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Testis development and gonadotropin secretion in broiler breeder males1
Testis development and gonadotropin secretion in broiler breeder males1 J. A. Vizcarra,*2 J. D. Kirby,† and D. L. Kreider‡ *Food and Animal Sciences, Alabama A&M University, Normal 35762; †Agricultural Experiment Station, Department of Animal and Range Sciences, South Dakota State University, Brookings 57007; and ‡Department of Animal Science, University of Arkansas, Fayetteville 72701 wk). After photostimulation, there was an exponential increase in testis weight (TW), FSH, and testosterone concentrations. At 28 wk, TW from broilerized males were significantly heavier than those from control birds, and concentrations of luteinizing hormone, FSH, and testosterone were maximal at that time. After 28 wk, there was a significant decrease in FSH and testosterone concentrations that were associated with reduced TW. No correlation was observed between BW and TW. However, TW was highly correlated with FSH concentrations and daily sperm production. Our data suggest that management and photoperiod had a profound effect on testicular function that was associated with FSH concentrations in male broiler breeders.
Key words: testis, gonadotropin, broiler breeder, management, photoperiod 2010 Poultry Science 89:328–334 doi:10.3382/ps.2009-00286
INTRODUCTION
laboratories have addressed the effect of male broiler breeders’ performance under controlled photoperiod and dietary manipulations. In fact, to the best of our knowledge, there is no information on the potential relationship between gonadotropin secretion and testis function using conventional and pedigree breeder feeding programs. Therefore, the objective of this experiment was to evaluate testis development and gonadotropin secretion in male broiler breeders raised under 2 different management systems.
Chickens have been selected for many generations to maximize growth, BW, and yield. Genetic selection over the past 55 yr has been associated with loss of reproductive efficiency in males and females (Rappaport and Soller, 1966; Reddy, 1996; Barbato, 1999). Several techniques have been developed to optimize reproduction in male broiler breeders. Among those, male broiler breeders are raised to maintain a BW that matches an optimized growth curve to prevent obesity. In addition, birds are subjected to long-day photostimulation to maintain their reproductive productivity throughout their adult life. However, when one examines the results of testes development, using present-day management systems, much variability in testis size is observed (Vizcarra et al., 2004). Despite the importance of male fertility to increase reproductive efficiency (Wilson et al., 1979), only a few
MATERIALS AND METHODS Birds and Sampling Procedure One-day-old male broiler breeders (n = 2,700) were banded, vaccinated, and randomly assigned to 2 management treatments (36 pens; n = 75 males per pen). Males on a conventional breeder program (control) were reared on a 15L:9D photoperiod and ad libitum food and water intake for 2 wk. On the third week, males were placed on a restricted diet to maintain a weight gain of approximately 0.75% of their initial BW per week, and the photoperiod was reduced to 8L:16D. Males on a pedigree breeder program (broilerized) were reared on a 23L:1D photoperiod and ad libitum food
©2010 Poultry Science Association Inc. Received June 10, 2009. Accepted October 24, 2009. 1 This work was partially supported by funds from the USDA/NRI/ CGP, the Arkansas Agricultural Experiment Station, and a grant from Cobb-Vantress Inc. (Siloam Springs, AR). 2 Corresponding author: [email protected]
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ABSTRACT One-day-old chicks were used to evaluate testis development and concentrations of luteinizing hormone, follicle-stimulating hormone (FSH), and testosterone during ontogenesis. Males on a conventional breeder program (control) were reared on a 15L:9D photoperiod and ad libitum food and water intake for 2 wk. On the third week, males were placed on a restricted diet and the photoperiod was reduced to 8L:16D. Males on a pedigree breeder program (broilerized) were reared on a 23L:1D photoperiod and unrestricted food and water intake for 6 wk. At 7 wk, males were placed on a restricted diet and the photoperiod was reduced to 8L:16D. On wk 18, both treatment groups were photostimulated (16L:8D) until the end of the experiment (50
TESTIS DEVELOPMENT AND GONADOTROPIN SECRETION
Assays Concentrations of LH and FSH were measured by RIA (Vizcarra et al., 2004) using reagents provided by the USDA-Agricultural Research Service Animal Hormone Program (USDA-pLH-B1, USDA-pFSH-B1, and their cognitive antibodies). Inter- and intraassay CV were 8.2 and 7.2% for LH and 2.4 and 9.7% for FSH, respectively. Testosterone was quantified using a solid phase RIA (ICN Testosterone Kit, Pharmaceuticals Inc., Costa Mesa, CA), as described previously (Vizcarra et al., 2004). Inter- and intraassay CV were 1.7 and 4.9%, respectively. Sperm production (Kirby et al., 1996) was determined in quadruplicates. Briefly, testicular parenchyma was homogenized in assay buffer in a semimicro blender, and elongated spermatid nuclei were counted on a hemocytometer. Daily sperm production per gram of testis (DSPG) was estimated by dividing the number of homogenization-resistant nuclei per gram of testis by a factor of 4.5. This factor represents the average number of days that elongated spermatids remain in the testis before their entry into the testis excurrent ducts (de Reviers, 1968).
Statistical Analyses Effects of treatment on testis weight (TW), BW, LH, FSH, testosterone concentrations, and DSPG in weekly samples were analyzed using repeated measurements over time (PROC MIXED, SAS Inst. Inc., Cary, NC).
The pen was considered the experimental unit, and at least 3 covariance structures were evaluated (compound symmetric, unstructured, and autoregressive). The autoregressive structure provided the best model-fit criteria for all of the variables. If a significant treatment × time interaction existed, the SLICE option in SAS was used to test for significant difference between treatments at each time. Analyses of regression and correlation between various variables were performed using least squares models. When appropriate, linear or nonlinear polynomial response curves were calculated and significant differences in the intercept and slope among treatments were computed by using dummy variables (Steel and Torrie, 1980). Logistic curves of the form Y = k/1 + [(k − n0)/n0] e−rt were used to describe the relationship between TW and time during the first 28 wk of the experimental period (PROC NLIN, SAS Inst. Inc.). The value of k represents the height of the horizontal asymptote, whereas n0 is the value of Y at time (t) = 0, and r is the slope or growth rate. Spline models with unknown knots (PROC NLIN, SAS Inst. Inc.) were used to evaluate the relationship between DSPG and TW. The spline procedure estimates different linear regressions for different ranges of the independent variable (i.e., TW), and the endpoints of the ranges are called knots (Freund and Littell, 2000). The knot, in our data, is an estimate of the TW at which DSPG plateaus.
RESULTS Restricted feed management was successful in achieving target BW for conventional and broilerized management programs (Figure 1). Quadratic regression equations best described a treatment × week interaction for BW, and weights were significantly different between wk 4 and 33 of the experimental period. Logistic curves were used to describe the relationship between TW and week for control and broilerized birds during the first 28 wk of the experimental period (Figure 2). There were no differences in the intercept (n0), indicating that treatments did not affect TW before photostimulation. After photostimulation (18 WOA), there was an exponential increase in TW, but no differences were observed in the slope (r) of control and broilerized birds. By wk 28, testis from broilerized males weighed 34% more than those from control birds (k = 21.1 ± 0.5 and 28.3 ± 0.3 g for control and broilerized birds, respectively, P < 0.01). Although no significant differences were observed in the intercept, testis from broilerized birds were 3-fold heavier than those from control birds at wk 7 (Figure 2, inset). Histological inspection of testes from 7-wk broilerized males showed precocious testicular maturation with zygotene or pachytene, or both, spermatocytes visible in all seminiferous tubules (data not shown). These structures were not present in control birds.
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and water intake for 6 wk. On the seventh week, males were placed on a restricted diet to maintain a weight gain of approximately 0.75% of their initial BW per week, and the photoperiod was reduced to 8L:16D. On wk 18, both treatment groups underwent an incremental step-up lighting regimen until they reached a 16L:8D photoperiod by 28 wk of age (WOA). Weekly, starting at 3 WOA and until the end of the experiment (50 WOA), 30 males were weighed and bled. Blood samples were taken via a brachial wing vein in 5-mL tubes containing EDTA to prevent clotting. Samples (n = 15/treatment per week) were placed on ice and centrifuged (1,800 × g for 20 min) within 4 h, and plasma was decanted and stored at −20°C until concentrations of testosterone, luteinizing hormone (LH), and follicle-stimulating hormone (FSH) were quantified by RIA. After blood samples were obtained, birds were killed and testes were removed and weighed. The left testis was fixed in Bouin’s fixative solution for histological evaluation. Additionally, at wk 27, 29, 35, 39, 45, and 50, the right testis was frozen and stored at −80°C for further determination of daily sperm production. All experimental procedures described were approved by the University of Arkansas Animal Care and Use Committee.
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From wk 29 to the end of the experiment, quadratic linear regressions best described the relationship between TW and week for broilerized and control birds (Figure 2). There was a significance difference in the intercept and in the slope (P < 0.01). From wk 29 to wk 34, testis were significantly heavier in broilerized males compared with control birds. However, after 35 WOA, there were no significant differences in TW between treatments. There was no treatment or treatment × week effect on LH, FSH, and testosterone concentrations (P > 0.1). However, a significant week effect (P < 0.01) was observed during the experimental period (Figure 3). Concentrations of LH, FSH, and testosterone averaged 1.19, 2.06, and 0.21 ng/mL from wk 3 to 20, respectively. By wk 27, concentrations were increased to 2.60, 13.75, and 1.60 ng/mL (LH, FSH, and testosterone, respectively). After wk 35, concentrations of LH, FSH, and testosterone averaged 1.95, 7.92, and 1.82 ng/mL, respectively.
No significant correlations were observed between TW and BW (Figure 4). On average, less than 21% of the variation in TW was explained by changes in BW after photostimulation. There was no treatment or week or treatment × week effect on DSPG. On average, 7.8 × 107 sperm cells per day per gram of testicular parenchyma were produced during wk 27 to 50 of the experimental period. The relationship between DSPG and TW was best described by 2 linear regressions (Figure 5A). The estimated knot derived from the spline model reveals that DSPG reached a maximum when testis weighed 8.6 ± 0.5 g. Quadratic regressions best described the relationship between FSH and TW (Figure 5B). More than 90% of the variation on TW was explained by changes in FSH concentrations. Similarly, quadratic regressions best described the relationship between LH and TW (Figure 5B). However, less than 35% of the variation in TW was explained by changes in LH concentrations. The
Figure 2. Least squares regression (lines) and means (symbols) for testis weight in control and broilerized male breeders. Nonlinear regressions were used during the first 28 wk of the experimental period. Quadratic linear regressions were computed from wk 29 to 50. Testis weight × week interaction (P < 0.01). Each symbol represents an average of 30 testes.
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Figure 1. Least squares regression (lines) and means (symbols) for BW in control and broilerized male breeders during the experimental period. Body weight × week interaction (P < 0.01). Each symbol represents an average of 15 birds.
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DISCUSSION
relationship between testosterone and TW was also described by a quadratic regression (Figure 5B). Eightyone percent of the variation of TW was explained by changes in testosterone concentrations.
Figure 4. Least squares regression (lines) and means (symbols) for the relationship between testis weight (TW) and BW in control and broilerized male breeders. Control males: TW = 7.2 + 0.002BW; r = 0.20; broilerized males TW = 4.15 + 0.003BW; r = 0.22.
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Figure 3. Least squares means for luteinizing hormone (LH), follicle-stimulating hormone (FSH), and testosterone (T) in male broiler breeders during the experimental period. Each symbol represents an average of 15 plasma samples.
Body weights for conventional and broilerized management programs were within the recommended limits suggested by the industry (Cobb-Vantress, 1998). The broilerized management program is typically used in grandparent and great-grandparent stock, to allow geneticists to select parents for the next generations. In contrast, conventional-reared control birds were raised using standard breeder management practices with feed and light restriction starting on wk 3. Differences in BW between wk 4 and 33 reflected the increased energy consumption in the broilerization system during the first 7 wk after hatching. The onset of active meiosis after Sertoli cell differentiation is characterized by the presence of pachytene and zygotene spermatocytes in the cockerel (Kirby and Froman, 2000). We observed that testis from broilerized males were heavier and underwent active meiosis at 7 WOA, whereas control birds did not complete Sertoli cell proliferation. The onset of meiosis can be significantly altered by manipulation of the photoperiod (Ingkasuwan and Ogasawara, 1996). In the present experiment, broilerized males were reared on a 23L:1D photoperiod and ad libitum food and water intake for 6 wk, whereas control birds were reared on a 15L:9D photoperiod and ad libitum food and water intake for only 2 wk. The increased TW and the presence of meiotic cells at 7 WOA in broilerized birds was associated with increased photoperiod and feed consumption. Additionally, TW was significantly higher in broilerized birds 8 wk after both treatment groups underwent the incremental step-up lightning regimen on wk 18. We rationalize that the early Sertoli cell differentiation and proliferation in broilerized males was responsible for the increased TW after photostimulation. However, further experiments need to be conducted to validate this hypothesis. Testicular weight, LH, FSH, and testosterone concentrations were significantly increased after photostimulation (Figures 2 and 3). In chickens, as in most
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Two chicken GnRH isoforms have been described (King and Millar, 1982; Miyamoto et al., 1982, 1984). Nonetheless, only indirect measurement of the GnRH pulse generator is available by assessing plasma LH and FSH concentrations in frequent samples. The pattern of LH and FSH secretion in intact male broiler breeders is pulsatile with a frequency of 0.54 and 0.38 pulses/h, respectively (Vizcarra et al., 2004). A significant lack of synchrony between episodic release of LH and FSH was also observed, suggesting that LH and FSH secretion are regulated independently (Vizcarra et al., 2004). In the present study, we also observed a differential pattern of LH and FSH secretion before and after photostimulation (Figure 3), further suggesting that gonadotropin secretion might be differentially regulated in broiler breeders. A possible autocrine-paracrine regulation of FSH release at the pituitary level by activins, inhibins, and follistatins cannot be overlooked (Baird et al., 1991; DePaolo et al., 1991; Mather et al., 1992). In fact, active immunization against inhibin (α subunit) in meat-type male chickens was associated with increased testosterone concentrations and increased TW (Satterlee et al., 2006). On the other hand, when a similar immuniza-
Figure 5. Least squares regressions (lines) and means (symbols) for the relationship between daily sperm production per gram of testis (DSPG), follicle-stimulating hormone (FSH), luteinizing hormone (LH), and testosterone against testis weight (TW) in male broiler breeders. A) Spline function: DSPG = −1.4 + 1.1 × TW − 1.1 × MAX(TW − 8.5, 0) (R = 0.98). Each symbol represents an average of 10 testes. B) FSH = 2.26 + 0.63TW − 0.01TW2; R = 0.95; LH = 1.24 + 0.08TW − 0.002TW2; R = 0.59; testosterone = 0.35 + 0.11TW − 0.001TW2; R = 0.92. Each symbol represents an average of 66 samples.
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seasonal breeders, the stimulation of the gonadotropic axis is influenced by the photoperiod. Hypothalamic photoreceptors transduce the energy from light into a biological signal such as a hormone that regulates the secretion of gonadotropin-releasing hormone (GnRH; Kuenzel, 1993; Etches, 1996). In male quails, plasma concentrations of LH and FSH increase sharply hours after a transfer from short days (8L:16D) to long days (20L:4D) (Follett et al., 1977). However, egg-type males that were raised at a constant short-day photoperiod (8L:16D) also exhibited an increase in LH, FSH, and, to a lesser degree, testosterone concentration after 15 WOA (Lovell et al., 2000). Encephalic photoreceptors are believed to be involved in the light-induced gonadotropic stimulation; however, the hypothalamic photoreceptors have not been identified. In avian species, the photoreceptors may be located within the medial basal hypothalamus (Kuenzel, 1993) in the same region where the GnRH pulse generator is located. In the chicken, the pineal gland expresses a circadian rhythm of melatonin under light-dark cycles, and it has been suggested that the pineal cells may have photoreceptive capabilities with a circadian rhythm (Nakahara et al., 1997).
TESTIS DEVELOPMENT AND GONADOTROPIN SECRETION
ed for testosterone production and respond rapidly to LH stimulation (Maung and Follett, 1977; Johnson, 1986). Testosterone is secreted in an episodic fashion, and pulses closely followed LH episodes in male broiler breeders and male turkeys (Bacon et al., 1994; Yang et al., 1998; Vizcarra et al., 2004). However, the association between LH and testosterone pulses was evident only in broiler males with TW greater than 10 g and relatively high testosterone concentrations (Vizcarra et al., 2004). Even though LH, FSH, and testosterone are essential for spermatogenesis in birds, it appears that FSH is the best indicator of testis function in males. We conclude that management and photoperiod had a profound effect on testicular function that was associated with FSH concentrations in male broiler breeders.
ACKNOWLEDGMENTS We acknowledge Marsha Rhoads (University of Arkansas) for her assistance in sample collection and handling and J. Proudman (USDA, Beltsville, MD) for LH and FSH reagents. Avian pituitary hormones and antisera were obtained through the USDA-Agricultural Research Service Animal Hormone Program.
REFERENCES Bacon, W. L., D. W. Long, K. Kurima, D. P. Chapman, and W. H. Burke. 1994. Coordinate pattern of secretion of luteinizing hormone and testosterone in mature male turkeys under continuous and intermittent photoschedules. Poult. Sci. 73:864–870. Baird, D. T., B. K. Campbell, G. E. Mann, and A. S. McNeilly. 1991. Inhibin and oestradiol in the control of FSH secretion in the sheep. J. Reprod. Fertil. 43:125–138. Barbato, G. F. 1999. Genetic relationships between selection for growth and reproductive effectiveness. Poult. Sci. 78:444–452. Brown, N. L., J. D. Bayle, C. G. Scanes, and B. K. Follett. 1975. Chicken gonadotrophins: Their effects on the testes of immature and hypophysectomized Japanese quail. Cell Tissue Res. 156:499–520. Cobb-Vantress. 1998. Breeder Management Guide. Cobb-Vantress, Siloam Springs, AR. de Reviers, M. 1968. Determination de la durée des processus spermatogenetiques chez le coq a l’aide de thymidine tritice. Proc. 6th Int. Congr. Anim. Reprod. 1:183–185. de Reviers, M., and J. B. Williams. 1984. Testis development and production of spermatozoa in the cockerel (Gallus domesticus). Pages 183–202 in Reproductive Biology of Poultry. F. J. Cunningham, P. E. Lake, and D. Hewitt, ed. British Poultry Science Ltd., Harlow, UK. DePaolo, L. V., T. A. Bicsak, G. F. Erickson, S. Shimasaki, and N. Ling. 1991. Follistatin and activin: A potential intrinsic regulatory system within diverse tissues. Proc. Soc. Exp. Biol. Med. 198:500–512. Etches, R. J. 1996. Photoperiodism. Pages 106–124 in Reproduction in Poultry. CABI, Wallingford, UK. Feigelson, M. 1986. Suppression of testicular maturation and fertility following androgen administration to neonatal male rats. Biol. Reprod. 35:1321–1332. Follett, B. K., D. T. Davies, and B. Gledhill. 1977. Photoperiodic control of reproduction in Japanese quail: Changes in gonadotrophin secretion on the first day of induction and their pharmacological blockade. J. Endocrinol. 74:449–460. Freund, R. J., and R. C. Littell. 2000. Systems for Regression. 3rd ed. SAS Institute Inc., Cary, NC. Huhtaniemi, I. T., N. Nevo, A. Amsterdam, and Z. Naor. 1986. Effect of postnatal treatment with a gonadotropin-releasing hor-
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tion protocol was used in egg-type male chickens, a significant increase in TW without changes in FSH or testosterone concentrations was observed (Lovell et al., 2000). After a maximum TW was obtained at wk 28, quadratic regressions showed that TW decreased over the next 7 wk until both treatments reached a plateau after 35 WOA (Figure 2). The decline in weight was more pronounced in broilerized males compared with control birds. The reason for the TW regression over time, as well as the differences between treatments, is not completely clear at this time. We hypothesize that decreased FSH concentrations might be responsible for TW regression (see below). The nonsignificant relationship between BW and TW in broilerized and control birds (Figure 4), suggests that the increased BW achieved by broilerized males was not responsible for the increased TW after photostimulation. In fact, the variation in TW at a given BW was extreme. For instance, cockerels with a BW within 4 to 5 kg had TW that varied from a minimum of 0.9 g to a maximum of 44 g. Despite the tremendous variation in TW, a close relationship between TW and DSPG was observed (Figure 5A). Our data strongly suggest that daily sperm production can be used to estimate TW of any given male. It appears that a TW of 8.6 g or more is required for the production of at least 7 to 8 × 107 sperm per gram of testis. Similarly, de Reviers and Williams (1984) reported that normal males released 8 to 12 × 107 sperm per day from the seminiferous epithelium into the lumen of the seminiferous tubules. The ability of the testis to produce sperm is regulated by the number of Sertoli cells per testis, and daily sperm production is correlated with the number of Sertoli cells (de Reviers and Williams, 1984; Sharpe, 1994). It is clear, at least in mammals, that FSH is the most important regulator of Sertoli cell proliferation and differentiation (Feigelson, 1986; Huhtaniemi et al., 1986). In addition, testosterone potentiates the effect of FSH on Sertoli cells in quails (Tsutsui and Ishii, 1978). Taken together, although sperm production depends on the number of Sertoli cells, the process is ultimately controlled by the hypothalamus-pituitary-gonadal axis. We found that plasma FSH concentrations accurately predicted TW (r = 0.95; Figure 5B). In contrast, plasma LH concentrations were not predictive of TW (r = 0.59; Figure 5B). Although the main effect of FSH is exerted upon Sertoli cells, LH affects mainly Leydig cells (Brown et al., 1975; Ishii and Yamamoto, 1976). Our data suggest that FSH has a profound effect in the variation of TW and sperm production. In fact, more than 90% of the variation on TW was explained by changes in FSH concentrations. We speculate that other factors such as chronic or acute stress, feed intake, social interactions, and subclinical diseases might also affect testicular development and sperm production. Testosterone, to a lesser degree, was also a good indicator of testis function (r = 0.92; Figure 5B). Chicken Leydig cells contain all of the enzymes that are need-
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Vizcarra et al. Miyamoto, K., Y. Hasegawa, M. Nomura, M. Igarashi, K. Kangawa, and H. Matsuo. 1984. Identification of the second gonadotropin-releasing hormone in chicken hypothalamus: Evidence that gonadotropin secretion is probably controlled by two distinct gonadotropin-releasing hormones in avian species. Proc. Natl. Acad. Sci. USA 81:3874–3878. Nakahara, K., N. Murakami, T. Nasu, H. Kuroda, and T. Murakami. 1997. Individual pineal cells in chick possess photoreceptive, circadian clock and melatonin-synthesizing capacities in vitro. Brain Res. 774:242–245. Rappaport, S., and M. Soller. 1966. Mating behavior, fertility and rate-of-gain in Cornish males. Poult. Sci. 45:997–1003. Reddy, R. P. 1996. Symposium: The effects of long-term selection on growth of poultry. Introduction. Poult. Sci. 75:1164–1167. Satterlee, D. G., S. A. Castille, and W. C. Fioretti. 2006. Active immunization of broiler breeder cockerels against chicken inhibin accelerates puberty and prevents age-induced testicular involution. Poult. Sci. 85:1087–1094. Sharpe, R. M. 1994. Regulation of spermatogenesis. Pages 1363– 1434 in The Physiology of Reproduction. 2nd ed. Vol 1. E. Knobil and J. D. Neill, ed. Raven Press, New York, NY. Steel, R. G. D., and J. H. Torrie. 1980. Principles and Procedures of Statistics: A Biometrical Approach. 2nd ed. McGraw-Hill, New York, NY. Tsutsui, K., and S. Ishii. 1978. Effects of follicle-stimulating hormone and testosterone on receptors of follicle-stimulating hormone in the testis of the immature Japanese quail. Gen. Comp. Endocrinol. 36:297–305. Vizcarra, J. A., D. L. Kreider, and J. D. Kirby. 2004. Episodic gonadotropin secretion in the mature fowl: Serial blood sampling from unrestrained male broiler breeders (Gallus domesticus). Biol. Reprod. 70:1798–1805. Wilson, H. R., N. P. Piesco, E. R. Miller, and W. G. Nesbeth. 1979. Prediction of the fertility potential of broiler breeder males. World’s Poult. Sci. J. 35:95–118. Yang, J., D. W. Long, N. Inpanbutr, and W. L. Bacon. 1998. Effects of photoperiod and age on secretory patterns of luteinizing hormone and testosterone and semen production in male domestic turkeys. Biol. Reprod. 59:1171–1179.
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mone antagonist on sexual maturation of male rats. Biol. Reprod. 35:501–507. Ingkasuwan, P., and F. Ogasawara. 1996. The effect of light and temperature and their interactions on the semen production of White Leghorn males. Poult. Sci. 45:1199–1204. Ishii, S., and K. Yamamoto. 1976. Demonstration of follicle stimulating hormone (FSH) activity in hypophyseal extracts of various vertebrates by the response of the Sertoli cells of the chick. Gen. Comp. Endocrinol. 29:506–510. Johnson, A. L. 1986. Reproduction in the male. Pages 432–451 in Avian Physiology. P. D. Sturkie, ed. Springer-Verlag, New York, NY. King, J. A., and R. P. Millar. 1982. Structure of chicken hypothalamic luteinizing hormone-releasing hormone. II. Isolation and characterization. J. Biol. Chem. 257:10729–10732. Kirby, J. D., and D. P. Froman. 2000. Reproduction in male birds. Pages 597–615 in Sturkie’s Avian Physiology. 5th ed. G. C. Whittow, ed. Academic Press, New York, NY. Kirby, J. D., M. V. Mankar, D. Hardesty, and D. L. Kreider. 1996. Effects of transient prepubertal 6-N-propyl-2-thiouracil treatment on testis development and function in the domestic fowl. Biol. Reprod. 55:910–916. Kuenzel, W. J. 1993. The search for deep encephalic photoreceptors within the avian brain, using gonadal development as a primary indicator. Poult. Sci. 72:959–967. Lovell, T. M., P. G. Knight, N. P. Groome, and R. T. Gladwell. 2000. Measurement of dimeric inhibins and effects of active immunization against inhibin α-subunit on plasma hormones and testis morphology in the developing cockerel. Biol. Reprod. 63:213–221. Mather, J. P., T. K. Woodruff, and L. A. Krummen. 1992. Paracrine regulation of reproductive function by inhibin and activin. Proc. Soc. Exp. Biol. Med. 201:1–15. Maung, Z. W., and B. K. Follett. 1977. Effects of chicken and ovine luteinizing hormone on androgen release and cyclic AMP production by isolated cells from the quail testis. Gen. Comp. Endocrinol. 33:242–253. Miyamoto, K., Y. Hasegawa, T. Minegishi, M. Nomura, Y. Takahashi, M. Igarashi, K. Kangawa, and H. Matsuo. 1982. Isolation and characterization of chicken hypothalamic luteinizing hormone-releasing hormone. Biochem. Biophys. Res. Commun. 107:820–827.
Key words: testis, gonadotropin, broiler breeder, management, photoperiod 2010 Poultry Science 89:328–334 doi:10.3382/ps.2009-00286
INTRODUCTION
laboratories have addressed the effect of male broiler breeders’ performance under controlled photoperiod and dietary manipulations. In fact, to the best of our knowledge, there is no information on the potential relationship between gonadotropin secretion and testis function using conventional and pedigree breeder feeding programs. Therefore, the objective of this experiment was to evaluate testis development and gonadotropin secretion in male broiler breeders raised under 2 different management systems.
Chickens have been selected for many generations to maximize growth, BW, and yield. Genetic selection over the past 55 yr has been associated with loss of reproductive efficiency in males and females (Rappaport and Soller, 1966; Reddy, 1996; Barbato, 1999). Several techniques have been developed to optimize reproduction in male broiler breeders. Among those, male broiler breeders are raised to maintain a BW that matches an optimized growth curve to prevent obesity. In addition, birds are subjected to long-day photostimulation to maintain their reproductive productivity throughout their adult life. However, when one examines the results of testes development, using present-day management systems, much variability in testis size is observed (Vizcarra et al., 2004). Despite the importance of male fertility to increase reproductive efficiency (Wilson et al., 1979), only a few
MATERIALS AND METHODS Birds and Sampling Procedure One-day-old male broiler breeders (n = 2,700) were banded, vaccinated, and randomly assigned to 2 management treatments (36 pens; n = 75 males per pen). Males on a conventional breeder program (control) were reared on a 15L:9D photoperiod and ad libitum food and water intake for 2 wk. On the third week, males were placed on a restricted diet to maintain a weight gain of approximately 0.75% of their initial BW per week, and the photoperiod was reduced to 8L:16D. Males on a pedigree breeder program (broilerized) were reared on a 23L:1D photoperiod and ad libitum food
©2010 Poultry Science Association Inc. Received June 10, 2009. Accepted October 24, 2009. 1 This work was partially supported by funds from the USDA/NRI/ CGP, the Arkansas Agricultural Experiment Station, and a grant from Cobb-Vantress Inc. (Siloam Springs, AR). 2 Corresponding author: [email protected]
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ABSTRACT One-day-old chicks were used to evaluate testis development and concentrations of luteinizing hormone, follicle-stimulating hormone (FSH), and testosterone during ontogenesis. Males on a conventional breeder program (control) were reared on a 15L:9D photoperiod and ad libitum food and water intake for 2 wk. On the third week, males were placed on a restricted diet and the photoperiod was reduced to 8L:16D. Males on a pedigree breeder program (broilerized) were reared on a 23L:1D photoperiod and unrestricted food and water intake for 6 wk. At 7 wk, males were placed on a restricted diet and the photoperiod was reduced to 8L:16D. On wk 18, both treatment groups were photostimulated (16L:8D) until the end of the experiment (50
TESTIS DEVELOPMENT AND GONADOTROPIN SECRETION
Assays Concentrations of LH and FSH were measured by RIA (Vizcarra et al., 2004) using reagents provided by the USDA-Agricultural Research Service Animal Hormone Program (USDA-pLH-B1, USDA-pFSH-B1, and their cognitive antibodies). Inter- and intraassay CV were 8.2 and 7.2% for LH and 2.4 and 9.7% for FSH, respectively. Testosterone was quantified using a solid phase RIA (ICN Testosterone Kit, Pharmaceuticals Inc., Costa Mesa, CA), as described previously (Vizcarra et al., 2004). Inter- and intraassay CV were 1.7 and 4.9%, respectively. Sperm production (Kirby et al., 1996) was determined in quadruplicates. Briefly, testicular parenchyma was homogenized in assay buffer in a semimicro blender, and elongated spermatid nuclei were counted on a hemocytometer. Daily sperm production per gram of testis (DSPG) was estimated by dividing the number of homogenization-resistant nuclei per gram of testis by a factor of 4.5. This factor represents the average number of days that elongated spermatids remain in the testis before their entry into the testis excurrent ducts (de Reviers, 1968).
Statistical Analyses Effects of treatment on testis weight (TW), BW, LH, FSH, testosterone concentrations, and DSPG in weekly samples were analyzed using repeated measurements over time (PROC MIXED, SAS Inst. Inc., Cary, NC).
The pen was considered the experimental unit, and at least 3 covariance structures were evaluated (compound symmetric, unstructured, and autoregressive). The autoregressive structure provided the best model-fit criteria for all of the variables. If a significant treatment × time interaction existed, the SLICE option in SAS was used to test for significant difference between treatments at each time. Analyses of regression and correlation between various variables were performed using least squares models. When appropriate, linear or nonlinear polynomial response curves were calculated and significant differences in the intercept and slope among treatments were computed by using dummy variables (Steel and Torrie, 1980). Logistic curves of the form Y = k/1 + [(k − n0)/n0] e−rt were used to describe the relationship between TW and time during the first 28 wk of the experimental period (PROC NLIN, SAS Inst. Inc.). The value of k represents the height of the horizontal asymptote, whereas n0 is the value of Y at time (t) = 0, and r is the slope or growth rate. Spline models with unknown knots (PROC NLIN, SAS Inst. Inc.) were used to evaluate the relationship between DSPG and TW. The spline procedure estimates different linear regressions for different ranges of the independent variable (i.e., TW), and the endpoints of the ranges are called knots (Freund and Littell, 2000). The knot, in our data, is an estimate of the TW at which DSPG plateaus.
RESULTS Restricted feed management was successful in achieving target BW for conventional and broilerized management programs (Figure 1). Quadratic regression equations best described a treatment × week interaction for BW, and weights were significantly different between wk 4 and 33 of the experimental period. Logistic curves were used to describe the relationship between TW and week for control and broilerized birds during the first 28 wk of the experimental period (Figure 2). There were no differences in the intercept (n0), indicating that treatments did not affect TW before photostimulation. After photostimulation (18 WOA), there was an exponential increase in TW, but no differences were observed in the slope (r) of control and broilerized birds. By wk 28, testis from broilerized males weighed 34% more than those from control birds (k = 21.1 ± 0.5 and 28.3 ± 0.3 g for control and broilerized birds, respectively, P < 0.01). Although no significant differences were observed in the intercept, testis from broilerized birds were 3-fold heavier than those from control birds at wk 7 (Figure 2, inset). Histological inspection of testes from 7-wk broilerized males showed precocious testicular maturation with zygotene or pachytene, or both, spermatocytes visible in all seminiferous tubules (data not shown). These structures were not present in control birds.
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and water intake for 6 wk. On the seventh week, males were placed on a restricted diet to maintain a weight gain of approximately 0.75% of their initial BW per week, and the photoperiod was reduced to 8L:16D. On wk 18, both treatment groups underwent an incremental step-up lighting regimen until they reached a 16L:8D photoperiod by 28 wk of age (WOA). Weekly, starting at 3 WOA and until the end of the experiment (50 WOA), 30 males were weighed and bled. Blood samples were taken via a brachial wing vein in 5-mL tubes containing EDTA to prevent clotting. Samples (n = 15/treatment per week) were placed on ice and centrifuged (1,800 × g for 20 min) within 4 h, and plasma was decanted and stored at −20°C until concentrations of testosterone, luteinizing hormone (LH), and follicle-stimulating hormone (FSH) were quantified by RIA. After blood samples were obtained, birds were killed and testes were removed and weighed. The left testis was fixed in Bouin’s fixative solution for histological evaluation. Additionally, at wk 27, 29, 35, 39, 45, and 50, the right testis was frozen and stored at −80°C for further determination of daily sperm production. All experimental procedures described were approved by the University of Arkansas Animal Care and Use Committee.
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From wk 29 to the end of the experiment, quadratic linear regressions best described the relationship between TW and week for broilerized and control birds (Figure 2). There was a significance difference in the intercept and in the slope (P < 0.01). From wk 29 to wk 34, testis were significantly heavier in broilerized males compared with control birds. However, after 35 WOA, there were no significant differences in TW between treatments. There was no treatment or treatment × week effect on LH, FSH, and testosterone concentrations (P > 0.1). However, a significant week effect (P < 0.01) was observed during the experimental period (Figure 3). Concentrations of LH, FSH, and testosterone averaged 1.19, 2.06, and 0.21 ng/mL from wk 3 to 20, respectively. By wk 27, concentrations were increased to 2.60, 13.75, and 1.60 ng/mL (LH, FSH, and testosterone, respectively). After wk 35, concentrations of LH, FSH, and testosterone averaged 1.95, 7.92, and 1.82 ng/mL, respectively.
No significant correlations were observed between TW and BW (Figure 4). On average, less than 21% of the variation in TW was explained by changes in BW after photostimulation. There was no treatment or week or treatment × week effect on DSPG. On average, 7.8 × 107 sperm cells per day per gram of testicular parenchyma were produced during wk 27 to 50 of the experimental period. The relationship between DSPG and TW was best described by 2 linear regressions (Figure 5A). The estimated knot derived from the spline model reveals that DSPG reached a maximum when testis weighed 8.6 ± 0.5 g. Quadratic regressions best described the relationship between FSH and TW (Figure 5B). More than 90% of the variation on TW was explained by changes in FSH concentrations. Similarly, quadratic regressions best described the relationship between LH and TW (Figure 5B). However, less than 35% of the variation in TW was explained by changes in LH concentrations. The
Figure 2. Least squares regression (lines) and means (symbols) for testis weight in control and broilerized male breeders. Nonlinear regressions were used during the first 28 wk of the experimental period. Quadratic linear regressions were computed from wk 29 to 50. Testis weight × week interaction (P < 0.01). Each symbol represents an average of 30 testes.
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Figure 1. Least squares regression (lines) and means (symbols) for BW in control and broilerized male breeders during the experimental period. Body weight × week interaction (P < 0.01). Each symbol represents an average of 15 birds.
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DISCUSSION
relationship between testosterone and TW was also described by a quadratic regression (Figure 5B). Eightyone percent of the variation of TW was explained by changes in testosterone concentrations.
Figure 4. Least squares regression (lines) and means (symbols) for the relationship between testis weight (TW) and BW in control and broilerized male breeders. Control males: TW = 7.2 + 0.002BW; r = 0.20; broilerized males TW = 4.15 + 0.003BW; r = 0.22.
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Figure 3. Least squares means for luteinizing hormone (LH), follicle-stimulating hormone (FSH), and testosterone (T) in male broiler breeders during the experimental period. Each symbol represents an average of 15 plasma samples.
Body weights for conventional and broilerized management programs were within the recommended limits suggested by the industry (Cobb-Vantress, 1998). The broilerized management program is typically used in grandparent and great-grandparent stock, to allow geneticists to select parents for the next generations. In contrast, conventional-reared control birds were raised using standard breeder management practices with feed and light restriction starting on wk 3. Differences in BW between wk 4 and 33 reflected the increased energy consumption in the broilerization system during the first 7 wk after hatching. The onset of active meiosis after Sertoli cell differentiation is characterized by the presence of pachytene and zygotene spermatocytes in the cockerel (Kirby and Froman, 2000). We observed that testis from broilerized males were heavier and underwent active meiosis at 7 WOA, whereas control birds did not complete Sertoli cell proliferation. The onset of meiosis can be significantly altered by manipulation of the photoperiod (Ingkasuwan and Ogasawara, 1996). In the present experiment, broilerized males were reared on a 23L:1D photoperiod and ad libitum food and water intake for 6 wk, whereas control birds were reared on a 15L:9D photoperiod and ad libitum food and water intake for only 2 wk. The increased TW and the presence of meiotic cells at 7 WOA in broilerized birds was associated with increased photoperiod and feed consumption. Additionally, TW was significantly higher in broilerized birds 8 wk after both treatment groups underwent the incremental step-up lightning regimen on wk 18. We rationalize that the early Sertoli cell differentiation and proliferation in broilerized males was responsible for the increased TW after photostimulation. However, further experiments need to be conducted to validate this hypothesis. Testicular weight, LH, FSH, and testosterone concentrations were significantly increased after photostimulation (Figures 2 and 3). In chickens, as in most
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Two chicken GnRH isoforms have been described (King and Millar, 1982; Miyamoto et al., 1982, 1984). Nonetheless, only indirect measurement of the GnRH pulse generator is available by assessing plasma LH and FSH concentrations in frequent samples. The pattern of LH and FSH secretion in intact male broiler breeders is pulsatile with a frequency of 0.54 and 0.38 pulses/h, respectively (Vizcarra et al., 2004). A significant lack of synchrony between episodic release of LH and FSH was also observed, suggesting that LH and FSH secretion are regulated independently (Vizcarra et al., 2004). In the present study, we also observed a differential pattern of LH and FSH secretion before and after photostimulation (Figure 3), further suggesting that gonadotropin secretion might be differentially regulated in broiler breeders. A possible autocrine-paracrine regulation of FSH release at the pituitary level by activins, inhibins, and follistatins cannot be overlooked (Baird et al., 1991; DePaolo et al., 1991; Mather et al., 1992). In fact, active immunization against inhibin (α subunit) in meat-type male chickens was associated with increased testosterone concentrations and increased TW (Satterlee et al., 2006). On the other hand, when a similar immuniza-
Figure 5. Least squares regressions (lines) and means (symbols) for the relationship between daily sperm production per gram of testis (DSPG), follicle-stimulating hormone (FSH), luteinizing hormone (LH), and testosterone against testis weight (TW) in male broiler breeders. A) Spline function: DSPG = −1.4 + 1.1 × TW − 1.1 × MAX(TW − 8.5, 0) (R = 0.98). Each symbol represents an average of 10 testes. B) FSH = 2.26 + 0.63TW − 0.01TW2; R = 0.95; LH = 1.24 + 0.08TW − 0.002TW2; R = 0.59; testosterone = 0.35 + 0.11TW − 0.001TW2; R = 0.92. Each symbol represents an average of 66 samples.
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seasonal breeders, the stimulation of the gonadotropic axis is influenced by the photoperiod. Hypothalamic photoreceptors transduce the energy from light into a biological signal such as a hormone that regulates the secretion of gonadotropin-releasing hormone (GnRH; Kuenzel, 1993; Etches, 1996). In male quails, plasma concentrations of LH and FSH increase sharply hours after a transfer from short days (8L:16D) to long days (20L:4D) (Follett et al., 1977). However, egg-type males that were raised at a constant short-day photoperiod (8L:16D) also exhibited an increase in LH, FSH, and, to a lesser degree, testosterone concentration after 15 WOA (Lovell et al., 2000). Encephalic photoreceptors are believed to be involved in the light-induced gonadotropic stimulation; however, the hypothalamic photoreceptors have not been identified. In avian species, the photoreceptors may be located within the medial basal hypothalamus (Kuenzel, 1993) in the same region where the GnRH pulse generator is located. In the chicken, the pineal gland expresses a circadian rhythm of melatonin under light-dark cycles, and it has been suggested that the pineal cells may have photoreceptive capabilities with a circadian rhythm (Nakahara et al., 1997).
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ed for testosterone production and respond rapidly to LH stimulation (Maung and Follett, 1977; Johnson, 1986). Testosterone is secreted in an episodic fashion, and pulses closely followed LH episodes in male broiler breeders and male turkeys (Bacon et al., 1994; Yang et al., 1998; Vizcarra et al., 2004). However, the association between LH and testosterone pulses was evident only in broiler males with TW greater than 10 g and relatively high testosterone concentrations (Vizcarra et al., 2004). Even though LH, FSH, and testosterone are essential for spermatogenesis in birds, it appears that FSH is the best indicator of testis function in males. We conclude that management and photoperiod had a profound effect on testicular function that was associated with FSH concentrations in male broiler breeders.
ACKNOWLEDGMENTS We acknowledge Marsha Rhoads (University of Arkansas) for her assistance in sample collection and handling and J. Proudman (USDA, Beltsville, MD) for LH and FSH reagents. Avian pituitary hormones and antisera were obtained through the USDA-Agricultural Research Service Animal Hormone Program.
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tion protocol was used in egg-type male chickens, a significant increase in TW without changes in FSH or testosterone concentrations was observed (Lovell et al., 2000). After a maximum TW was obtained at wk 28, quadratic regressions showed that TW decreased over the next 7 wk until both treatments reached a plateau after 35 WOA (Figure 2). The decline in weight was more pronounced in broilerized males compared with control birds. The reason for the TW regression over time, as well as the differences between treatments, is not completely clear at this time. We hypothesize that decreased FSH concentrations might be responsible for TW regression (see below). The nonsignificant relationship between BW and TW in broilerized and control birds (Figure 4), suggests that the increased BW achieved by broilerized males was not responsible for the increased TW after photostimulation. In fact, the variation in TW at a given BW was extreme. For instance, cockerels with a BW within 4 to 5 kg had TW that varied from a minimum of 0.9 g to a maximum of 44 g. Despite the tremendous variation in TW, a close relationship between TW and DSPG was observed (Figure 5A). Our data strongly suggest that daily sperm production can be used to estimate TW of any given male. It appears that a TW of 8.6 g or more is required for the production of at least 7 to 8 × 107 sperm per gram of testis. Similarly, de Reviers and Williams (1984) reported that normal males released 8 to 12 × 107 sperm per day from the seminiferous epithelium into the lumen of the seminiferous tubules. The ability of the testis to produce sperm is regulated by the number of Sertoli cells per testis, and daily sperm production is correlated with the number of Sertoli cells (de Reviers and Williams, 1984; Sharpe, 1994). It is clear, at least in mammals, that FSH is the most important regulator of Sertoli cell proliferation and differentiation (Feigelson, 1986; Huhtaniemi et al., 1986). In addition, testosterone potentiates the effect of FSH on Sertoli cells in quails (Tsutsui and Ishii, 1978). Taken together, although sperm production depends on the number of Sertoli cells, the process is ultimately controlled by the hypothalamus-pituitary-gonadal axis. We found that plasma FSH concentrations accurately predicted TW (r = 0.95; Figure 5B). In contrast, plasma LH concentrations were not predictive of TW (r = 0.59; Figure 5B). Although the main effect of FSH is exerted upon Sertoli cells, LH affects mainly Leydig cells (Brown et al., 1975; Ishii and Yamamoto, 1976). Our data suggest that FSH has a profound effect in the variation of TW and sperm production. In fact, more than 90% of the variation on TW was explained by changes in FSH concentrations. We speculate that other factors such as chronic or acute stress, feed intake, social interactions, and subclinical diseases might also affect testicular development and sperm production. Testosterone, to a lesser degree, was also a good indicator of testis function (r = 0.92; Figure 5B). Chicken Leydig cells contain all of the enzymes that are need-
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