Effects of progesterone injection on performance, plasma hormones and ovarian morphology of ad libitum and restricted fed broiler breeder hens

An experiment was conducted to evaluate responses of feed-satiated and feedrestricted breeder hens to daily injection of progesterone (P4). A total of 64 Cobb 500 hens were fed either restricted or ad libitum from 27 to 38 wk of age. Fourteen laying hens from each group were selected to conduct P4 injection assay. Half of birds in each group (n = 7) were injected daily by 2.5 mg P4/kg BW and remaining birds were used as control. The P4 was injected subcutaneously, at the base of the neck daily (at 0900 h) for 21 d. Settable and abnormal eggs were recorded daily. Blood samples were taken just before initiation of injections, 10-d and 20-d after initiation of injection. Plasma samples were analyzed for glucose, cholesterol, triacylglycerol (TAG), P4, estradiol (E2), testosterone, T3 and T4 concentration. Settable egg production declined following P4 injection, whereas total egg production (including soft shell egg) remain at high levels in injected birds during the first week after initiation of P4 injection. Progesterone injection in feed-satiated and feed-restricted birds resulted in ovary regression; the ovary of these birds had no hierarchical follicle. Progesterone injection increased incidence of holding hardshelled eggs in the uterus. Plasma E2 concentrations were affected both by feeding pattern and P4 injection. Progesterone injection depressed plasma E2 concentration in both ad libitum and restricted fed hens. Hens with free access to feed had significantly lower plasma E2 levels compared to restricted fed hens. Our results revealed that whereas injection of P4 induced frequent ovulation early in the injection period in both feedsatiated and feed-restricted breeder hens; however this higher ovulation rate did not result in more settable egg production.


INTRODUCTION
Progesterone is the major hormone secreted by granulosa cells of large mature hierarchical follicles (F1) in birds. Progesterone acts at the level of the ovary and at the hypothalamus to stimulate the LH surge for ovulation. The progesterone production by F1 follicle peaks 6 to 8 h prior to ovulation (Johnson and van rinhoven, 1980) and triggers gonadotropin-releasing hormone (GnRH) release by the hypothalamus. This in turn is followed by an increase in LH and FSH release from the anterior pituitary. The LH stimulates an even greater output of progesterone by the granulosa cells of the F1 follicle (Etches, *Corresponding author. E-mail: rezataherkhani@yahoo.com. Tel/Fax: +982612248082. 1990), completing the positive feedback loop producing the LH peak 4 to 6 h prior to ovulation (Johnson et al., 1985). It has been shown that chronic or acute injection of P 4 may affect bird in different manner. Administration of an acute dose of exogenous P 4 has been shown to induce premature ovulation of a mature follicle at a specific time during ovulatory cycles in normal laying chicken hens (Nakada et al., 1994). Single injection of an acute dose of P 4 in laying hens during the preovulatory open period has been shown to have positive effect on inducing a preovulatory LH surge and ovulation Sharp, 1975, 1976;Johnson et al., 1985).
However, chronic injection of P 4 has been shown to increases baseline concentrations of P 4 and result in arrested laying and disrupted distribution of hierarchical follicles in turkeys (Liu et al., 2001b;Bacon and Liu, 2004). High baseline concentrations of P 4 in arrested hens might negatively feedback on the ability of the hypothalamus to secret surges of GnRH and subsequently surges of LH, or on the ability of the pituitary to respond to surges of GnRH secretion if they occur (Liu et al., 2001b;Bacon and Liu, 2004;Liu and Bacon, 2005).
Feed satiated hens have been shown to have more large yellow follicles than their restricted fed counterparts, so the objective of this study was to evaluate the effects of injection of a high dose of P 4 to simulate the essential P 4 surge required for establishment of LH surge in feedsatiated and feed-restricted broiler breeder hens during post peak production period (when ovulation rates are decreased) on egg production, ovulation rate and ovarian morphology. We also measured plasma glucose, TAG, cholesterol, P 4 , E 2 , testosterone, T 3 and T 4 concentration to know how P 4 injection affects these metabolites and hormones and consequently the bird's performance.

Birds and management
A total of 64 Cobb 500 broiler breeder pullets with similar body weight (2050 ± 40 g) were selected from a commercial flock (20 week of age) and moved to individual cages (0.6 × 0.4; 0.24 m 2 / bird) to conduct the research. Soft plastic wires were placed at cage floor to minimize bird's foot damages. Each cages had an individual feeder and a nipple waterer. Birds did not have access to each others feed. Pullets were fed restricted amounts of feed to provide a standard BW according to the Cobb Breeder Management Guide (Vantress, 2005) until 27 wk of age. Birds fed a pre-lay diet from 21 wk of age to first egg production and a broiler breeder layer diet thereafter (2700 kcal AMEn and 15.20% crude protein). Photostimulation occurred at 22 wk of age by providing 16 h of light (lights on at 0700 h) and this photoperiod was maintained until experiment termination. At 27 wk of age (50% egg production), hens were selected on the basis of their egg production and BW and assigned to two feeding regimens from 27 to 38 wk of age: i) ad libitum feeding and ii) restricted feeding. At 38 wk of age, 14 laying hen (hens produced at least one egg during last 10 d) from ad libitum and restricted fed hens were selected to conducted progesterone injection assay. At this time, hens from ad libitum group were 500 g heavier than their restricted counterparts. Half of birds in each group (n = 7) were injected daily by 2.5 mg P4/kg BW and remaining birds were used as control. The P4 injections were injected subcutaneously, at the base of the neck, daily (at 0900 h) for 21 d. The P4 vials were diluted by canola oil and 0.15 ml/ kg BW were injected each time.

Laying performance
Eggs were manually collected 2 times per day. Hen-day egg production was calculated weekly from daily egg counts and the numbers of abnormally double-yolked and soft shell egg were also recorded daily. Ovulation rate was calculated as (normal eggs + soft shell eggs + 2×double yolk eggs).

Necropsy and tissue collection
At the end of experimental period, 4 hens per treatment were slaughtered for necropsy. Liver, abdominal fat pad and ovary were collected at necropsy. Weight of liver, ovary and abdominal fat pad were divided by (BW/100) to estimate their fractional contribution. Ovaries were weighed (after removing hierarchical follicles) and follicles were classified into 3 groups: hierarchical follicles (large yellow follicles, > 8 mm), small yellow follicles (2 to 8 mm) and large white follicles (2 to 5 mm) according to the system devised by Gilbert et al. (1983).

Blood sampling
To evaluate plasma hormones and metabolites, first bleeding was carried out before initiation of injections and two subsequent bleedings were repeated every 10 d. Blood samples were obtained just before P4 injection. Blood samples were collected in EDTA coated tubes from the brachial vein of all hens per group. Blood samples were immediately centrifuged at 2000 × g for 15 min to collect blood plasma. Plasma samples were stored at -20°C until assayed for glucose, TAG, cholesterol, as well as T3, T4, progesterone, estradiol and testosterone.

Plasma metabolites and hormones
Plasma glucose, total triacylglycerol and cholesterol (CHO) were determined enzymatically using an automated analyzer (Hitachi 902, Japan). Specific radioimmunoassay was used to determine plasma hormone concentrations. All samples were analyzed within 1 assay in order to avoid inter-assay variations. Plasma P4 and E2 were determined by commercial RIA kits (ICN, Biochemichals, Cleveland, OH) with intra-assay CV of 6.46 and 7.15, respectively. The use of the P4 and E2 kits for chicken has previously validated by Onagbesan et al. (2006). Plasma testosterone was determined by using RIA kit (DRG, Germany) according to manufacturer's procedure with an intraassay CV of 6.39. Total T3 and T4 concentration were determined using commercial RIA kits (DRG, Germany). The intra-assay CV for T3 and T4 were 3.58 and 4.62, respectively.

Statistical analysis
The experimental birds were housed in individual cages. The experimental design was completely randomized with factorial arrangement using individual broiler breeder hens as experimental unit. There were 2 method of feed allocation with or without P4 injection. Performance data as well as plasma metabolites and hormones data were analyzed as repeated measures using Proc Mixed of SAS software (SAS Institute, Cary, NC). Differences between means were evaluated using least square means procedure. Data obtained in necropsy were analyzed using GLM procedure of SAS software and means were separated by Duncan multiple range test. Significant differences were considered to be P < 0.05.

Performance data
Progesterone injection significantly affected settable egg production. Progesterone injected bird produced less settable egg compared to non-injected birds both in restricted and ad libitum fed groups ( Figure 1). Drop in egg production continued through experimental period and reached to zero, 3 wk after P 4 injections. Egg production Zaghari et al. in ad libitum non-injected birds was significantly lower than their restricted fed counterparts. Ovulation rate in hens from different treatment are presented in Figure 2. Although egg production declined following P 4 injection, however ovulation rate remain at high levels in injected birds in the first week after initiation of P 4 injection. Ad libitum fed hens without P 4 injection had significantly lower ovulation rate compared to birds from remaining treatment. Whereas ovulation rate in injected birds was numerically higher than non-injected birds in the first week after initiation of P 4 injection, a dramatic decline occurred during the second and third wk post P 4 injections. The decline in ovulation rate in injected birds was more severe in restricted birds compared to ad libitum fed hens.
The number of soft shell eggs in P 4 injected birds was significantly higher than their non-injected counterparts in the first week post injections; however along with decline in ovulation rate in the later weeks, numbers of soft shell eggs tend to decrease (Table 1).
Hens with P 4 injection produced more soft shell eggs compared to non-injected hens (Table 1). Ad libitum fed birds also produced more (P < 0.05) double yolk eggs than restricted fed hens (Table 1).

Carcass and ovary data
Abdominal fad pad fractional weight in ad libitum fed birds were significantly higher than restricted fed birds (Table 1); furthermore, feed-satiated hens had significantly heavier liver compared to feed-restricted birds. However P 4 injection had no significant effect on abdominal fat and liver fractional weight (Table 1).
Ovary fractional weight in hens with P 4 injection both in feed-satiated and feed-restricted birds were significantly lower than non-injected birds (Table 1).
Progesterone injection in feed-satiated and feed-restricted birds resulted in ovary regression; the ovary of these birds had no hierarchical follicle, SYF and also LWF ( Table 1). The numbers of LYF in ad libitum fed hens were significantly higher than feed-restricted birds. Progesterone injection increased incidence of holding hardshelled eggs in the uterus. Three hens from ad libitum and 2 hens from restricted fed birds (42 and 28% of necropsied birds, respectively) hold a hard-shelled egg in their uterus (data not shown).

Hormones and metabolites
Plasma glucose, TAG and CHO concentrations of hens from different treatments are presented in Table 2. Ad libitum fed hens had significantly (P < 0.0001) higher plasma glucose, TAG and CHO levels than their restrictted fed counterparts. However, P 4 injection did not have any significant effect on these metabolites.
Progesterone injection significantly (P < 0.0001) reduced plasma P 4 concentration both in ad libitum or restricted fed birds 10 and 20 d after initiation of P 4 injection ( Figure 3). Plasma P 4 levels were not different between feed-satiated and feed-restricted birds.
Plasma E 2 concentrations were affected both by feeding pattern and P 4 injection (Figure 4). Progesterone injection depressed (P < 0.0001) plasma E 2 concentration in both ad libitum and restricted fed hens. Hens with free access to feed had significantly lower (P < 0.0001) plasma E 2 levels compared to restricted fed hens.
Plasma testosterone concentrations of hens from different treatments are presented in figure 5. Ad libitum fed birds had significantly higher (P < 0.0001) plasma testosterone levels compared to their restricted fed counterparts. Hens with P 4 injection had significantly lower (P < 0.001) plasma testosterone concentrations than noninjected birds.
Ad libitum fed birds had significantly higher (P < 0.0001) plasma T 3 ( Figure 6) and lower (P < 0.0001) plasma T 4 concentration (Figure 7) than their restricted fed counterparts. Progesterone injection did not change plasma T 3 or T 4 concentration neither in ad libitum fed hens nor in restricted fed birds.

DISCUSSION
Progesterone acts on ovary and hypothalamus to stimulate the LH surge for ovulation. The rise in progesterone concentration stimulates a rise in LH concentration, which in turn stimulates a further rise in progesterone. This positive feedback loop is what causes the progesterone and ultimately LH surge (Johnson et al., 1985).
The current study was designed to examine the effects of a simulated progesterone surge (by injection of a high dose of P 4 ) on broiler breeder hens performance, ovary morphology, plasma metabolites and hormones concentrations in either feed-satiated or feed-restricted hens. Although P 4 injection resulted in increased ovulation rate during the first week post injections, however, settable egg number decreased along with initiation of injections because of high number of soft shell and double yolk egg production. During the second and third wk after injections both ovulation rate and egg production decreased in feed-satiated or feed-restricted birds. Increased ovulation rate during first week after injection may be due to simultaneity of P 4 injection with open period of LH. High dose of P 4 acts as a progesterone surge and drives follicles to ovulation. Inability of oviduct to direct all ovulated follicles resulted in higher incidence of soft shell and double yolk eggs. Liu and Bacon (2005) observed higher incidence of soft shell egg after P 4 injections in broiler breeder. Similar to results obtained in the current study, egg production decreased after P 4 injection in turkey , broiler breeder (Liu and Bacon, 2005) and in Japanese quail (Tell et al., 1999;Liu and Bacon, 2004). Egg production ceased 3 wk after injecting daily P 4 . Necropsy data shown a regressed ovary with no LYF, SYF or LWF in all hens with progesterone injection. Taken together, these data suggest that P 4 treatment has a strong negative effect on egg production rate and hierarchical follicular maintenance and development. Liu and Bacon (2005) came to similar conclusion.
Ad libitum fed broiler breeders are known to have more hierarchical follicles than their restricted fed counterparts (Yu et al., 1992;Chen et al., 2006;Sun et al., 2006). It seems that higher ovulation rate in ad libitum fed hens with P 4 injection compared to restricted fed birds may be due to more LYF number in their ovary. Progesterone injection significantly lowered E 2 concentration both in feed-satiated and feedrestricted hens. Plasma concentrations of E 2 are increased after follicle stimulating hormone (FSH) injection in laying hens late in the reproductive period (Palmer and Bahr, 1992), suggesting that the secretion of FSH is also decreased after P 4 injection. Liu and Bacon (2005) reported a decline in LH concentration after P 4 injection. Taken together, these data suggest P 4 injection may have negative effect on secretion of GnRH. High concentrations of E 2 are required to sensitize the hypothalamic pituitary axis to the positive feed back effects of progesterone (Wilson and Sharp, 1976) to stimulate vitellogenin formation in the liver (Redshaw and Foliett, 1972), to regulate calcium metabolism (Etches, 1987), to stimulate and maintain a functional oviduct and to maintain secondary sexual characteristic. The lower concentration of E 2 may thus lead to regression of the oviduct and impaired follicles development after P 4 injection. Similar to our results, Liu and Bacon (2005) observed a depression in plasma E 2 along with regressed ovary after injection of P 4 in broiler breeder hens. Lower E 2 concentration of ad libitum fed hens compared to restricted fed hens observed in this research is in agreement with Onagbesan et al. (2006). Plasma P 4 concentrations in hens with daily injection of P 4 decreased 10 and 20 d after initiation of injections. Injection of acute dose of P 4 will result in higher P 4 surge concentration which in turn lowers P 4 production through negatively affecting LH and FSH production. Liu and Bacon (2005) concluded that higher P 4 surge concentration and lower LH baseline concentration may be associated with decline in egg production in P 4 injected breeder hens. Progesterone concentration decreased following P 4 injections in broiler breeder hens in the study of Liu and Bacon (2005).
Besides E 2 and P 4 , progesterone injection lowered plasma testosterone levels both in feed-satiated and restricted fed hens. It seems that P 4 injection negatively Week after initiation of injection affect ovarian steroidogenesis.
Whereas P 4 injection had no significant effect on plasma metabolites, feeding pattern significantly affected plasma glucose, TAG and CHO. Ad libitum fed hens had higher levels of plasma glucose, TAG and CHO compared to restricted fed birds. These results are consistent with those reported by Chen et al. (2006) and Sun et al. (2006).
Progesterone injection increased incidence of holding a hard-shelled eggs in the uterus. Similar to results observed in our study, Liu and Bacon (2005) reported a high incidence of holding hard-shelled eggs in broiler breeder hen's uterus. Holding a hard-shelled egg in the uterus was also induced by exogenous P 4 in turkey  and quail hens (Lin et al., 1993;Tell et al., 1999;Liu and Bacon, 2004), suggesting that P 4 may be associated with inhibition of muscular contraction of the uterus at oviposition in these species. Progesterone injection had no significant effect on plasma T 3 and T 4 concentrations; however ad libitum fed hens had lower T 3 and higher T 4 level compared to restricted fed hens. Numerous studies suggest that feed restriction decrease T 3 and increase T 4 levels in broiler breeders (Darras et al., 1995;Bruggeman et al., 1997).
In conclusion, whereas injecting an high dose of P 4 once a day for 3 wk induced frequent ovulation early in the injection period in both feed-satiated and feed-restricted breeder hens; however this higher ovulation rate did not result in more settable egg production. Egg production decreased with P 4 injection and ceased at the termination of experiment. Decreased egg production in P 4 injected bird was associated with lower levels of sex steroid hormones (P 4 , E 2 and testosterone) and regressed ovary. Progesterone injection increased numbers of hens holding a hard-shelled egg in their uterus. Progesterone injection had no significant effect on glucose home ostasis and lipid metabolism. Restricted fed and laying ad libitum fed breeder hens respond in a similar way to a high dose of P 4 injection.