start time, and peak duration time of LH, progesterone, and testosterone are given in Table 1. For comparison, time 0 was arbitrarily set at the beginning the scotophase. Bleedings were started 150 min before the scotophase. Mean values of basal levels, maximum levels, and peak duration times of all three hormones are listed in Table 2. All peak levels of LH preceded those of progesterone. In seven of nine sets of samples, the concentration of progesterone increased first in one of nine sets at the same time and in one of nine sets later

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than the initial increase of LH. In eight of nine sets of samples, the testosterone peaks associated with LH ovulatory surges started earlier than LH and in one of nine sets at the same time as the LH surge (Table 3).

DISCUSSION The changes in concentration and temporal relationships among the plasma levels of LH, progesterone, and testosterone have been determined in finer resolution in laying turkey hens than previously reported. The hens were cannulated using a jugular cannulation

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Plasma Concentration Changes during the Ovulatory Cycle

TABLE 2 The Mean (6SD) Values of Basal Level, Peak Amplitude, Relative Increase, and Peak Duration of Luteinizing Hormone (LH), Progesterone (P), and Testosterone (T) in Plasma of Laying Turkey Hens during the Ovulatory Cycle Hormones

Basal level (ng/ml)

Peak amplitude (ng/ml)

Relative increase

Peak duration (min)

LH P T

3.03 6 0.53 a 1.48 6 0.18 b 0.07 6 0.01 c

12.71 6 5.28 a 8.34 6 2.18 a 0.24 6 0.06 b

4.32 6 1.97 ab 5.54 6 1.08 a 3.16 6 0.64 b

455.0 6 113.8 a 632.5 6 79.7 b 502.2 6 266 (142 6 78) ab*

(a–c) Means within columns with different superscript letters are different (P , 0.05). * Peaks associated with LH and P peaks were compared.

system and serially sampled every 10 min for 26 hr. The secretion pattern of each hormone included the prominent feature of the preovulatory surges superimposed on the basal level of the hormone in circulation. The pattern of peaks of LH and progesterone are characterized by only one major peak, the preovulatory surge, which was very repeatable in profile for each hormone. The pattern of testosterone peaks was much more variable in number and profile. The results obtained are in general agreement with previous studies in turkeys and other avian species. In turkey hens, Mashaly et al. (1976) suggested that LH and progesterone increase at the same time, between 8 and 2 hr before ovulation. In studies by Furr et al. (1973) using chicken hens, the increase of progesterone either preceded or occurred simultaneously with the LH increase, but the increase in LH never preceded the increase in progesterone. Other studies showed similar results in chicken hens (Duplaix et al., 1981). In ducks, the plasma concentrations of LH and progesterone increase between 7 and 4 hr prior to ovulation or oviposition (Tanabe et al., 1980; Wilson et al., 1982). In all of these studies cited, intervals between samples

TABLE 3 Time Ranks of the Amplitude Occurrence and Peak Initiation of Progesterone (P), Testosterone (T), and Luteinizing Hormone (LH) during the Ovulatory Cycle in Turkey Hens Set no.

Hormone

1

2

3

4

5

6

7

8

9

P T LH

3—3 1—1 2—2

3—1 1—2 2—1

2—1 1—2 2—2

3—2 1—2 2—3

3—1 1—1 2—3

3—2 1—2 2—3

3—1 1—1 2—2

3—2 1—1 2—3

3—2 1—1 2—3

Numbers before dash indicate amplitude occurrence order, after dash indicate peak initiation order.

were relatively long ($1 hr) and thus a precise conclusion concerning temporal changes in the hormones associated with ovulation was not possible. In 7 of 9 sets of samples the progesterone surge occurred first, in 1 of 9 at same time, and in 1 of 9 progesterone was later than LH. In 8 of 9 sets of samples, peaks of testosterone started earlier than those of LH and progesterone, in 1 of 9 later than progesterone and at the same time as LH. The relationships suggest that secretion of LH, progesterone, and testosterone may be under the control of different hormonal systems. Here, it is difficult to say that LH stimulates in vivo the increase in progesterone or vise versa. Johnson et al. (1985) proposed that progesterone plays a positive feedback role on LH secretion, which results in the occurrence of the preovulatory surge of LH. In the present study, the changing pattern of LH and progesterone did not show a positive feedback relationship. A single injection of progesterone did not induce an LH surge in ovariectomized chicken hens (Wilson and Sharp, 1976). In ovariectomized turkey hens, injection of progesterone decreased the LH concentration in plasma (El Halawani et al., 1983). In a study by Wilson and Sharp (1975), injection of progesterone at different stages during the ovulatory cycle induced different responses of LH secretion. It could be deducted that if progesterone is an initiator, it could induce LH surge at any time during the ovulatory cycle. This result suggests that the LH surge probably has its own rhythm related to ovulation independently of progesterone. Taken together, it is reasonable to assume that progesterone is not likely to be the single proximate factor to induce the preovulatory surge of LH in turkey hens, but it could influence the secretion of the preovulatory surge of LH by some other mechanism(s).

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The increase of testosterone associated with peaks of LH and progesterone occurred earlier than LH. In chickens the increase of testosterone concentration in plasma occurred in general 4 to 2 hr before those of LH and progesterone (Johnson and van Tienhoven, 1980a). Various peaks of testosterone were detected in the present study. The peaks of testosterone associated with the peaks of LH and progesterone were of higher amplitude and of longer duration than those occurring independently of the LH and progesterone surges. What the temporal relationships of these various peaks of testosterone are to changes in LH, and progesterone, is not apparent. Although increases of testosterone concentration occurred earlier than LH, some studies show that testosterone probably is not an initiator of the LH surge. Testosterone can partly restore ovulation blocked by inhibiting steroidogenesis and induce nonsignificant LH secretion (Lang et al., 1984). Johnson and van Tienhoven (1981) used a high dose of testosterone injection im to induce an LH increase and premature ovulation, but could not induce LH secretion and ovulation by injecting testosterone in the third ventricle area. Testosterone is secreted mainly from small follicles. Progesterone is mainly produced from the largest follicle ready to be ovulated (Etches et al., 1981; Robinson and Etches, 1986). Relative increases in progesterone and testosterone were significantly different. Different changes in progesterone and testosterone reflected that the large and small follicles have different temporal sensitivity to stimulations. This stimulation probably is not due to the increase in LH because LH increases were later than testosterone or progesterone increases. But some studies have shown that LH can induce the largest follicle to produce progesterone (Imai and Nalabandov, 1978). Whether LH induces the preovulatory surges of progesterone and testosterone remains unanswered. The crepuscular peak of LH in chicken hens studied by Johnson and van Tienhoven (1980a, 1984) was not observed in the present study. One bird showed that the preovulatory surges of LH, progesterone, and testosterone may occur during the photophase. This indicates that the preovulatory surge may not be strictly associated with the photoperiod. In the study by Duplaix et al. (1981), an intermittent lighting sched-

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Yang, Long, and Bacon

ule changed the egg-laying pattern, but could not change the interval time between the surge of LH and oviposition. Other studies have demonstrated that the preovulatory surges of LH and progesterone are associated with ovulation, as no ovulations are detected without LH and progesterone surges (Mashaly et al., 1976; Sharp et al., 1981; for review, Cunningham, 1987). This association indicates the preovulatory surges of LH or progesterone may be proximal initiators of ovulation. In the present study, all hens laid an egg during serial bleeding, and all also presented the preovulatory surges for the hormones. The induction of the preovulatory surges of LH and progesterone may be controlled by other unknown hormone factors secreted by the ovary. The exact times of occurrence of ovulation were not determined in this study, but the time of ovulation could be estimated from the changes of concentrations of the hormones. According to other studies, ovulation occurs around the point when LH or progesterone concentrations decline to baseline. Since concentrations of LH and progesterone decreased to basal level at different times, it is difficult to deduct the exact time of ovulation. But it could be estimated by choosing LH or progesterone as a reference. If ovulation occurs when the LH concentration returns to basal level, the increase of LH and progesterone would occur 6–10 hr before ovulation; if based on progesterone changes, the increase of LH would occur at 7–12 hr before ovulation. Also, the data from other studies are based on oviposition time rather than ovulation time. Since oviposition intervals change during a sequence (for review, Etches, 1990), estimation of time of ovulation based on oviposition time is not precise. Some studies on effects of the hormones on ovulation have repeated controversial results. Progesterone induced ovulation in the perfused fowl ovary in vitro without the presence of LH (Tanaka et al., 1987). Using hypophysectomized chicken hens, Nakada et al. (1994) suggested that progesterone probably can induce ovulation alone without a preovulatory surge of LH. This indicates that if progesterone can directly induce ovulation, the surge of LH is not necessary for ovulation unless it controls progesterone secretion. But in their studies, no plasma concentrations of LH or progesterone were determined, so it is difficult to know the exact effects of LH and progesterone. In contrast, it is

Plasma Concentration Changes during the Ovulatory Cycle

suggested in the study by Lang et al. (1984) that LH could induce ovulation without gonadal steroid increases. But in the study by Furr and Smith (1975), the injection of antibodies against progesterone and testosterone blocked ovulation. Thus, many details of the mechanisms controlling ovulation in avian species remain unknown. In conclusion, the surges of LH, progesterone, and testosterone were temporally associated with ovulation. The present data delineated a clear temporal relationship, with preovulatory increases in progesterone occurring before (7/9), later than (1/9) or at the same time as (1/9) those of LH. The increase in testosterone preceded LH or was at the same time as LH. Testosterone increased either earlier (8/9) or later (1/9) than progesterone. The surges of LH, progesterone, and testosterone occurred between 7 and 12 hr before ovulation. The interaction among LH, progesterone, and testosterone and effects on ovulation still remain elusive.

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