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Age-related changes in gonadal and serotonergic axes of broiler breeder roosters
Domestic Animal Endocrinology 44 (2013) 145–150
Contents lists available at SciVerse ScienceDirect
Domestic Animal Endocrinology journal homepage: www.domesticanimalendo.com
Age-related changes in gonadal and serotonergic axes of broiler breeder roosters N. Avital-Cohen a, *, R. Heiblum a, N. Argov-Argaman a, A. Rosenstrauch b, Y. Chaiseha c, N. Mobarkey a, I. Rozenboim a a b c
Department of Animal Science, Robert H. Smith Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem, Rehovot 76100, Israel Department of Life Sciences, Achva Academic College, MP Shikmim, 79800, Israel School of Biology, Institute of Science, Suranaree University of Technology, Nakhon Ratchasima, Thailand
a r t i c l e i n f o
a b s t r a c t
Article history: Received 23 May 2012 Received in revised form 1 January 2013 Accepted 1 January 2013
Fertility of domestic roosters decreases at w50 wk of age. In a previous study on aging white leghorn roosters, low fertility was accompanied by low levels of both hypothalamic vasoactive intestinal peptide (VIP) and pituitary prolactin (PRL) mRNA expression; however, their role in aging broiler breeder rooster reproduction is still unclear. In this study we compared reproductive activities of young (35-wk-old) and aging (73-wk-old) broiler breeder roosters. Weekly semen volume; concentration and ejaculation grade; and concentrations of plasma testosterone, estradiol, and PRL were examined. Every other week, 10 roosters from each group were euthanized, their testes weighed, and hypothalamus and pituitary removed to determine mRNA expression of hypothalamic GnRH-I, pituitary FSH, pituitary LH, hypothalamic VIP, and pituitary PRL. Aging roosters had significantly lower testis weight and semen volume, sperm concentration, ejaculation grade and plasma testosterone and low hypothalamic GnRH-I, pituitary FSH, and pituitary LH mRNA expression than young roosters (P 0.05). Aging roosters had higher concentrations of plasma estradiol and PRL and higher hypothalamic VIP and pituitary PRL mRNA expression than young roosters (P 0.05). We suggest that PRL, which is known to inhibit the gonadal axis, and its releasing factor, VIP, play an important role in the reproductive failure associated with age in broiler breeder roosters. Ó 2013 Elsevier Inc. All rights reserved.
Keywords: Reproduction Semen quality Prolactin Vasoactive intestinal peptide Aging
1. Introduction Fertility of domestic roosters peaks at 32 wk of age and declines at w45 wk of age. The aging phenomenon is manifested in several avian species by a significant reduction in gonadal axis activity [1–5] associated with a decline in reproductive behavior [6]. Previous studies have shown a significant reduction in the amplitude of release, number of cells, and concentration of hypothalamic GnRH-I in aging male quail [2] and a subsequent reduction in the synthesis and release of pituitary LH and pituitary FSH [1,5]. * Corresponding author. Tel.: þ972-8-948-9045; fax: þ972-8-9465763. E-mail address: [email protected] (N. Avital-Cohen). 0739-7240/$ – see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.domaniend.2013.01.002
Follicle-stimulating hormone has a major role in the proliferation and differentiation of Sertoli cells, which regulate sperm production in the testis. Decreased FSH concentration in broiler breeders is suggested to be responsible for testis regression and decreased daily sperm production [7]. Luteinizing hormone activates Leydig cells through specific membrane receptors, stimulating steroidogenesis and testosterone secretion [8,9]. The number of testicular LH receptors and plasma testosterone concentration decrease in aging birds [3], whereas estradiol concentration increases [10]. Aging roosters that show low fertility display regular spermatogenesis [11]. However, the spermatozoa remain trapped in the Sertoli cells because of reduction in the number of Leydig cells and in the concentration of circulating androgen [3]. Consequently, sperm count in the semen is reduced [12]. Studies in
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other species have also shown aging-related decline in semen volume and reduced sperm motility [13]. The lactotrophic axis, which produces pituitary prolactin (PRL), also plays an important role in reproduction. Synthesis and secretion of PRL are under the control of hypothalamic vasoactive intestinal peptide (VIP), the major avian PRL-releasing factor [14,15]. In early experiments with male chickens, high plasma PRL concentrations were found to induce testicular regression [16,17]. In addition, PRL is well documented to inhibit secretion of hypothalamic GnRH-I and pituitary LH and to diminish gonadal activity [18,19]. In contrast, in rats, PRL was found to have a permissive role by increasing the sensitivity of LH receptors on Leydig cells [20,21]. Previous studies in our laboratory on young white leghorn (WL) roosters actively immunized against VIP showed a decrease in pituitary PRL expression and an increase in the examined reproductive variables and in pituitary LH expression, indicating a negative role for VIP and PRL in reproduction [22]. Furthermore, active immunization against VIP in aging WL roosters resulted in reduced mRNA expression of genes that encode hypothalamic VIP, pituitary PRL, hypothalamic GnRH, pituitary LH, and pituitary FSH and decreased semen-quality variables. Administration of PRL to these roosters abolished the negative effects of VIP immunization, indicating the requirement for VIP and PRL in aging WL roosters [23]. In view of our results that show a permissive role for PRL in aging WL roosters, the objective of the present study was to examine the relation of PRL and its releasing factor, VIP, with reproduction in young and aging broiler breeder roosters. 2. Materials and methods
PRL concentrations. Plasma samples were stored at 20 C until assay. 2.3. Hormone analysis Steroid hormones were extracted from 0.5 mL of plasma with 5 mL of diethylether. Recovery after extraction was 90% for estradiol and testosterone. Plasma estradiol and testosterone were measured in a single ELISA according to a previously described method [24] that used primary antibody and tracer dilutions as described previously [22]. The assay was conducted in duplicates; minimal detectable dose was 0.78 pg/mL and the interassay CV were <5% for estradiol and testosterone. Plasma PRL was assayed by competitive ELISA with the use of biotinylated PRL tracers as described previously [25]. The assay was conducted in duplicates; intra-assay CV with pooled chicken plasma was 7%. Interassay CVs for estradiol, testosterone, and PRL were not computed because all samples were analyzed in a single assay. Absorbance at 405 nm was read in a Tecan Sunrise Microplate reader (Männedorf, Switzerland). Poolextracted plasma samples were run in duplicates in each plate as internal quality control. 2.4. Semen-quality variables Semen was sampled once a week with the use of the “abdominal massage method” [26]. Semen volume, sperm concentration, and ejaculation grade [27] were determined. Ejaculation was graded as the response to massage procedure from 0 to 8 as follows: 0, no erection of the phallus; l, erection with no fluid; 2, erection with secretion of fluid only; 3 to 8, secretion of semen with increasing amounts of spermatozoa.
2.1. Experimental animals 2.5. Tissue sampling All procedures were approved by the Animal Care and Welfare Committee of The Hebrew University of Jerusalem. Young broiler breeder roosters at 35 wk of age and aging roosters at 73 wk of age (Cobb; n ¼ 50) were housed in individual cages under photostimulatory conditions (16L:8D) at a light intensity of 0.1 W/m2 from white fluorescent lamps. Birds were subjected to a commercial restricted feeding program with daily administration of feed, as recommended by Cobb 500 Breeder Management Guide with modification for birds reared in cages. The roosters’ diet contained 11.5% crude protein and 2,700 kcal/kg. The experiment was conducted over a 10-wk period, which means the young roosters started at 35 wk and ended at 45 wk and the aging roosters started at 73 wk and ended at 83 wk. At the first day of each week brachial vein blood was collected, and on the second day semen was collected. Every other week 10 roosters from each age group were killed; tissue samples were collected from the brain for analysis of mRNA expression. 2.2. Blood sampling Weekly heparinized blood samples were drawn from the brachial vein for determination of plasma steroid and
Every other week, 10 roosters from each group were killed (injection of pentobarbitone sodium at 1 mL/1.5 kg BW; Chemical and Technical Supplies, Chemical Industries Ltd, Kiryat Malachi, Israel). A block of tissue that encompassed the hypothalamus and preoptic area was removed from the floor of the brain. It was 4 mm wide (2 mm on each side of the midline) and extended from the septomesencephalic tract rostrally to the mammillary bodies caudally. It was 2 mm high at its rostral end and 4 mm high at its caudal end. All hypothalamus, pituitary, and testis tissues in all groups were removed at the same time and stored at 80 C until analysis for mRNA expression. 2.6. RNA extraction and real-time PCR Total RNA was isolated from the tissues with the use of TRI reagent (MRC, Cincinnati, OH) according to the manufacturer’s protocol. Total RNA (1 mg) was reversetranscribed to cDNA in a total volume of 20 mL with the use of 200 U of reverse transcriptase, 50 pmol of random hexamer, and 0.075 nmol of oligo-dT. Gene-specific primers for hypothalamic GnRH-I, pituitary FSH-b, pituitary LH-b, hypothalamic VIP, pituitary PRL,
N. Avital-Cohen et al. / Domestic Animal Endocrinology 44 (2013) 145–150
glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and b-actin were designed with Primer Express software Oligo. net (Molecular biology insights, CO, USA) for SYBR Green detection according to the published cDNA sequences for each of the studied genes (Table 1). Real-time PCR was performed with an Mx3000P QPCR system (Stratagene, Foster City, CA, USA) with the SYBR Green I PCR kit (Eurogenetec, Seraing, Belgium). Each realtime reaction (18 mL) contained SYBR Green Master Mix that comprised ROX passive reference (200 mM dNTPs including dUTP, 5 mM MgCl2 uracil N-glycosylase, and AmpliTaq HotGoldStar DNA polymerase), 0.54 mL of a 1:10,000 dilution of SYBR Green stock solution, 1.5 mM dNTPs, 10 nM of each primer, and 25 to 50 ng of cDNA. The geometric mean of b-actin and GAPDH housekeeping genes was used as a standard. A dissociation curve analysis was run after each real-time experiment to confirm the presence of only one product and the absence of primer-dimer formation. The threshold cycle number (Ct) for each tested gene X was used to quantify the relative abundance of the gene: 2(Ct geneXCt [geometric mean of bactin and GAPDH]) 1000. 2.7. Statistical analysis All data were subjected to 2-way ANOVA. Hormone concentrations and semen-quality variables were analyzed with 2-way repeated measurement ANOVA. JMP software version 7 was used for all analyses (SAS Institute, Cary, NC). Differences between means were tested by Tukey-Kramer test. Differences were considered significant at P 0.05. Data are presented as mean SEM. 3. Results 3.1. Semen-quality variables and percentage of testis weight No significant interaction was observed between time in experiment and age on sperm-quality variables and percentage of testis weight (Fig. 1A–D). Young roosters exhibited significantly higher sperm concentration, ejaculation grade, semen volume, and testis weight (as percentage of BW) than aging roosters throughout the 9 wk of the experiment (Fig. 1A–D, respectively; P < 0.05) but with no significant effect between experiment duration.
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3.2. Plasma steroid concentrations Concentration of plasma testosterone was significantly higher in young roosters than in aging roosters (Fig. 2A, P < 0.05). Concentration of plasma estradiol was significantly lower in young roosters than in aging roosters (Fig. 2B, P < 0.05). No significant interaction was observed between time in experiment and age and no significant effect between experiment duration. 3.3. Gonadal axis gene expression Aging roosters exhibited significantly lower concentrations of hypothalamic GnRH-I (Fig. 3A, P < 0.05), pituitary LH (Fig. 3B, P < 0.05), and pituitary FSH (Fig. 3C, P < 0.05) mRNA expression than young roosters. The differences between the groups were significant, except for the third week of the experiment (young: 38 wk of age; aging: 76 wk of age) for GnRH-I mRNA expression, and the fifth week of the experiment (young: 40 wk of age; old: 78 wk of age) for LH and FSH mRNA expressions. No significant effect of experiment duration was observed, and no interaction was observed between time in experiment and age on hypothalamic GnRH-I, pituitary LH, and pituitary FSH mRNA expression (Fig. 3A–C). 3.4. Lactotrophic axis gene expression Hypothalamic VIP (Fig. 4A, P < 0.05) and pituitary PRL (Fig. 4B, P < 0.05) mRNA expressions were lower in young roosters than in aging roosters in all measurements taken during the study. In addition, concentration of plasma PRL was significantly lower in young roosters than in aging roosters (Fig. 4C, P < 0.05). In the duration of 9 wk of the experiment no significant interactions were observed between time in experiment and age and no significant effect between experiment duration. 4. Discussion This is the first study to show an inverse relation between the gonadal axis and VIP-PRL in broiler breeder roosters at different ages. Aging roosters had lower testis weight, semen-quality variables, plasma testosterone
Table 1 Primers used in real-time PCR. Gene
Sequence
Product length (bp)
Accession number
b-actin
F: 50 -CCGCAAATGCTTCTAAACCG-30 R: 50 -AAAGCCATGCCAATCTCGTC-30 F: 50 -CTCCTGTTCACCGCATCT-30 R: 50 -CTGCCCTTCTCCTAGACTTTC-30 F: 50 -AAGAAGCTCCAGATACCATTCTCT-30 R: 50 -GAGAGTAATTTCATTTCCAGCAT-30 F: 50 -CCACGTGGTGCTCAGGATACT-30 R: 50 -AGGTACATATTTGCTGAACAGATGAGA-30 F: 50 -AACGTAACGGTGGCGGTG-30 R: 50 -AGGCCGTGGTGGTCACAG-30 F: 50 -TGAGGTTAAGTATTTCTTCACAGCCATTTGCTT-30 R: 50 -GACCGCGCCCATGGGTCCCTAAAGTC-30 F: 50 -GGCACGCCATCACTATC-30 R: 50 -CCTGCATCTGCCCATTT-30
100
l08165
144
X_69491
129
J04614
GnRH-I PRL FSH-b LH-b VIP GAPDH
50
NM_204257
84
S_70834
205 61
NM_205366 K01458
Abbreviations: F, forward; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PRL, prolactin; R, reverse; VIP, vasoactive intestinal peptide.
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A
C
B
D
Fig. 1. Sperm concentration (cells/mL 109; A), ejaculation grade (0–8: 0, no erection of the phallus and 8, secretion of semen with high amounts of spermatozoa; B), sperm volume (in mL; C), and testis weight (as percentage of BW; D) of young (35- to 44-wk-old) and aging (73- to 82-wk-old) broiler breeder roosters (Cobb). Data are presented as mean SEM (A–C, n ¼ 50 at the beginning of the experiment, with 10 less birds every other week; n ¼ 10 at the end of the experiment in panel D). Significant differences were observed between the age groups at each experimental time point, but with no significant time by age interaction.
concentration, and mRNA expression of hypothalamic GnRH-I, pituitary FSH, and pituitary LH genes than young roosters. Furthermore, aging roosters exhibited higher concentrations of plasma estradiol and PRL, hypothalamic VIP, and pituitary PRL mRNA expression.
These results are in agreement with previous studies conducted on aging male quails [4] and WL roosters [23], showing that low fertility is accompanied by a reduction in hypothalamic GnRH expression, causing a reduction in pituitary LH and pituitary FSH mRNA expressions. These
A
B
Fig. 2. Concentrations of plasma testosterone (in ng/mL; A) and estradiol (in pg/mL; B) of young (35- to 44-wk-old) and aging (73- to 82-wk-old) broiler breeder roosters (Cobb). Plasma steroid concentrations were determined by enzyme-linked immunosorbent assay. Data are presented as mean SEM (n ¼ 50 at the beginning of the experiment, with 10 less birds every other week; n ¼ 10 at the end of the experiment). Significant differences were observed between the age groups at each experimental time point, but with no significant time by age interaction.
Fig. 3. Chicken hypothalamic GnRH-I (A), pituitary FSH-b (B), and pituitary LH-b (C) mRNA expression of young (35- to 44-wk-old) and aging (73- to 82wk-old) broiler breeder roosters (Cobb). Expression of GnRH-I, FSH-b, and LH-b mRNA was determined by real-time PCR, and densitometric analysis of GnRH-I, FSH-b, and LH-b mRNA was performed relative to b-actin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA expression. AU, arbitrary units. Data are presented as mean SEM (n ¼ 10). Values with different letters are significantly different (P < 0.05).
N. Avital-Cohen et al. / Domestic Animal Endocrinology 44 (2013) 145–150
Fig. 4. Chicken hypothalamic vasoactive intestinal peptide (VIP; A) and pituitary prolactin (PRL; B) mRNA expression and plasma PRL concentration (in ng/mL; C) of young (35- to 44-wk-old) and aging (73- to 82-wk-old) broiler breeder roosters (Cobb). Expression of VIP and PRL mRNA was determined by real-time PCR, and densitometric analysis of VIP and PRL mRNA was performed relative to b-actin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA expression. AU, arbitrary units. Data are presented as mean SEM (n ¼ 10). Values with different letters are significantly different (P < 0.05).
reductions result in low-quality semen, low plasma testosterone concentration, and testis regression. Our results are also supported by findings from a study on broiler breeders which showed that decreased FSH and testosterone concentration is accompanied by decreased sperm production and testis regression [7]. In our study, hypothalamic VIP and pituitary PRL mRNA expression increased in aging compared with young roosters, accompanied by a decrease in reproduction. This suggests an inhibitory effect of the lactotrophic axis on reproduction of aging broiler breeder roosters. Plasma PRL concentration did not differ between young and aging roosters at the beginning of the experiment, even though PRL mRNA expression and semen amounts were significantly lower in aging males. The lack of a significant difference in plasma PRL concentration was probably associated with the relatively large variation within groups at the beginning of the experiment, as frequently encountered in hormones studies. Studies on turkey hens have shown that high concentrations of PRL decrease reproductive activity via inhibition of hypothalamic GnRH release [18,28,29], reduce LH mRNA expression and LH secretion from the pituitary [30], and inhibit ovarian steroid hormone production [31]. In addition, the same inhibitory effect of VIP-PRL was observed in young WL roosters actively immunized against VIP, resulting in decreased plasma PRL concentration and pituitary PRL mRNA expression and increased reproductive activities [22]. A previous study conducted on rats also showed an increase in PRL mRNA expression with age [32,33], as recorded in our study.
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Findings derived from the present study suggest that PRL has an inhibitory effect on the reproductive system of broiler breeder roosters. This seems to contradict our earlier study on WL roosters, in which we found that PRL enhances the reproductive variables of aging roosters [23]. The latter findings were supported because active immunization against VIP reduced spermatogenesis and PRL administration restored reproductive variables. In a comparative analysis with our previous work, we found that basal concentrations of PRL are higher in broiler breeder roosters than in WL roosters [22,23]. It is therefore possible that PRL reduces reproductive performance at both high and low concentrations. These results are in agreement with previous studies conducted on rats which show differences in basal PRL concentrations of several strains [34]. From the present study, we can conclude that gonadal axis function deteriorates in aging broiler breeder roosters, and the increased concentrations of VIP-PRL, as inhibitors of the hypothalamic-pituitary-gonadal axis, suggest their involvement in the reproductive failure associated with age. In addition, this study suggested that PRL concentrations in broiler breeder roosters are opposite those in aging WL roosters, indicating different mechanisms of secretion and differential involvement of PRL in reproduction. Further investigation is warranted to confirm our suggestions about the involvement of VIP and PRL in the reproductive failure associated with aging broiler breeder roosters. References [1] Ishii S. The molecular biology of avian gonadotropin. Poult Sci 1993; 72:856–66. [2] Ottinger MA, Abdelnabi M, Li Q, Chen K, Thompson N, Harada N, Viglietti-Panzica C, Panzica GC. The Japanese quail: a model for studying reproductive aging of hypothalamic systems. Exp Gerontol 2004;39:1679–93. [3] Ottinger MA, Kubakawa K, Kikuchi M, Thompson N, Ishii S. Effects of exogenous testosterone on testicular luteinizing hormone and follicle-stimulating hormone receptors during aging. Exp Biol Med 2002;227:830–6. [4] Ottinger MA, Thompson N, Viglietti-Panzica C, Panzica GC. Neuroendocrine regulation of GnRH and behavior during aging in birds. Brain Res Bull 1997;44:471–7. [5] Sharp PJ, Talbot RT, Main GM, Dunn IC, Fraser HM, Huskisson NS. Physiological roles of chicken LHRH-I and -II in the control of gonadotrophin release in the domestic chicken. J Endocrinol 1990; 124:291–9. [6] Limonta P, Dondi D, Maggi R, Martini L, Piva F. Effects of aging on pituitary and testicular luteinizing hormone-releasing hormone receptors in the rat. Life Sci 1988;42:335–42. [7] Vizcarra JA, Kirby JD, Kreider DL. Testis development and gonadotropin secretion in broiler breeder males. Poult Sci 2010;89:328–34. [8] Catt KJ, Harwood JP, Clayton RN, Davies TF, Chan V, Katikineni M, Nozu K, Dufau ML. Regulation of peptide hormone receptors and gonadal steroidogenesis. Recent Prog Horm Res 1980;36:557–662. [9] Fuentes LB, Calvo JC, Charreau EH, Guzman JA. Seasonal variations in testicular LH, FSH, and PRL receptors; in vitro testosterone production; and serum testosterone concentration in adult male vizcacha (Lagostomus maximus maximus). Gen Comp Endocrinol 1993;90:133–41. [10] Weil S, Rozenboim I, Degen AA, Dawson A, Friedlander M, Rosenstrauch A. Fertility decline in aging roosters is related to increased testicular and plasma levels of estradiol. Gen Comp Endocrinol 1999;115:23–8. [11] Muncher Y, Sod-Moriah UA, Weil S, Rosenstrauch AW, Friedlander M. Intratesticular retention of sperm and premature decline in fertility in the domestic rooster, Gallus domesticus. J Exp Zool 1995;273:76–81.
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[12] Rosenstrauch A, Degen AA, Friedlander M. Spermatozoa retention by Sertoli cells during the decline in fertility in aging roosters. Biol Reprod 1994;50:129–36. [13] Wagner L. Fertility of the aging male. Prog Urol 2004;14:577–82. [14] Sharp PJ, Dawson A, Lea RW. Control of luteinizing hormone and prolactin secretion in birds. Comp Biochem Physiol Part C Pharmacol Toxicol Endocrinol 1998;119:275–82. [15] Youngren O, Chaiseha Y, Phillips R, El Halawani M. Vasoactive intestinal peptide concentrations in turkey hypophysial portal blood differ across the reproductive cycle. Gen Comp Endocrinol 1996;103:323–30. [16] Lofts B, Marshall AJ. The effects of prolactin administration on the internal rhythm of reproduction in male birds. J Endocrinol 1956; 13:101–6. [17] Mazzi V, Vellano C, Toscano C. Antigonadal effects of prolactin in adult male crested newt (Triturus cristatus carnifex Laur). Gen Comp Endocrinol 1967;8:320–4. [18] Rozenboim I, Silsby JL, Tabibzadeh C, Pitts GR, Youngren OM, el Halawani ME. Hypothalamic and posterior pituitary content of vasoactive intestinal peptide and gonadotropin-releasing hormones I and II in the turkey hen. Biol Reprod 1993;49:622–6. [19] Weick RF, Stobie KM. Role of VIP in the regulation of LH secretion in the female rat. Neurosci Biobehav Rev 1995;19:251–9. [20] Aragona C, Friesen HG. Specific prolactin binding sites in the prostate and testis of rats. Endocrinology 1975;97:677–84. [21] Huhtaniemi IT, Catt KJ. Induction and maintenance of gonadotropin and lactogen receptors in hypoprolactinemic rats. Endocrinology 1981;109:483–90. [22] Avital-Cohen N, Heiblum R, Argov N, Rosenstrauch A, Chaiseha Y, Mobarkey N, Rozenboim I. The effect of active immunization against vasoactive intestinal peptide and inhibin on reproductive performance of young White Leghorn roosters. Poult Sci 2011;90:2321–31. [23] Avital-Cohen N, Heiblum R, Argov N, Rosenstrauch A, Chaiseha Y, Mobarkey N, Rozenboim I. The effect of active immunization against vasoactive intestinal peptide (VIP) and inhibin on reproductive performance of aging White Leghorn roosters. Poult Sci 2012;91: 161–74. [24] Nash JP, Cuisset BD, Bhattacharyya S, Suter HC, Le Menn F, Kime DE. An enzyme linked immunosorbant assay (ELISA) for testosterone,
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Contents lists available at SciVerse ScienceDirect
Domestic Animal Endocrinology journal homepage: www.domesticanimalendo.com
Age-related changes in gonadal and serotonergic axes of broiler breeder roosters N. Avital-Cohen a, *, R. Heiblum a, N. Argov-Argaman a, A. Rosenstrauch b, Y. Chaiseha c, N. Mobarkey a, I. Rozenboim a a b c
Department of Animal Science, Robert H. Smith Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem, Rehovot 76100, Israel Department of Life Sciences, Achva Academic College, MP Shikmim, 79800, Israel School of Biology, Institute of Science, Suranaree University of Technology, Nakhon Ratchasima, Thailand
a r t i c l e i n f o
a b s t r a c t
Article history: Received 23 May 2012 Received in revised form 1 January 2013 Accepted 1 January 2013
Fertility of domestic roosters decreases at w50 wk of age. In a previous study on aging white leghorn roosters, low fertility was accompanied by low levels of both hypothalamic vasoactive intestinal peptide (VIP) and pituitary prolactin (PRL) mRNA expression; however, their role in aging broiler breeder rooster reproduction is still unclear. In this study we compared reproductive activities of young (35-wk-old) and aging (73-wk-old) broiler breeder roosters. Weekly semen volume; concentration and ejaculation grade; and concentrations of plasma testosterone, estradiol, and PRL were examined. Every other week, 10 roosters from each group were euthanized, their testes weighed, and hypothalamus and pituitary removed to determine mRNA expression of hypothalamic GnRH-I, pituitary FSH, pituitary LH, hypothalamic VIP, and pituitary PRL. Aging roosters had significantly lower testis weight and semen volume, sperm concentration, ejaculation grade and plasma testosterone and low hypothalamic GnRH-I, pituitary FSH, and pituitary LH mRNA expression than young roosters (P 0.05). Aging roosters had higher concentrations of plasma estradiol and PRL and higher hypothalamic VIP and pituitary PRL mRNA expression than young roosters (P 0.05). We suggest that PRL, which is known to inhibit the gonadal axis, and its releasing factor, VIP, play an important role in the reproductive failure associated with age in broiler breeder roosters. Ó 2013 Elsevier Inc. All rights reserved.
Keywords: Reproduction Semen quality Prolactin Vasoactive intestinal peptide Aging
1. Introduction Fertility of domestic roosters peaks at 32 wk of age and declines at w45 wk of age. The aging phenomenon is manifested in several avian species by a significant reduction in gonadal axis activity [1–5] associated with a decline in reproductive behavior [6]. Previous studies have shown a significant reduction in the amplitude of release, number of cells, and concentration of hypothalamic GnRH-I in aging male quail [2] and a subsequent reduction in the synthesis and release of pituitary LH and pituitary FSH [1,5]. * Corresponding author. Tel.: þ972-8-948-9045; fax: þ972-8-9465763. E-mail address: [email protected] (N. Avital-Cohen). 0739-7240/$ – see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.domaniend.2013.01.002
Follicle-stimulating hormone has a major role in the proliferation and differentiation of Sertoli cells, which regulate sperm production in the testis. Decreased FSH concentration in broiler breeders is suggested to be responsible for testis regression and decreased daily sperm production [7]. Luteinizing hormone activates Leydig cells through specific membrane receptors, stimulating steroidogenesis and testosterone secretion [8,9]. The number of testicular LH receptors and plasma testosterone concentration decrease in aging birds [3], whereas estradiol concentration increases [10]. Aging roosters that show low fertility display regular spermatogenesis [11]. However, the spermatozoa remain trapped in the Sertoli cells because of reduction in the number of Leydig cells and in the concentration of circulating androgen [3]. Consequently, sperm count in the semen is reduced [12]. Studies in
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other species have also shown aging-related decline in semen volume and reduced sperm motility [13]. The lactotrophic axis, which produces pituitary prolactin (PRL), also plays an important role in reproduction. Synthesis and secretion of PRL are under the control of hypothalamic vasoactive intestinal peptide (VIP), the major avian PRL-releasing factor [14,15]. In early experiments with male chickens, high plasma PRL concentrations were found to induce testicular regression [16,17]. In addition, PRL is well documented to inhibit secretion of hypothalamic GnRH-I and pituitary LH and to diminish gonadal activity [18,19]. In contrast, in rats, PRL was found to have a permissive role by increasing the sensitivity of LH receptors on Leydig cells [20,21]. Previous studies in our laboratory on young white leghorn (WL) roosters actively immunized against VIP showed a decrease in pituitary PRL expression and an increase in the examined reproductive variables and in pituitary LH expression, indicating a negative role for VIP and PRL in reproduction [22]. Furthermore, active immunization against VIP in aging WL roosters resulted in reduced mRNA expression of genes that encode hypothalamic VIP, pituitary PRL, hypothalamic GnRH, pituitary LH, and pituitary FSH and decreased semen-quality variables. Administration of PRL to these roosters abolished the negative effects of VIP immunization, indicating the requirement for VIP and PRL in aging WL roosters [23]. In view of our results that show a permissive role for PRL in aging WL roosters, the objective of the present study was to examine the relation of PRL and its releasing factor, VIP, with reproduction in young and aging broiler breeder roosters. 2. Materials and methods
PRL concentrations. Plasma samples were stored at 20 C until assay. 2.3. Hormone analysis Steroid hormones were extracted from 0.5 mL of plasma with 5 mL of diethylether. Recovery after extraction was 90% for estradiol and testosterone. Plasma estradiol and testosterone were measured in a single ELISA according to a previously described method [24] that used primary antibody and tracer dilutions as described previously [22]. The assay was conducted in duplicates; minimal detectable dose was 0.78 pg/mL and the interassay CV were <5% for estradiol and testosterone. Plasma PRL was assayed by competitive ELISA with the use of biotinylated PRL tracers as described previously [25]. The assay was conducted in duplicates; intra-assay CV with pooled chicken plasma was 7%. Interassay CVs for estradiol, testosterone, and PRL were not computed because all samples were analyzed in a single assay. Absorbance at 405 nm was read in a Tecan Sunrise Microplate reader (Männedorf, Switzerland). Poolextracted plasma samples were run in duplicates in each plate as internal quality control. 2.4. Semen-quality variables Semen was sampled once a week with the use of the “abdominal massage method” [26]. Semen volume, sperm concentration, and ejaculation grade [27] were determined. Ejaculation was graded as the response to massage procedure from 0 to 8 as follows: 0, no erection of the phallus; l, erection with no fluid; 2, erection with secretion of fluid only; 3 to 8, secretion of semen with increasing amounts of spermatozoa.
2.1. Experimental animals 2.5. Tissue sampling All procedures were approved by the Animal Care and Welfare Committee of The Hebrew University of Jerusalem. Young broiler breeder roosters at 35 wk of age and aging roosters at 73 wk of age (Cobb; n ¼ 50) were housed in individual cages under photostimulatory conditions (16L:8D) at a light intensity of 0.1 W/m2 from white fluorescent lamps. Birds were subjected to a commercial restricted feeding program with daily administration of feed, as recommended by Cobb 500 Breeder Management Guide with modification for birds reared in cages. The roosters’ diet contained 11.5% crude protein and 2,700 kcal/kg. The experiment was conducted over a 10-wk period, which means the young roosters started at 35 wk and ended at 45 wk and the aging roosters started at 73 wk and ended at 83 wk. At the first day of each week brachial vein blood was collected, and on the second day semen was collected. Every other week 10 roosters from each age group were killed; tissue samples were collected from the brain for analysis of mRNA expression. 2.2. Blood sampling Weekly heparinized blood samples were drawn from the brachial vein for determination of plasma steroid and
Every other week, 10 roosters from each group were killed (injection of pentobarbitone sodium at 1 mL/1.5 kg BW; Chemical and Technical Supplies, Chemical Industries Ltd, Kiryat Malachi, Israel). A block of tissue that encompassed the hypothalamus and preoptic area was removed from the floor of the brain. It was 4 mm wide (2 mm on each side of the midline) and extended from the septomesencephalic tract rostrally to the mammillary bodies caudally. It was 2 mm high at its rostral end and 4 mm high at its caudal end. All hypothalamus, pituitary, and testis tissues in all groups were removed at the same time and stored at 80 C until analysis for mRNA expression. 2.6. RNA extraction and real-time PCR Total RNA was isolated from the tissues with the use of TRI reagent (MRC, Cincinnati, OH) according to the manufacturer’s protocol. Total RNA (1 mg) was reversetranscribed to cDNA in a total volume of 20 mL with the use of 200 U of reverse transcriptase, 50 pmol of random hexamer, and 0.075 nmol of oligo-dT. Gene-specific primers for hypothalamic GnRH-I, pituitary FSH-b, pituitary LH-b, hypothalamic VIP, pituitary PRL,
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glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and b-actin were designed with Primer Express software Oligo. net (Molecular biology insights, CO, USA) for SYBR Green detection according to the published cDNA sequences for each of the studied genes (Table 1). Real-time PCR was performed with an Mx3000P QPCR system (Stratagene, Foster City, CA, USA) with the SYBR Green I PCR kit (Eurogenetec, Seraing, Belgium). Each realtime reaction (18 mL) contained SYBR Green Master Mix that comprised ROX passive reference (200 mM dNTPs including dUTP, 5 mM MgCl2 uracil N-glycosylase, and AmpliTaq HotGoldStar DNA polymerase), 0.54 mL of a 1:10,000 dilution of SYBR Green stock solution, 1.5 mM dNTPs, 10 nM of each primer, and 25 to 50 ng of cDNA. The geometric mean of b-actin and GAPDH housekeeping genes was used as a standard. A dissociation curve analysis was run after each real-time experiment to confirm the presence of only one product and the absence of primer-dimer formation. The threshold cycle number (Ct) for each tested gene X was used to quantify the relative abundance of the gene: 2(Ct geneXCt [geometric mean of bactin and GAPDH]) 1000. 2.7. Statistical analysis All data were subjected to 2-way ANOVA. Hormone concentrations and semen-quality variables were analyzed with 2-way repeated measurement ANOVA. JMP software version 7 was used for all analyses (SAS Institute, Cary, NC). Differences between means were tested by Tukey-Kramer test. Differences were considered significant at P 0.05. Data are presented as mean SEM. 3. Results 3.1. Semen-quality variables and percentage of testis weight No significant interaction was observed between time in experiment and age on sperm-quality variables and percentage of testis weight (Fig. 1A–D). Young roosters exhibited significantly higher sperm concentration, ejaculation grade, semen volume, and testis weight (as percentage of BW) than aging roosters throughout the 9 wk of the experiment (Fig. 1A–D, respectively; P < 0.05) but with no significant effect between experiment duration.
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3.2. Plasma steroid concentrations Concentration of plasma testosterone was significantly higher in young roosters than in aging roosters (Fig. 2A, P < 0.05). Concentration of plasma estradiol was significantly lower in young roosters than in aging roosters (Fig. 2B, P < 0.05). No significant interaction was observed between time in experiment and age and no significant effect between experiment duration. 3.3. Gonadal axis gene expression Aging roosters exhibited significantly lower concentrations of hypothalamic GnRH-I (Fig. 3A, P < 0.05), pituitary LH (Fig. 3B, P < 0.05), and pituitary FSH (Fig. 3C, P < 0.05) mRNA expression than young roosters. The differences between the groups were significant, except for the third week of the experiment (young: 38 wk of age; aging: 76 wk of age) for GnRH-I mRNA expression, and the fifth week of the experiment (young: 40 wk of age; old: 78 wk of age) for LH and FSH mRNA expressions. No significant effect of experiment duration was observed, and no interaction was observed between time in experiment and age on hypothalamic GnRH-I, pituitary LH, and pituitary FSH mRNA expression (Fig. 3A–C). 3.4. Lactotrophic axis gene expression Hypothalamic VIP (Fig. 4A, P < 0.05) and pituitary PRL (Fig. 4B, P < 0.05) mRNA expressions were lower in young roosters than in aging roosters in all measurements taken during the study. In addition, concentration of plasma PRL was significantly lower in young roosters than in aging roosters (Fig. 4C, P < 0.05). In the duration of 9 wk of the experiment no significant interactions were observed between time in experiment and age and no significant effect between experiment duration. 4. Discussion This is the first study to show an inverse relation between the gonadal axis and VIP-PRL in broiler breeder roosters at different ages. Aging roosters had lower testis weight, semen-quality variables, plasma testosterone
Table 1 Primers used in real-time PCR. Gene
Sequence
Product length (bp)
Accession number
b-actin
F: 50 -CCGCAAATGCTTCTAAACCG-30 R: 50 -AAAGCCATGCCAATCTCGTC-30 F: 50 -CTCCTGTTCACCGCATCT-30 R: 50 -CTGCCCTTCTCCTAGACTTTC-30 F: 50 -AAGAAGCTCCAGATACCATTCTCT-30 R: 50 -GAGAGTAATTTCATTTCCAGCAT-30 F: 50 -CCACGTGGTGCTCAGGATACT-30 R: 50 -AGGTACATATTTGCTGAACAGATGAGA-30 F: 50 -AACGTAACGGTGGCGGTG-30 R: 50 -AGGCCGTGGTGGTCACAG-30 F: 50 -TGAGGTTAAGTATTTCTTCACAGCCATTTGCTT-30 R: 50 -GACCGCGCCCATGGGTCCCTAAAGTC-30 F: 50 -GGCACGCCATCACTATC-30 R: 50 -CCTGCATCTGCCCATTT-30
100
l08165
144
X_69491
129
J04614
GnRH-I PRL FSH-b LH-b VIP GAPDH
50
NM_204257
84
S_70834
205 61
NM_205366 K01458
Abbreviations: F, forward; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PRL, prolactin; R, reverse; VIP, vasoactive intestinal peptide.
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A
C
B
D
Fig. 1. Sperm concentration (cells/mL 109; A), ejaculation grade (0–8: 0, no erection of the phallus and 8, secretion of semen with high amounts of spermatozoa; B), sperm volume (in mL; C), and testis weight (as percentage of BW; D) of young (35- to 44-wk-old) and aging (73- to 82-wk-old) broiler breeder roosters (Cobb). Data are presented as mean SEM (A–C, n ¼ 50 at the beginning of the experiment, with 10 less birds every other week; n ¼ 10 at the end of the experiment in panel D). Significant differences were observed between the age groups at each experimental time point, but with no significant time by age interaction.
concentration, and mRNA expression of hypothalamic GnRH-I, pituitary FSH, and pituitary LH genes than young roosters. Furthermore, aging roosters exhibited higher concentrations of plasma estradiol and PRL, hypothalamic VIP, and pituitary PRL mRNA expression.
These results are in agreement with previous studies conducted on aging male quails [4] and WL roosters [23], showing that low fertility is accompanied by a reduction in hypothalamic GnRH expression, causing a reduction in pituitary LH and pituitary FSH mRNA expressions. These
A
B
Fig. 2. Concentrations of plasma testosterone (in ng/mL; A) and estradiol (in pg/mL; B) of young (35- to 44-wk-old) and aging (73- to 82-wk-old) broiler breeder roosters (Cobb). Plasma steroid concentrations were determined by enzyme-linked immunosorbent assay. Data are presented as mean SEM (n ¼ 50 at the beginning of the experiment, with 10 less birds every other week; n ¼ 10 at the end of the experiment). Significant differences were observed between the age groups at each experimental time point, but with no significant time by age interaction.
Fig. 3. Chicken hypothalamic GnRH-I (A), pituitary FSH-b (B), and pituitary LH-b (C) mRNA expression of young (35- to 44-wk-old) and aging (73- to 82wk-old) broiler breeder roosters (Cobb). Expression of GnRH-I, FSH-b, and LH-b mRNA was determined by real-time PCR, and densitometric analysis of GnRH-I, FSH-b, and LH-b mRNA was performed relative to b-actin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA expression. AU, arbitrary units. Data are presented as mean SEM (n ¼ 10). Values with different letters are significantly different (P < 0.05).
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Fig. 4. Chicken hypothalamic vasoactive intestinal peptide (VIP; A) and pituitary prolactin (PRL; B) mRNA expression and plasma PRL concentration (in ng/mL; C) of young (35- to 44-wk-old) and aging (73- to 82-wk-old) broiler breeder roosters (Cobb). Expression of VIP and PRL mRNA was determined by real-time PCR, and densitometric analysis of VIP and PRL mRNA was performed relative to b-actin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA expression. AU, arbitrary units. Data are presented as mean SEM (n ¼ 10). Values with different letters are significantly different (P < 0.05).
reductions result in low-quality semen, low plasma testosterone concentration, and testis regression. Our results are also supported by findings from a study on broiler breeders which showed that decreased FSH and testosterone concentration is accompanied by decreased sperm production and testis regression [7]. In our study, hypothalamic VIP and pituitary PRL mRNA expression increased in aging compared with young roosters, accompanied by a decrease in reproduction. This suggests an inhibitory effect of the lactotrophic axis on reproduction of aging broiler breeder roosters. Plasma PRL concentration did not differ between young and aging roosters at the beginning of the experiment, even though PRL mRNA expression and semen amounts were significantly lower in aging males. The lack of a significant difference in plasma PRL concentration was probably associated with the relatively large variation within groups at the beginning of the experiment, as frequently encountered in hormones studies. Studies on turkey hens have shown that high concentrations of PRL decrease reproductive activity via inhibition of hypothalamic GnRH release [18,28,29], reduce LH mRNA expression and LH secretion from the pituitary [30], and inhibit ovarian steroid hormone production [31]. In addition, the same inhibitory effect of VIP-PRL was observed in young WL roosters actively immunized against VIP, resulting in decreased plasma PRL concentration and pituitary PRL mRNA expression and increased reproductive activities [22]. A previous study conducted on rats also showed an increase in PRL mRNA expression with age [32,33], as recorded in our study.
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Findings derived from the present study suggest that PRL has an inhibitory effect on the reproductive system of broiler breeder roosters. This seems to contradict our earlier study on WL roosters, in which we found that PRL enhances the reproductive variables of aging roosters [23]. The latter findings were supported because active immunization against VIP reduced spermatogenesis and PRL administration restored reproductive variables. In a comparative analysis with our previous work, we found that basal concentrations of PRL are higher in broiler breeder roosters than in WL roosters [22,23]. It is therefore possible that PRL reduces reproductive performance at both high and low concentrations. These results are in agreement with previous studies conducted on rats which show differences in basal PRL concentrations of several strains [34]. From the present study, we can conclude that gonadal axis function deteriorates in aging broiler breeder roosters, and the increased concentrations of VIP-PRL, as inhibitors of the hypothalamic-pituitary-gonadal axis, suggest their involvement in the reproductive failure associated with age. In addition, this study suggested that PRL concentrations in broiler breeder roosters are opposite those in aging WL roosters, indicating different mechanisms of secretion and differential involvement of PRL in reproduction. Further investigation is warranted to confirm our suggestions about the involvement of VIP and PRL in the reproductive failure associated with aging broiler breeder roosters. References [1] Ishii S. The molecular biology of avian gonadotropin. Poult Sci 1993; 72:856–66. [2] Ottinger MA, Abdelnabi M, Li Q, Chen K, Thompson N, Harada N, Viglietti-Panzica C, Panzica GC. The Japanese quail: a model for studying reproductive aging of hypothalamic systems. Exp Gerontol 2004;39:1679–93. [3] Ottinger MA, Kubakawa K, Kikuchi M, Thompson N, Ishii S. Effects of exogenous testosterone on testicular luteinizing hormone and follicle-stimulating hormone receptors during aging. Exp Biol Med 2002;227:830–6. [4] Ottinger MA, Thompson N, Viglietti-Panzica C, Panzica GC. Neuroendocrine regulation of GnRH and behavior during aging in birds. Brain Res Bull 1997;44:471–7. [5] Sharp PJ, Talbot RT, Main GM, Dunn IC, Fraser HM, Huskisson NS. Physiological roles of chicken LHRH-I and -II in the control of gonadotrophin release in the domestic chicken. J Endocrinol 1990; 124:291–9. [6] Limonta P, Dondi D, Maggi R, Martini L, Piva F. Effects of aging on pituitary and testicular luteinizing hormone-releasing hormone receptors in the rat. Life Sci 1988;42:335–42. [7] Vizcarra JA, Kirby JD, Kreider DL. Testis development and gonadotropin secretion in broiler breeder males. Poult Sci 2010;89:328–34. [8] Catt KJ, Harwood JP, Clayton RN, Davies TF, Chan V, Katikineni M, Nozu K, Dufau ML. Regulation of peptide hormone receptors and gonadal steroidogenesis. Recent Prog Horm Res 1980;36:557–662. [9] Fuentes LB, Calvo JC, Charreau EH, Guzman JA. Seasonal variations in testicular LH, FSH, and PRL receptors; in vitro testosterone production; and serum testosterone concentration in adult male vizcacha (Lagostomus maximus maximus). Gen Comp Endocrinol 1993;90:133–41. [10] Weil S, Rozenboim I, Degen AA, Dawson A, Friedlander M, Rosenstrauch A. Fertility decline in aging roosters is related to increased testicular and plasma levels of estradiol. Gen Comp Endocrinol 1999;115:23–8. [11] Muncher Y, Sod-Moriah UA, Weil S, Rosenstrauch AW, Friedlander M. Intratesticular retention of sperm and premature decline in fertility in the domestic rooster, Gallus domesticus. J Exp Zool 1995;273:76–81.
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