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BOR - Papers in Press, published online ahead of print May 12, 2004.
Biol Reprod 2004, 10.1095/biolreprod.104.027672
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BIOLOGY OF REPRODUCTION 71, 878–886 (2004)
DOI: 10.1095/biolreprod.104.027672
© 2004 by the Society for the Study of Reproduction, Inc.

Ovary

Progesterone Injection and Egg Production in Turkey Hens1

Wayne L. Bacon2, and Han-Ken Liu

Department of Animal Sciences, Ohio Agricultural Research and Development Center, The Ohio State University, Wooster, Ohio 44691


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
An arrest in laying associated with either a polyovarian follicle (POF) or a polycystic ovarian follicle (PCOF) syndrome has been reported in turkey hens photostimulated at an early age with a constant-light photoperiod. Hens expressing the POF or PCOF syndrome had stopped laying for several weeks, but the ovary contained an increased number of mature-size and larger follicles (POF hens), which were cystic (PCOF) in some of the hens. Hens with the POF or PCOF syndrome had plasma progesterone (P4) concentrations that were relatively high and without surges. We hypothesized that high plasma P4 concentrations may block ovulatory surges of LH but not the growth or maintenance of hierarchical follicles leading to development of the POF or PCOF syndrome in turkey hens. In the first six studies, hens were photostimulated with either a 14L:10D or a 24L:0D photoperiod and, after laying for 1–38 wk, were then injected daily for up to 14 days with P4 (up to 1.50 mg kg–1 day–1) and necropsied. At all ages, the oviposition rate was reduced at a P4 dosage of 0.17 mg kg–1 day–1. With dosages of 0.33 mg kg–1 day–1 or greater, however, ovipositions stopped in most hens within approximately 2 days. For hens laying for less than 15 wk, oviductal weight and number of hierarchical follicles of P4-injected hens were not different from control vehicle-injected hens, but the numbers of mature, cystic, and atretic follicles were increased. For hens laying for 38 wk, when treated with P4, oviductal weight and number of hierarchical follicles decreased, but number of atretic follicles increased. No effect of photoperiod was found on egg production, oviductal weight, or follicle number, and none of the hens developed POF or PCOF syndrome in these experiments. Two additional experiments were conducted with hens early in the reproductive period that had been photostimulated with 14L:10D or 24L:0D and injected with P4 (0.33 mg kg–1 day–1) for 10 or 12 days but not necropsied until 3 wk after the last injection. Most of the hens photostimulated with the 24L:0D photoperiod and injected with P4, and a few of the hens photostimulated with the 14L:10D photoperiod and injected with P4, had developed the PCOF syndrome when necropsied. The hens with the PCOF syndrome had high levels of P4 when necropsied. From these studies, we concluded that the PCOF syndrome can be induced early in the reproduction period by photostimulating turkey hens with a 24L: 0D photoperiod, injecting them for 10 to 12 days with P4 at a dosage of 0.33 mg kg–1 day–1, and then waiting 3 wk for the PCOF syndrome to develop.

luteinizing hormone, ovary, ovulation, pituitary, progesterone


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
An arrest in laying associated with a polyovarian follicle (POF) or polycystic ovarian follicle (PCOF) syndrome has been observed in Egg line turkey hens [1, 2]. In these studies, hens were divided into three different classes based on laying history and types of ovarian follicles present at necropsy. Laying hens had a normal laying history, and their ovary had only hierarchical follicles arranged in a single follicular hierarchy. Arrested-laying POF hens had ceased laying for 2–3 wk, but their ovary contained hierarchical follicles arranged in a single follicular hierarchy as well as five or more mature (F1) or slightly heavier-weight follicles. Arrested-laying PCOF hens had ceased laying for 2–3 wk, but their ovary contained hierarchical follicles arranged in a single follicular hierarchy as well as five or more mature or heavier-weight follicles, some of which were cystic. Some of the laying, POF, and PCOF hen ovaries also contained atretic follicles. The oviducts of the POF and PCOF hens were similar in weight to those of the laying hens, although these hens had not laid eggs for up to 3 wk [1, 2]. Hens of both POF and PCOF classes would evert their oviducts on application of abdominal pressure as applied to turkey hens during artificial insemination. Many of the POF and PCOF hens also retained hard-shelled eggs in the uterus for several days without ovipositions. The commercial turkey industry utilizes heavy-weight, sire-line males crossed with medium-weight, dam-line females to produce poults. Egg production rate of the sire-line females is generally much lower than that for the dam-line females [3]. The ovaries of sire-line females, however, contain an increased number of hierarchical follicles [4–7], which is associated with an increase in multiple follicular hierarchy sets, in comparison to dam-line females [3–7]. The number and type of ovarian follicles in hens expressing POF or PCOF syndrome were thus different from those in sire-line hens. The former had an increase in the number of mature and larger-size follicles and a normal hierarchy, whereas the latter had an increase in the incidence of multiple follicular hierarchy sets within the hierarchy.

In POF and PCOF hens, the concentration of LH was relatively low and without ovulatory surges; the concentration of progesterone (P4) was relatively high, approaching levels observed during ovulatory surges but without ovulatory surges; and the concentration of estradiol-17ß (E2) was similar to that in laying hens [1]. These plasma concentrations and peripheral patterns of LH, P4, and E2 in the POF and PCOF hens are similar to concentrations reported for laying chicken hens that had been treated with eCG for several days [8–10]. Chicken and turkey hens treated with eCG [11, 12] also have an arrest in egg production, and their ovaries contain numerous mature-size follicles similar to those in POF or PCOF hens [1, 2]. Multiple mature-size follicles of hens injected with eCG could be ovulated by injection of LH [13, 14] or chicken anterior pituitary extract [15]. Hens treated with eCG did not ovulate, however, after injection of P4 [16].

Acute administration of exogenous LH to hypophysectomized laying hens [7] and intact laying hens [17], of P4 to intact laying hens [18–20], or of GnRH to intact laying hens [21] has been shown to induce premature ovulation if administered when plasma levels of LH and P4 had been at baseline levels between surges for several hours. In these studies, only the largest and most mature follicle was ovulated after hormonal injection.

The effect of continuously high P4 levels on serial ovulations has received little recent attention, but laying chicken hens treated with daily injections of P4 for up to 1 wk ceased egg production [13]. Chronic treatment with P4 has been reported to depress egg production in chickens [22] and turkeys [23]. Also, turkey hens treated with P4 implants, either with or without estrogen treatment, had a greatly decreased egg production rate [24]. Thus, chronic treatment with P4 has been reported to depress the rate of egg production in both turkeys and chickens.

Age when photostimulated and photoperiod have been shown to be important variables concerning the incidence of POF and PCOF syndromes [2] in Egg line turkey hens. The incidence of the POF and PCOF syndromes was greater when photosensitive turkey hens were photostimulated at relatively young ages (age, <31 wk) and when exposed to a constant-light photoperiod of 24L:0D in comparison to a diurnal photoperiod of 14L:10D. Thus, age when photostimulated and photoperiod are associated with the incidence of POF and PCOF syndromes.

Acute injection of P4 in laying chicken hens can arrest the movement of an egg in the oviduct [25, 26]. Chronic treatment of laying quail hens with P4 can result in fully formed eggs being retained in the uterus for several weeks [27]. These studies indicate that continuously high P4 levels might inhibit uterine smooth muscle contractions and, thus, block ovipositions.

Acute injection of P4 might also feed back negatively on the ability of the hypothalamus to secrete surges of GnRH or, alternatively, on the ability of the pituitary to secrete surges of LH if surges of GnRH secretion occur. In the present study, we hypothesized that chronic high plasma concentrations of P4, resulting from daily injections of the hormone, may block ovulatory surges of LH but not recruitment of follicles into the hierarchy or their development, thus resulting in development of the POF or PCOF syndrome. Because the POF and PCOF syndromes have only been observed relatively early during egg production, and at a higher rate with a constant-light photoperiod, we also hypothesized that the age of the hen, the duration of egg production, and the photoperiod may alter the response to P4 injection. The objectives of the present experiments were to determine the effects of P4 injection, photoperiod, and duration of egg production on ovarian and oviductal morphology, egg production, and incidence of POF and PCOF syndromes in turkey hens.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Birds and Experiments

Turkey hens of the Egg line [28] were exposed to short-day lighting of 6L:18D at 16 wk of age to induce photosensitivity and delay puberty. Age when photostimulated, photoperiod used for photostimulation, duration of egg production when P4 injections began, duration of P4 injections, dosages of P4 injected, and duration of the postinjection period before experiment termination and necropsy are given in Table 1 for the eight experiments. When photostimulated, the hens were housed in light-tight rooms in individual cages (60 x 60 x 80 cm) bedded with wood shavings [1] except in experiments 2 and 6. The hens used in experiments 2 and 6 were initially housed in floor pens for 36 wk of egg production [28] and then in the cages for 1 wk before initiation of injections. Light intensity varied from 20 lux from the bottom tier inside the cages to 100 lux at the top tier outside the cages. Progesterone (Sigma, St. Louis, MO) was injected s.c. daily at the dorsal region of the neck. Progesterone was prepared in canola oil at dilutions so that 0.20 ml was injected for each 1.00 kg of body weight.


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  		TABLE 1. Experimental parameters for the experiments

During injection periods and for the 3-wk postinjection periods of experiments 7 and 8, the hens were checked daily for oviposited eggs and digitally palpated for uterine eggs. In the various experiments, egg production was also measured for 7–14 days before initiation of P4 injections. Eggs were stored at 4°C until processed, within 1 wk of laying, to measure egg weight and yolk weight. Blood samples (1.5 ml) were collected at various times during experiments 2, 6, and 8. Blood samples were usually taken just before a daily P4 injection during the period of injections. In experiment 6, however, samples were additionally collected at various times after a single daily injection, and in experiment 8, samples were collected weekly for the 3 wk between the last injections and experiment termination. All animal procedures were approved by the Institutional Animal Care and Use Committee (Protocol No. 01-AG001).

Morphological Measurements

Hens were killed by an overdose of pentobarbital and necropsied at the end of the P4 injections (experiments 1–6) or 3 wk after the end of the P4 injections (experiments 7 and 8). At necropsy, the following morphological measurements were made: 1) presence and location of any eggs in the oviduct, 2) weight of the ovary after removal of all yolky follicles heavier than 0.80 g, 3) weight of the oviduct after removal of any eggs, and 4) weight and classification of all follicles heavier than 0.80 g. Follicles were classified as previously described [1, 2] by the following criteria: Type I follicles were turgid, yolky follicles that increased hierarchically in weight from 0.80 to 1.00 g above the mean yolk weight of the last three eggs laid by a hen before necropsy. Type II and type III follicles were turgid, yolky follicles that were heavier than the type I follicles. Type III follicles were differentiated from type I and type II follicles by containing a less viscous, whitish fluid just under the follicle wall, which was interspersed with the viscous yellow yolk. Type III follicles were also whitish in color in comparison to type I and type II follicles. Type I and type II follicles were easily differentiated from type III follicles by puncturing them and examining the extruded contents for the presence of the whitish fluid [1]. Hens were classified as either not expressing or expressing the POF or PCOF syndrome [1, 2]. Laying hens had a normal laying history and an ovary with only type I follicles at autopsy. The POF hens had ceased laying for 2–3 wk, and their ovary had type I and at least five type II follicles at autopsy. The PCOF hens had ceased laying for 2–3 wk, and their ovary had type I and at least five type II and/or type III ovarian follicles at autopsy.

Hormone Assays

Progesterone was assayed as previously described [29]. All samples from individual experiments were assayed in single assays. The mean intraassay coefficient of variation (CV) was 9.5% for a pool of plasma with a mean P4 concentration of 7.12 ng/ml.

Estradiol-17ß was assayed as previously described [30]. Only samples from experiment 8 were assayed in a single assay. The mean intraassay CV was 8.7% for a pool of plasma with a mean E2 concentration of 0.51 ng/ml.

Luteinizing hormone was assayed as previously described [31]; only samples from experiment 8 were assayed. The mean intraassay CV was 8.5%, and the interassay CV for the two assays was 11.0%, for a pool of plasma with a mean LH concentration of 2.94 ng/ml.

Data Analyses

Egg production and morphological data for experiments 1, 2, 5, and 6 were analyzed by one-way ANOVA for the effect of P4 dose. Means were separated when appropriate using the Tukey procedure. Egg production and morphological data for experiments 3, 4, 7, and 8 were analyzed by factorial ANOVA. The model included main effects of photoperiod and P4 dose as well as their interaction. Main-effect means were separated when appropriate using the Tukey procedure. The differences in incidence of the POF and PCOF syndromes in experiments 7 and 8 were examined by chi-square analysis.

The P4 data from laying hens were first separated into baseline and surge classes as follows: the P4 concentrations of laying hens were transformed to ln P4 concentration, and the distribution of ln P4 concentration was then plotted as a histogram and fitted with a five-degree polynomial. Histograms and polynomial fitting of the data from experiments 2, 6, and 8 are presented in Figure 1. Note the bimodal distribution of ln P4 concentration, with a break between the lower (baseline) and the upper (surge) populations at ln P4 (ng/ml) = 1.6 (4.95 ng/ml) for each experiment. All concentrations of 4.95 ng/ml or less were considered to be baseline concentrations; all concentrations greater than 4.95 ng/ml were considered to be surge concentrations. Hens that had not laid an egg for 2 days or longer were assumed to have baseline (nonsurge) concentrations of P4, although some values were greater than 4.95 ng/ml. For experiments 2 and 6, the baseline P4 concentrations among treatment groups within experimental day were compared using ANOVA, with dosage of P4 as main effect. In experiment 8, preliminary analyses showed no effect of photoperiod on hormone (LH, P4, and E2) concentrations. The data were then regrouped into control and P4-injected hens. The P4-injected hens were retrospectively divided into two groups: Laying hens were those that stopped laying during P4 injection but returned to laying before necropsy 3 wk after the last injection, and PCOF hens were those classified as presenting with the PCOF syndrome at necropsy. All P4-injected hens fell into one or the other of these two groups. Main-effect means for LH, P4, and E2 analyses were separated using the Tukey procedure when appropriate. All statistical analyses were conducted using the MINITAB (Release 13.1; MINITAB, State College, PA) statistical package.



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  		FIG. 1. Histograms of P4 concentration of laying hens. The smooth lines graph the five-degree polynomial fit of the histograms. In each experiment, two populations of samples were observed. The baseline population included concentrations from ln P4 = 0 ± 0.1 to 1.5 ± 0.1, and the surge population included all concentrations ln P4 ≥ 1.6. a) Experiment 2. b) Experiment 6. c) Experiment 8


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Experiment 1

Egg production (Table 2) was not different among treatment groups before initiation of P4 injections, but it declined with the 0.17 mg kg–1 day–1 dosage and stopped with the 0.50 and 1.50 mg kg–1 day–1 dosages. More of the hens treated with 0.50 and 1.50 mg kg–1 day–1 held hard-shelled eggs for 5 days or longer than did hens in the control and 0.17 mg kg–1 day–1 treatment groups. No differences were observed in oviductal weight, number of ovarian yolky follicles, or type I follicles among the treatment groups, but the hens treated with 0.50 mg kg–1 day–1 had more type II follicles. No type III follicles were found, and none of the hens had developed either the POF or PCOF syndrome when necropsied. The number of atretic follicles increased with the dosage of P4.


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  		TABLE 2. Responses to 7 days of P4 injection; experiment 1.a

Experiment 2

Egg production (Table 3) was not different among treatment groups before initiation of P4 injections, but it declined with the 0.17 mg kg–1 day–1 dosage and stopped within 2 days with the 0.33 and 0.50 mg kg–1 day–1 dosages. More of the hens treated with 0.33 and 0.50 mg kg–1 day–1 dosages held hard-shelled eggs for 5 days or longer than did hens in the control and 0.17 mg kg–1 day–1 treatment groups. Oviductal weight was lighter in the 0.50 mg kg–1 day–1 treatment group than in the control group. Ovarian yolky follicle number and number of type I follicles were both lower with the 0.50 mg kg–1 day–1 treatment group than in the control group or other P4 treatment groups. No type II or type III follicles were found, and none of the hens had developed either the POF or PCOF syndrome when necropsied. The number of atretic follicles was lowest in the control group and highest in the 0.33 and 0.50 mg kg–1 day–1 treatment groups.


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  		TABLE 3. Responses to 14 days of P4 injection; experiment 2.a

Plasma baseline P4 concentrations 24 h after injection were associated with dosage (Fig. 2a) and were relatively stable during the 14 days of injections. Mean plasma baseline P4 concentrations 24 h after injection were 1.77, 2.99, 4.57, and 5.92 ng ml–1 for the 0.00, 0.17, 0.33, and 0.50 mg kg–1 day–1 dosages, respectively.



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  		FIG. 2. Baseline P4 concentrations of turkey hens. Only baseline samples were included from hens that were laying or had laid within 48 h of sample collection. a) Experiment 2. Blood samples were collected 24 h after P4 injection except for Day 0, when samples were collected before the first injection. b) Experiment 6. Blood samples were collected 24 h after P4 injection except for Day 0, when samples were collected before the first injection, and after the Day 7 sample, when additional samples were collected 2.5, 6, and 17 h after injection

Experiment 3

Egg production (Table 4) was not different among P4 dosage groups or photoperiod groups before initiation of injections, but it declined with the 0.17 mg kg–1 day–1 dosage and stopped with the 0.33 and 0.50 mg kg–1 day–1 dosages. Photoperiod had no effect on egg production. Treatment with P4 had no effect on eggs held for 5 days or longer, but more hens photostimulated with the 24L:0D photoperiod than with the 14L:10D photoperiod held eggs. Oviductal weight was not affected by either P4 treatment or lighting treatment. Total ovarian yolky follicle number and number of type I follicles were increased in the 0.33 and 0.50 mg kg–1 day–1 dosage groups but were not different between photoperiods. The number of type II follicles was increased at the 0.50 mg kg–1 d–1 dosage and with the 14L:10D photoperiod. The number of type III follicles was not affected by P4 injection or photoperiod. None of the hens had developed either POF or PCOF syndrome when necropsied. The number of atretic follicles was lowest in the control group and highest in the 0.33 and 0.50 mg kg–1 day–1 dosage groups, but the number was not different between photoperiods. No interactions between P4 dosages and photoperiods were noted for any of the traits.


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  		TABLE 4. Responses to 14 days of P4 injection of hens photostimulated with a diurnal or constant photoperiod; experiment 3.a

Experiments 4–6

Egg production and morphological measurements for experiments 4 and 5 were similar to those for experiment 3 (data not shown). Egg production and morphological measurements for experiment 6 were similar to those for experiment 2 (morphological data not shown). For experiment 6, plasma baseline P4 concentrations 24 h after injections were associated with dosage (Fig. 2b), and the concentrations were in agreement with those of experiment 2. On Experimental Day 7, blood was collected 2.5, 6, and 17 h after P4 injection. For each dosage, plasma concentrations of P4 peaked at a level approximately 3.3-fold above the baseline P4 concentration for that dosage between 2.5 and 6 h after injection.

Experiment 7

Egg production (Table 5) was not different between the P4-injected and the photoperiod groups before initiation of P4 injections, but egg production stopped in the P4-injected hens. Photoperiod had no effect on egg production during treatment with P4. The control hens continued to lay during the 3 wk after the P4 treatment ceased. More of the hens treated with P4 and given the 24L:0D photoperiod held hard-shelled eggs for 5 days or longer than did hens in the other treatment groups. An interaction was noted between P4 treatment and photoperiod: more of the 24L:0D photoperiod and P4-injected hens (83%) held eggs than did hens in the 24L:0D control and in both 14L:10D photoperiod groups (range, 0–16%). Oviductal weights, measured 3 wk after the last P4 injections, were not affected by P4 treatment or photoperiod. Total ovarian yolky follicle number was higher with P4 treatment and in the 24L:0D photoperiod groups. No difference was observed in the number of type I follicles between P4 dosage or photoperiod groups. The number of type II and type III follicles was greater in the P4 treatment and 24L:0D photoperiod groups. An interaction was noted between P4 treatment and photoperiod, with more type II and type III follicles (means 6.8 and 3.7, respectively) present in the P4 and 24L:0D photoperiod group than in the control and 14L:10D photoperiod groups (ranges of means, 0–2.6 and 0–0.8, respectively). The number of atretic follicles was greater in the P4-treated hens, but this number was not affected by photoperiod.


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  		TABLE 5. Responses to 10 days of P4 injection followed by 3 wk of no injections of hens photostimulated with a diurnal or constant photoperiod; experiment 7.a

When necropsied, more of the hens treated with P4 in the 24L:0D photoperiod group had developed PCOF syndrome than did hens treated with P4 in the 14L:10D photoperiod group (P ≤ 0.05), and none of the control hens in either photoperiod had developed POF or PCOF syndrome (Table 6). Weight distribution of follicles for a control and a P4-injected, PCOF syndrome hen from the 24L:0D photoperiod group are given in Figure 3a and for a control and a P4-injected, PCOF syndrome hen from the 14L:10D photoperiod group in Figure 3b. Both control hens had similar patterns of follicle weight distribution, and both PCOF hens had similar patterns of follicle weight distribution.


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  		TABLE 6. Incidence of the POF or PCOF syndrome in arrested-laying turkey hens



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  		FIG. 3. Weight distribution of follicles from control or P4-treated (0.33 mg kg–1 day–1) hens classified as presenting with PCOF syndrome from experiment 7. The control hen ovaries contained only type I follicles, whereas the PCOF hen ovaries contained type I, type II, type III, and atretic follicles. a) Weight distribution of follicles from a control and a PCOF hen photostimulated with a 24L:0D photoperiod. b) Weight distribution of follicles from a control and a PCOF hen photostimulated with a 14L:10D photoperiod

Experiment 8

Egg production (Table 7) was not different between P4 or photoperiod groups before initiation of P4 injections, but it stopped in all but one of the P4-injected hens: One of the P4-injected hens in the 14L:10D photoperiod group continued laying during the 12 days of injections. Photoperiod had no effect on egg production during P4 injection. The control hens continued laying during the 3 wk after the P4 treatment ceased. More of the hens treated with P4 and given the 24L:0D photoperiod held hard-shelled eggs for 5 days or longer than did hens in the control and 14L:10D photoperiod groups. Oviductal weights, measured 3 wk after the last P4 injections, were not affected by P4 treatment or by photoperiod. Total ovarian yolky follicle number was higher with P4 treatment and in the 24L:0D photoperiod groups. No differences were observed in the number of type I or type II follicles between P4 treatment or photoperiod groups. The number of type III follicles was greater in the P4-injected group, but the number was not different between photoperiod groups. The number of atretic follicles was not affected by P4 treatment or by photoperiod.


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  		TABLE 7. Responses to 12 days of P4 injection followed by 3 wk of no injections of hens photostimulated with a diurnal or constant photoperiod; experiment 8.a

When necropsied, more of the hens in the P4-treated and 24L:0D photoperiod group had developed PCOF syndrome compared with hens in the P4-treated and 14L:10D photoperiod group (P ≤ 0.05) (Table 6). None of the control hens had developed POF or PCOF syndrome when necropsied.

Plasma baseline P4 concentrations were increased during P4 injection (Fig. 4a). For the hens that had developed PCOF syndrome at necropsy 3 wk after last P4 injections, the level of P4 remained high until autopsy, whereas in the hens that had returned to laying, the plasma level of P4 declined to levels similar to those of the control laying hens. More of the hens photostimulated with the 14L:10D than with 24L:0D photoperiod and injected with P4 resumed laying (Table 6). Plasma baseline LH concentrations were minimally affected by either injection of P4 or laying state of the hens injected with P4 (Fig. 4b), but the level tended to be slightly higher in the control laying hens. Plasma E2 concentrations were minimally affected by either injection of P4 or laying state of the hens injected with P4 (Fig. 4c), but the level tended to be slightly higher in the control laying hens.



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  		FIG. 4. Baseline concentrations of P4 and LH and concentrations of E2 in control laying or P4-injected hens. The P4-injected hens were subdivided into groups that were classified as those presenting with PCOF syndrome (P4 PCOF) or those laying (P4 Laying) at necropsy. Means within experimental day with different letters are different (P, 0.05). a) Baseline concentrations of P4. The arrows ({downarrow}) indicate days when individual hens resumed laying. b) Baseline concentrations of LH. c) Concentrations of E2. The heavy bar at the top represents the duration of P4 injections


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The POF and PCOF syndromes in Egg line turkey hens have been defined previously [1]. None of the hens injected with P4 for 7–14 days and then immediately necropsied met these criteria, although some had not laid for 12 days and had held a fully formed egg in the shell gland for 5 days or longer after laying had ceased. An increase in the number of atretic follicles was noted for these hens, suggesting that follicles may enter the hierarchy but become atretic during P4 injections. Thus, continuous P4 injection for up to 14 days did not induce either POF or PCOF syndrome, even though it appeared to initiate the process by inducing an arrest in laying without ovarian or oviductal regression.

In experiments 7 and 8, it was decided to alter the experimental protocol by waiting for 3 wk after the last P4 injections to terminate the experiments and necropsy the hens. It was hypothesized that the hens would not resume laying during this postinjection period and that this period without daily injections of P4 and associated 3.3-fold increases in plasma concentration might allow additional follicles to accumulate in the ovary as type II and type III follicles. It was also hypothesized that continuous lighting would be more effective at inducing the PCOF syndrome than 14L:10D diurnal lighting, because continuous lighting is more effective at inducing the PCOF syndrome in turkey hens younger than those used in the present experiments without P4 injection [2]. Most of the hens in the 24L:0D photoperiod groups (12 of 13 [92%]) that had stopped laying for at least 10 days had developed the PCOF syndrome at 3 wk after the last daily P4 injection. Only a few hens in the 14L:10D photoperiod group (3 of 13 [23%]) that had stopped laying for at least 10 days had developed the PCOF syndrome at 3 wk after the last daily P4 injection. Of the remaining 10 hens in the 14L:10D photoperiod group that had stopped laying for at least 10 days, 7 resumed laying by 3 wk after the last P4 injection. Thus, injection of P4 at a dosage of 0.33 mg kg–1 day–1 was effective at stopping egg production of hens photostimulated with either photoperiod, but the 24L:0D photoperiod in conjunction with P4 injection was much more effective at inducing the PCOF syndrome during the 3 wk following P4 injections. In experiments 7 and 8, the hens had been laying for approximately 6 wk when P4 injections started but were of different ages (age, 34 and 48 wk, respectively) when photostimulated. The results of the two experiments were similar, indicating that this difference in age at photostimulation had little influence on the ability to induce the PCOF syndrome by injection of P4 relatively early in the egg production period. The injection of P4 can thus be considered as an initiator for development of the PCOF syndrome relatively early in the reproductive period, especially in turkey hens photostimulated with the 24L:0D photoperiod.

In turkey hens with either POF or PCOF syndrome, P4 levels were previously reported [1] to be relatively high and without surges, whereas the LH level was relatively low and without surges. In the present studies, the turkey hens were not serially bled, so the development of these nonsurge patterns of LH and P4 secretion previously reported for the hens presenting with POF or PCOF remains unknown. Because surges of LH are associated with ovulations, the data suggest that surge secretion of LH is blocked by daily injections of P4 at dosages of 0.33 mg kg–1 day–1 or greater.

The concentrations of FSH were not measured in the current or previous experiments [1] because of the lack of a validated assay for turkey FSH. In chicken hens, E2 levels increase with FSH treatment [32], indicating that peripheral levels of E2 may be closely associated with FSH. It is suggested that FSH secretion did not change with P4 treatment, because levels of E2 did not change during P4 treatment or development of the PCOF syndrome in experiment 8. For laying hens late in the egg production period, the level of E2 has been reported to decline [33]; thus, FSH secretion may also be lower at this time. In older hens, a lower rate of FSH secretion may lead to atresia of ovarian follicles during P4 injection (experiments 2 and 6) instead of ovarian follicle maintenance (experiments 1, 3, 4, and 5) or development of the PCOF syndrome (experiments 7 and 8) subsequent to P4 injection.

In all the present studies, a P4 dosage of 0.17 mg kg–1 day–1 reduced egg production by approximately 50%. With dosages of 0.33 mg kg–1 day–1, egg production ceased within approximately 2 days in most hens. Thus, the decline in egg production rate was equally sensitive to the dosage of P4 injected in young hens early during the egg production period as in old hens late during the egg production period. Age when hens were treated, however, did affect other responses to P4 treatment. In five of the present experiments, hens were photostimulated at 41 wk of age, then injected with P4 after 1–38 wk of egg production and immediately necropsied. If injected with P4 at 0.33–1.50 mg kg–1 day–1 before reaching 15 wk of egg production, the hens maintained a fully stimulated oviduct and a normal number of type I (i.e., hierarchical) follicles. The ovaries in these hens additionally had a few type II and type III follicles and an increased number of atretic follicles. If the hens were injected at 38 wk of egg production with 0.33 or 0.50 mg kg–1 day–1, their ovaries also had an increased number of atretic follicles. The oviduct had decreased in weight, however, and the ovary had few type I and no type II or type III follicles. It was concluded in the older hens with increased duration of the egg production period that egg production had stopped and that both the ovarian follicles and the oviduct were regressing after 14 days of P4 injection. In the younger hens, however, the oviduct was maintained at a weight comparable to that of laying hens, whereas the ovary was maintained in a stimulated state that was different from that of laying hens.

Ovulatory surge levels of P4 in plasma of laying turkey hens range from between approximately 5 and 8 ng/ml, whereas baseline levels between surges range between 1 and 2 ng/ml [1, 34]. The data from experiments 2 and 6 indicate that baseline levels of P4 24 h after injection were associated with dosage injected and peaked between 2.5- and 6-h postinjection at levels approximately 3.3-fold higher than the baseline associated with each dosage. Concentrations of P4 24 h after injection of P4 at dosages of 0.33 or 0.50 mg kg–1 day–1 were similar to ovulatory surge levels in laying turkey hens. It has previously been reported [1, 34] in turkey hens that ovulatory surges of LH occur only when P4 levels have been at baseline for several hours. This suggests that if P4 does not drop below ovulatory surge levels, then ovulatory surges of LH may be blocked, thereby blocking ovulation. Hens injected with 0.17 mg kg–1 day–1 had a level of P4 24 h after injection that was intermediate (2.9 ng/ml) between baseline and ovulatory surge levels and an egg production rate that was approximately 50% that of control hens. This suggests that if P4 remains above laying-hen baseline levels but declines to less than ovulatory surge levels between injections, then the interval between ovulatory surges may be longer, resulting in a lower rate of egg production. These effects of P4 injection on blocking or increasing the interval between ovulatory surges of LH occurred at all ages and durations of egg production studied and, thus, appear to be independent of hen age or duration of egg production.

From these studies, we conclude that PCOF syndrome can be induced early in the reproduction period by photostimulating hens with a 24L:0D photoperiod, injecting them with P4 for 10–12 days at a dosage of 0.33 mg kg–1 day–1, and then waiting 3 wk for the syndrome to develop. In older hens near the end of their reproductive period, however, injection of P4 induces both the ovary and the oviduct to regress.


   ACKNOWLEDGMENTS
 
The authors thank Dr. Karl E. Nestor for kindly providing the Egg line turkey hens used in these experiments. The authors also thank John A. Anderson for helping with injections and necropsies. The LH assay reagents were kindly supplied by Dr. John Proudman (U.S. Department of Agriculture, Beltsville, MD).


   FOOTNOTES
 
1 Salaries and research support provided by state and federal funds appropriated to the Ohio Agricultural Research and Development Center (Ohio State University, Wooster, OH). The present study was also supported, in part, by the George and Edna Jaap Endowment Fund for Poultry Research (Ohio State University). Back

2 Correspondence: Wayne L. Bacon, Department of Animal Sciences, Ohio Agricultural Research and Development Center, Wooster, Ohio 44691. FAX: 330 263 3949; bacon.2{at}osu.edu Back

Received: 22 January 2004.

First decision: 16 February 2004.

Accepted: 3 May 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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