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Biology of Reproduction 65, 1594-1600 (2001)
© 2001 Society for the Study of Reproduction, Inc.

Regular Article

Effects of Active Immunization with Inhibin {alpha} Subunit on Reproductive Characteristics of Turkey Hens1

Jaeyong Ahna, Seungkwon Youb, Hyunggee Kima, Yupaporn Chaisehac, and Mohamed El Halawani2,,a

a Department of Animal Science, University of Minnesota, St. Paul, Minnesota 55108 b School of Agricultural Biotechnology, Seoul National University, Suwon 441-744, Korea c School of Biology, Institute of Science, Suranaree University of Technology, Nakornrachasima 3000, Thailand

ABSTRACT

The hypothesis for the present study is that the active immunization of female turkeys with inhibin (INH) would neutralize endogenous INH, and increase levels of circulating follicle stimulating hormone (FSH) and the number of preovulatory follicles, and subsequently enhance egg production. Two experiments were conducted with female turkeys in their first (30 wk of age) and second (62 wk of age) laying cycles. Treatment groups included control turkeys immunized with keyhole limpet hemocyanine (KLH) and experimental turkeys immunized with recombinant turkey inhibin{alpha} conjugated to KLH (rtINH), vasoactive intestinal peptide (VIP) conjugated to KLH or rtINH+VIP. Egg production increased (P < 0.05) in VIP and rtINH+VIP immunized birds, but not in rtINH immunized hens in comparison with a control group. A similar number of ovarian follicles, arranged in the follicular hierarchy of laying hens, was observed in all experimental groups. However, there was a larger number of nongraded yellow follicles in rtINH-immunized (62.5%) and rtINH+VIP-immunized (73.5%) groups compared with that of controls, suggesting overstimulation by FSH. Anterior pituitary FSHß subunit, LHß subunit, and prolactin (PRL) mRNA contents were determined by Northern blot analysis and reverse transcriptase-polymerase chain reaction (RT-PCR) in laying hens at the end of the experimental period. Hens immunized with rtINH showed increased FSHß subunit mRNA content, but no change in the content of LHß subunit or PRL mRNA. Hens immunized with VIP or rtINH+VIP had significant increases in both pituitary LHß subunit and FSHß subunit mRNA contents, accompanied by a decline in PRL mRNA abundance. The magnitude of the increase in FSHß subunit to INH immunoneutralization was greater in first-cycle hens than in second-cycle hens. These data suggest that active immunization of female turkeys with INH neutralizes endogenous INH and increases both circulating FSH and the number of preovulatory follicles. However, no significant increase in egg production was observed in INH-immunized hens. The data confirm previous reports that VIP immunoneutralization increases egg production in turkey hens and shows for the first time that it also increases FSHß subunit and LHß subunit gene expression.

follicle-stimulating hormone, follicular development, hypothalamic hormones, hypothalamus, inhibin, luteinizing hormone, pituitary hormones, prolactin

INTRODUCTION

Several lines of evidence support the importance of inhibins (INHs) in the regulation of the hypothalamo-pituitary-gonadal axis. First, negative correlation between FSH and levels of INH B have been reported in humans [1, 2]. Second, infusion of INH A suppresses FSH levels in castrated rams and monkeys without demonstrating any effect on LH secretion [3, 4]. Third, active immunization against INH neutralizes endogenous INH and increases the ovulation rate in several mammalian species (sheep [5], gilt [6], rat [7], and heifer [8]). Plasma levels of FSH are raised in INH-immunoneutralized ewes [9], rats [7], and heifers [10].

Inhibins act within the ovary as paracrine factors. Inhibin A enhances LH-stimulated androgen secretion by human thecal cells cultured in vitro [11]. Direct injection of INH A into immature rat ovaries stimulates follicle maturation [12], and binding sites for INHs have been found in the ovary [13]. In mammals, circulating levels of INHs A and B change during the menstrual cycle. Inhibin A appears to be secreted mainly by luteal cells during the luteal phase and seems to be involved in the ovarian negative FSH feedback [14], whereas INH B is produced by granulosa cells during follicle development and may be a marker of follicle growth [15, 16]. In chickens, granulosa layers of large preovulatory follicles are the major source of INH [17–19]. Inhibin has been shown to have a modulatory role on gonadal steroidogenesis [20]. Studies on the role of INHs in turkey reproduction are lacking.

The hypothesis for this study is that active immunization of female turkeys using recombinant turkey INH{alpha} (rtINH{alpha}) would neutralize endogenous INH and increase the level of circulating FSH and the number of preovulatory follicles, thereby enhancing egg production. It is well established that enhanced hypothalamic vasoactive intestinal peptide (VIP) expression and secretion cause a marked elevation in circulating prolactin (PRL) levels, which induces incubation behavior in female turkeys [21–23]. The induction of incubation behavior is associated with cessation of ovulation, ovarian regression, and a decline in gonadotropic hormone secretion [24]. It was a concern that these chain of events would interfere or mask the effect of INH{alpha} immunoneutralization on ovarian function. It was deemed essential in this study to immunize turkey hens against both INH{alpha} and VIP, because VIP immunoneutralization prevents both the rise in circulating PRL and the induction of incubation behavior [25].

MATERIALS AND METHODS

Animals

In the first experiment, Nicolas large white female turkeys (30 wk old) were used. The hens were divided into two groups (10 hens/group), housed in floor pens with trap nests, and exposed to a lighting regimen of 15L:9D, with lights-on at 0400 h and lights-off at 1900 h, for 21 wk. Hens were photostimulated in the month of December.

In the second experiment, Nicholas large white female turkeys (62 wk old; recycled breeder hens) were used. Birds were divided into four treatment groups (32 hens/group). Each floor pen contained eight birds, two from each treatment group. Sixteen floor pens held a total of 128 birds. The lighting schedule was the same as in experiment 1. The experiment was initiated in May and continued for 26 wk.

Feed and water were always available. Trap nests were inspected six times daily from 0800 to 1600 h. Birds and any accompanying eggs were removed from the nests at each nest check. The number of times each bird entered the nest per day and daily egg production were tabulated. The birds were considered to be incubating if they were found in the nest more than four times without laying an egg.

Active Immunization with rtINH{alpha} and Vasoactive Intestinal Peptide

In experiment 1, rtINH{alpha} was conjugated by the glutaldehyde method to keyhole limpet hemocyanin (KLH; Peninsula Laboratories, Belmont, CA; [21]). The first dose of the immunogen contained 125 µg rtINH{alpha} and was given in a 250 µl ISA 70 adjuvant made up to 500 µl/bird with 0.85% (w/v) NaCl (saline). The mixture was emulsified and injected intradermally into the lateral thoracic wall under the wing. Booster immunizations were given with KLH-rtINH{alpha} conjugate containing 25 µg rtINH{alpha} in ISA 70 adjuvant. Control turkeys were injected with KLH and adjuvant only. Immunization began on the first day of photostimulation and was repeated at 4-wk intervals for a total of five injections.

In experiment 2, Freund complete adjuvant (Sigma Chemical Co., St. Louis, MO) was used (instead of ISA 70 adjuvant) for first immunization and Freund incomplete adjuvant was used for subsequent immunizations. Immunization schedule and doses of the immunogen were the same as those in experiment 1. The hens were immunized with 1) KLH and adjuvant, 2) synthetic turkey VIP conjugated to KLH, 3) rtINH{alpha}, or 4) rtINH{alpha}+VIP. Treatment groups were given six booster injections.

Blood Sampling

Blood samples ({approx}3 ml) were collected 2 wk after each immunization. Samples were centrifuged at 2000 x g for 30 min and plasma samples were maintained at -20°C until assayed for PRL and antibody titers for VIP and INH.

PRL Radioimmunoassay

Plasma was assayed for PRL content using the homologous radioimmunoassay described by Proudman and Opel [26].

VIP Antibody Titer

Plasma samples were prediluted (1:1000) in 0.5 M EDTA-PBS buffer and used as a source of primary antibody in a tVIP-binding assay [27]. 125I-mono-iodinated tVIP was obtained through use of the Iodogen method and purified with reverse-phase high-pressure liquid chromatography [28].

INH{alpha} Antibody Titer

Diluted plasma samples (1:100 to 1:20 000) were used as a primary antibody in an immuno-dot blotting assay. One hundred nanograms of each rtINH{alpha} protein was transferred onto a nitrocellulose membrane (Schicher & Schuell Inc., Keene, NH), and immobilized using the immuno blot procedure described by Hawkes [29]. Sliced membranes were transferred into 48-well microtiter plates (Millipore, Bedford, MA). Excess sites on the nitrocellulose membrane were blocked with 3% normal goat serum, 1% fish gelatin, 0.05% Tween-20, 0.01% Antifoam A in Tris-HCl buffer (TBA, 10 mM Tris-HCl pH 7.5), and 150 mM NaCl overnight; washed; and incubated with the diluted plasma samples (1:100, 1:500, 1:1000, 1:10 000, and 1:20 000) with gentle shaking for 2 h. After washing and repeating blocking steps, the membranes were incubated with horseradish-conjugated goat anti-turkey immunoglobulin (Ig) G (KPL, Gaithersburg, MD) diluted 1:1000 in TBS for 2 h. Thereafter, the membranes were rinsed for color development using 30 mg of 4-chloro-1-naphthol (4CN) in 3 mg/ml of ice-cold methanol with 0.01% (v/v) 30% hydrogen perioxidase in 5 volumes of TBS [29].

Northern Blot Analysis

Total cellular RNA from pituitaries of laying birds (control, VIP, rtINH{alpha} and rtINH{alpha}+VIP immunized groups) was isolated using TRIzol (Gibco, Gland Island, NY) according to the manufacturer's procedure. RNA samples (20 µg) were separated by electrophoresis on 1% agarose gels containing formaldehyde and blotted onto a positively charged nylon membrane (Schleicher & Schuell). Membranes were hybridized to each turkey-specific FSHß, LHß, and PRL cDNA fragments amplified by reverse transcriptase-polymerase chain reaction (RT-PCR) with corresponding primer sets (see the next paragraph) and verified by sequencing analysis. Probes were labeled with [{alpha}-32P]dATP using a Prime-it II Random Primer Labeling Kit (Stratagene, La Jolla, CA). To determine the equal amount of RNA loading, either RNA gel was stained with ethidium bromide or membranes were stained with methylene blue solution (0.02% methylene blue, 0.3 M sodium acetate pH 5.5), by which 28S and 18S rRNA were specifically stained. Specific hybridization signals were analyzed by the National Institutes of Health (NIH) Image program.

Reverse Transcription-Polymerase Chain Reaction

Total cellular RNA was extracted using TRIzol (Gibco) following the manufacturer's instruction. Single-strand cDNA was synthesized from 3 µg of total cellular RNA from each turkey anterior pituitary. RT-PCR was performed with the Superscript II kit (Gibco) following the manufacturer's instructions.

Oligonucleotides used as PCR primers are listed below. The FSHß primer pair was sense 5'-GCTCTAGACTACAGCTGTGAGCTCACCAAT-3', antisense 5'-CCATCGATTCTGCATTCTATACGTCCGTGG-3'; this primer sequence was based on the quail FSHß cDNA nucleotide sequence [30]. The LH-ß primer pair was sense 5'-GCTCTAGAACAGGTGTTGG TGCTGATGA-3', antisense 5'-CCATCGATGGCGACTGTAATGGAGCTGTGG-3'; this primer sequence was based on the turkey LHß nucleotide sequence [31]. The PRL primer pair was sense 5'-ACCTCCTTGCCAATCTGCTCCAGT-3', antisense 5'-TCCTCAATCTCTACAGCCTTCCAG-3'; this primer sequence was based on the turkey PRL cDNA nucleotide sequence [32]. The GAPDH primer pair was sense 5'-TGCAGGTGCTGAGTATGTTGTGGA-3', antisense 5'-CCACAACACGGTTGCTGTATCCAA-3'; this primer sequence was based on the chicken GAPDH cDNA nucleotide sequence [33].

Two milliliters of RT reaction was added to 18 µl of total master mixture, which included PCR buffer (final concentration 50 mM KCl, 10 mM Tris-HCl pH 8.3, 2.5 mM Mgcl2), 1 mM each of dNTP (dGTP, dATP, dTTP, and dCTP; Boehringer-Mannheim, Indianapolis, IN), 1 pmol of each corresponding primer, and 2.5 units Amplitag DNA polymerase (Perkin-Elmer/Cetus, Norwalk, CT). The PCR programs were as follows: GAPDH, 95°C for 2 min, 95°C for 30 sec, 60°C for 30 sec, and 72°C for 30 sec (21 cycles); FSH, 95°C for 2 min, 95°C for 30 sec, 60°C for 30 sec, and 72°C for 30 sec (23 cycles); LH, 95°C for 2 min, 95°C for 30 sec, 55°C for 30 sec, and 72°C for 30 sec (23 cycles); and PRL, 95°C for 2 min, 95°C for 30 sec, 60°C for 30 sec, and 72°C for 30 sec (18 cycles). The PCR reactions were carried out in a DNA Thermal Cycler 480 (Perkin Elmer). All RT-PCR cycles used to amplify FSHß, LHß, PRL, and GAPDH cDNA fragments were in the middle of linear ranges as verified by multicycle PCR analysis. Specific band intensities were analyzed by the NIH Image program.

Statistical Analysis

All statistical tests were carried out using the Statistical Analysis System [34]. Treatment differences in egg production, nest visits, and PRL content were observed by means of the general linear models procedure performed by repeated measures and individual treatment analysis. Statistical analysis for differences between ovary and oviduct weights; follicle number; and LHß, FSHß, and PRL mRNA were performed by a one-way ANOVA followed by the Duncan multiple range test. Significant differences are reported as P < 0.05, and the results are expressed as means ± SEM.

RESULTS

Experiment 1

A progressive increase in nesting activity was observed in all hens during the reproductive cycle. The average nesting frequency of hens immunized with rtINH{alpha} during the 147-day experimental period was similar to that of KLH-immunized controls (Table 1). Two of ten (20%) KLH-immunized controls and one of 10 hens (10%) immunized against rtINH{alpha} exhibited incubation behavior (Table 1). There was no significant difference in egg production between KLH-immunized controls and hens immunized against rtINH{alpha} (Table 1).


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  		TABLE 1. Reproduction characteristics of turkey hens actively immunized with recombinant turkey inhibin {alpha}

Anterior pituitary FSHß subunit and LHß subunit and PRL mRNAs from KLH-immunized controls and rtINH{alpha}-immunized hens were analyzed using Northern blot hybridization (Table 2, Fig. 1). After quantification by densitometry and normalization with 28S, the mean ± SEM relative intensity of FSHß, in rtINH{alpha}-immunized hens (0.68 ± 0.07) was significantly greater than that of the control group (0.26 ± 0.02; Table 2). No difference in either PRL or LHß subunit mRNA was observed between the control group and birds immunized with rtINH{alpha}. Comparable results were obtained using RT-PCR analysis (Table 2).


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  		TABLE 2. Effect of active immunization with recombinant turkey inhibin {alpha} subunit on FSHß subunit, LHß subunit and PRL mRNA levels, as determined by Northern blot and RT-PCR analyses



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  		FIG. 1. Representative autoradiograph of Northern blot (a) and agarose gel electrophoresis of PCR products (b) of turkey FSHß subunit, LHß subunit, and PRL mRNAs. Laying hens were immunized with INH{alpha}

Experiment 2

Immunological response to rtINH{alpha} and VIP immunization Antibodies to rtINH{alpha} were present in plasma collected from rtINH{alpha}-immunized and rtINH-{alpha}+VIP-immunized hens (Fig. 2a). The maximum binding shown by arbitrary density units (ADUs) was 146.3 ± 23.6 ADU for hens immunized with rtINH{alpha} and 146.6 ± 30.9 ADU for hens immunized with rtINH{alpha}+VIP. No rtINH{alpha} antibody titer was detected in plasma from KLH- or VIP-immunized hens (Fig. 2a).



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  		FIG. 2. Plasma antibody titer of INH{alpha} (ADU; a) and VIP (% binding; b) of turkey hens actively immunized with KLH (CON), VIP, rtTNH{alpha}, and VIP+rtINH{alpha}. Each point represents the mean values from 32 turkeys, and the vertical line represents the SEM

125I-Mono-iodinated tVIP binding to plasma indicated the presence of antibodies to tVIP in plasma collected from VIP-immunized and rtINH{alpha}+VIP-immunized hens. The percent binding of the mono-iodinated VIP averaged 13.8% for VIP-immunized hens and 11.3% for rtINH+tVIP-immunized hens. No VIP antibody titer was detected in plasma from control or INH{alpha}-immunized hens (Fig. 2b).

Plasma PRL levels Circulating PRL levels in nonimmunized control and rtINH{alpha}-immunized hens increased 20.8-fold and 19-fold by Week 9 of photostimulation, respectively. Thereafter, PRL levels continued to decline until the termination of the experiment (Fig. 3a). The mean plasma PRL values in the VIP- and rtINH+VIP-immunized groups remained significantly (P < 0.05) below those of control or rtINH{alpha}-immunized hens except on the first day of photostimulation. Overall, the mean PRL levels of control and rtINH{alpha}-immunized hens were similar (P > 0.05) and significantly (P < 0.05) greater than that of VIP- and rtINH{alpha}+VIP-immunized hens (Table 3).



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  		FIG. 3. Plasma PRL levels (a), number of nest visits (b), and egg production (c) of turkey hens immunized with VIP, rtINH{alpha}, or VIP+rtINH{alpha} during a photo-induced reproductive cycle


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  		TABLE 3. Reproductive characteristics of turkey hens actively immunized with vasoactive intestinal peptide (VIP), recombinant turkey inhibin {alpha} (tINH{alpha}), or VIP + tINH{alpha} during a 26-wk reproductive period

Nesting activity profile and the incidence of incubation behavior The nesting activity of control and rtINH{alpha}-immunized groups increased to a maximum of 33.3 ± 2.2 visits/hen per week by Week 11 and 32.5 ± 2.6 visits/hen per week by Week 10, respectively, following the onset of photostimulation (Fig. 3b). Thereafter, nesting activity continued to decline until the termination of the experiment. There was no significant difference (P > 0.05) in overall nesting activity between the two groups (Table 3). The VIP- and the rtINH{alpha}+VIP-immunized groups had significantly (P < 0.05) lower overall mean nesting activity when compared with controls or rtINH{alpha}-immunized birds (Table 3).

The incident of incubation behavior was 3.1% for VIP- and rtINH{alpha}+VIP-immunized hens, whereas 62.5% of controls and rtINH{alpha}-immunized hens were considered to be incubating (Table 3).

Egg production Peak egg production was comparable among treatment groups in Weeks 2, 3, and 4 of photo-stimulation. Thereafter, there were sharp declines in egg production in the control and the rtINH{alpha}-immunized groups, and these achieved significance by Week 6 of photostimulation (Fig. 3c).

There were significant differences in egg production between control/rtINH{alpha}-immunized and VIP-/rtINH{alpha}+VIP-immunized groups during Weeks 6–24 of photostimulation. The overall mean egg production during the 26-wk experimental period was significantly greater in VIP-/rtINH{alpha}+VIP-immunized birds compared with controls or rtINH{alpha}-immunized groups (Table 3). There was no significant difference between controls and rtINH{alpha}-immunized groups nor between VIP-immunized and rtINH{alpha}+VIP-immunized birds (Table 3).

At the end of the experiment, 10 laying hens from each group were killed and ovarian and oviduct weights were determined, as were the number of preovulating yellow follicles. There were no significant differences in ovarian or oviduct weights among experimental birds compared with controls (data not shown). A similar number of ovarian follicles, arranged in the follicular hierarchy of laying hens, was observed in all experimental groups. However, there was a greater number of nongraded, yellow follicles in rtINH{alpha}-immunized (62.5%) and rtINH{alpha}+VIP-immunized (73.5%) hens compared with controls or the VIP-immunized group.

Expression of reproductive hormones Quantitative RT-PCR analysis of anterior pituitary FSHß subunit, LHß subunit, and PRL mRNAs normalized to GAPDH is presented in Table 4. Active immunization against VIP increased (P < 0.05) the relative intensity of FSHß subunit (2.7-fold), LHß subunit (1.3-fold) and decreased (P < 0.05) PRL (34%). Recombinant turkey INH{alpha} immunoneutralization increased (P < 0.05) FSHß subunit expression by 97% with no effect on expression of either LHß subunit or PRL. There was no additive effect of coimmunization with rtINH{alpha}+VIP on the expression of the three hormones.


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  		TABLE 4. Effect of active immunization with rtINH{alpha}, VIP, and rtINH{alpha}+VIP on FSHß, LHß, and PRL mRNA content of the anterior pituitary

DISCUSSION

The mature encoding region of rtINH{alpha} (12.6 kDa) was subcloned into a thioredoxin (TRX) expression vector system [35]. This recombinant fusion protein, pTRX-rtINH{alpha} (24.6 kDa), was used for active immunization against INH{alpha} in the turkey. Active immunization of female turkeys with rtINH{alpha} induced antibodies against endogenous INH, elevated pituitary levels of FSHß subunit mRNA, and increased the number of nongraded preovulatory yellow follicles. Immunization against INH had no effect on egg production in turkeys, in contrast to an earlier study in which INH immunoneutralization increased egg production in quails [36].

Active immunization against INH neutralizes endogenous INH and increases the ovulation rate in mammals [6, 7, 10, 37–39]. The results of the present study showed no significant difference in egg production between birds that were actively immunized against INH and control hens, even though immunization significantly increased the number of preovulatory yellow follicles and significantly increased FSHß subunit mRNA content in the anterior pituitary. It is not apparent why egg production did not increase, particularly in view of the increase in FSHß subunit expression and in the number of preovulatory yellow follicles. This may be explained by the paracrine effect of INH on follicular maturation, as direct injection of rhINH A into immature rat ovaries enhances follicle maturation, which is indicated by follicular size [40]. It still remains to be proven that the ovarian response to rtINH{alpha} immunization is precipitated by a rise in FSH secretion (no FSH assay is available that measures circulating turkey FSH), because several studies have found plasma FSH to be unaffected by immunization despite an increased ovulation rate [9, 37, 39].

Immunization of hens in their first reproductive cycle with rtINH{alpha} increased (7.6-fold) FSHß subunit mRNA content more than it did in second-reproductive-cycle hens (twofold). Obviously, the expression level of INH is mutable. Intracellular levels of INH are not detectable, implying that INH is not accumulating within the cell, but is being secreted shortly after synthesis [41]. High INH levels are reported in young rats, which decrease with advancing age [42]. In women, reproductive aging is marked by decreased secretion of dimeric INH [43]. After menopause, levels of both forms of INH (A and B) decline and become undetectable [44]; this fall is precious for levels of INH B. Moreover, aging cycling women have lower INH B levels during the follicular phase [45] and lower levels of INH A in the luteal phase [46]. These findings, taken together with the present results, may explain the greater effect of INH immunization on FSHß subunit expression in young hens compared with older ones.

Active immunization with VIP suppressed circulating PRL, inhibited the expression of incubation behavior, and resulted in a substantial increase in egg production (present study, [25]). Egg production of hens immunized with rtINH{alpha}+VIP increased slightly compared with that of VIP-immunized hens. FSHß subunit mRNA pituitary content of the rtINH{alpha}-immunized group was similar to that of VIP-immunized birds, and both immunized groups showed significantly greater FSHß mRNA content than controls. LHß mRNA pituitary content of VIP-immunized and rtINH{alpha}+VIP-immunized groups was also significantly increased over controls and those animals immunized with rtINH{alpha}. These results clearly show for the first time that VIP acts as an antagonist toward both LH and FSH secretion. However, the mechanisms by which VIP mediates its effect is not known; it may act directly at the pituitary level or indirectly via INH, because VIP appears to have a stimulatory effect on INH synthesis or secretion [47], or via PRL, as the antigonadotropic effect of PRL has been well established [48]. As expected, pituitary PRL mRNA content and PRL plasma levels of VIP-immunized (present study, [49]); and rtINH{alpha}+VIP-immunized groups were significantly lower than the control group.

In summary, active immunization of female turkeys with KLH-rtINH{alpha} neutralized endogenous INH and increased both FSH ß subunit mRNA and the number of preovulatory yellow follicles. Immunization against INH had no effect on turkey egg production. VIP-rtINH{alpha} immunization slightly increased egg production compared with that of VIP-immunized birds, but the difference was not significant.

FOOTNOTES

First decision: 6 June 2001.

1 Supported by grant US-2846-97 from BARD. Back

2 Correspondence: Mohamed El Halawani, 495 Animal Science, Veterinary Medicine, 1988 Fitch Avenue, University of Minnesota, St. Paul, MN 55108. FAX: 612 625 2743;elhal001{at}tc.umn.edu Back

Accepted: July 5, 2001.

Received: May 11, 2001.

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Y. Chaiseha, O. M. Youngren, and M. E. El Halawani
Expression of Vasoactive Intestinal Peptide Receptor Messenger RNA in the Hypothalamus and Pituitary Throughout the Turkey Reproductive Cycle
Biol Reprod, March 1, 2004; 70(3): 593 - 599.
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