Research Note. The effects of incubation temperature on the sex of Japanese quail chicks

Transcription

1 Research Note The effects of incubation temperature on the sex of Japanese quail chicks A. Yılmaz, 1 C. Tepeli, M. Garip, and T. Çağlayan Department of Animal Science, Faculty of Veterinary Medicine, University of Selcuk, 42003, Selçuklu, Konya, Turkey ABSTRACT The effects of incubation temperature on the sex of Japanese quail chicks were investigated in this study. The study was conducted on Japanese quail. In all, 4500 eggs obtained from 2 generations were used. At the beginning of the study, a new flock was formed from available hatching eggs. Hatching eggs were gathered at 3 different ages (8 to 10 weeks, 16 to 18 weeks and 22 to 24 weeks of age) from the laying period in this flock. These eggs were exposed to 5 different incubation temperatures (36.7, 37.2, 37.7, 38.2, and 38.7 C). The hatching results were evaluated for each group. Chicks obtained from these temperature groups were reared separately to obtain quail for breeding. Eggs for incubation were gathered from these breeding quail when they were between 15 and 18 weeks of age. These eggs were placed in an incubator at a standard (37.7 C) temperature, separated by F 1 -generation temperature groups. The chicks in all groups were reared separately, and the sex of the chicks was determined at maturity. Statistical differences (P < 0.05) were found for the sex of the chicks in the third group (22 to 24 weeks) of the F 1 generation, compared with other groups. This result confirmed the hypothesis that different incubation temperatures for the first generation (at the embryo stage) might influence the sex of the next generation of chicks. Further studies are needed to investigate the effects of incubation temperature on chicks from different perspectives. Key words: incubation, temperature, sex determination, chick 2011 Poultry Science 90 : doi: /ps INTRODUCTION Some investigators (Fleming and Crews, 2001; Blumberg et al., 2002; Pace and Brenner, 2003; Juliana et al., 2004) have reported that the sex of turtles, birds, and some lizards in tropical areas is determined strictly according to genotype. Accordingly, they have concluded that incubation temperature, especially in birds and tropical reptiles, does not have a phenotypic effect on sex differentiation. In this respect, tropical reptiles differ from other reptiles. However, additional reports have suggested that enzyme and molecular factors related to incubation temperature may affect sex determination in poultry, reptiles, and especially in snakes (Graves and Shetty, 2001; Crews, 2003; Göth and Booth, 2005). For instance, it has been reported that different temperatures applied with different levels of the histidine triad (HIT), P450 arom, and P450 17α affected gene expression, and that expression of these genes influenced the development of different sexes (specifically, the rates of production of male and female embryos) (Dawson, 1998; O Neill et al., 2000; Pace and Brenner, 2003) Poultry Science Association Inc. Received March 9, Accepted June 19, Corresponding author: ayilmaz@selcuk.edu.tr Several studies of sex determination in poultry have been published in recent years (Ishimaru et al., 2008; Mattsson et al., 2008a,b; Alekseevich et al., 2009; Brunström et al., 2009; Lee et al., 2009; Schoenmakers et al., 2009; Smith et al., 2009). In studies of poultry, quail are commonly used because these birds are considered to represent appropriate experimental models; therefore, they serve as models for commercially produced poultry such as chicken and turkey (Takada et al., 2006; Mattsson et al., 2008a,b; Brunström et al., 2009). Conway and Martin (2000) have stated that embryonic development occurs without any defect as long as the air that surrounds the eggs during the incubation period remains within a critical temperature range (26 C to 40.5 C). These investigators also stated that any increase or decrease of the temperatures outside the critical range increases the death rate of the embryos. Appropriate incubation temperatures for many poultry species are reported to be from 37.0 to 37.8 C (Ensminger, 1992; Harvey, 1993; Woodard et al., 1993; Robbins, 1998). In some studies of poultry (Van Krey, 1993; Dawson, 1998; Kraak and Pen, 2002; Sundström, 2003; Miller et al., 2004), the data collected suggest that the sex of the individual becomes distinct in the first days of incubation, yet oogonia of the individuals differentiating as females develop during the last third of the incubation period. New data have been obtained 2402

2 on the mechanism of sex determination of egg-laying vertebrates, especially as the result of molecular studies (Crews et al., 1996; Willingham et al., 2000; Broderick et al., 2001; Gabriel et al., 2001; Milnes et al., 2002). These molecular studies suggest both similarities and differences in the sex determination mechanisms of egg-laying nonmammalian vertebrates and of mammals (Kraak and Pen, 2002; Miller et al., 2004). A recent report has produced the first evidence that incubation temperature has an effect on sex composition in the Australian brush turkey (Alectura lathami) (Göth and Booth, 2005). We are not aware of any other studies examining incubation temperature and its effect on sex determination in poultry. The purpose of this study was to investigate the possible effects on sex determination of different temperatures during the incubation of quail eggs. MATERIALS AND METHODS Experimental Materials In all, 4500 hatching eggs obtained from the quail were used in the study. To feed the birds, an appropriate ratio of chick growing feed to laying quail feed was determined according to the age and production period of the birds in the experiment. The feed used in the study was obtained from a commercial firm. Hatching eggs were obtained from a quail farm and were placed into an incubator. The chicks that hatched from the eggs were reared to 6 wk of age, after which a total of 160 female and 40 male quail were selected from this stock. These birds constituted the first maternal group (grandparents, F 0 ) in the study. The chicks in the parental (F 1 ) generation were kept in a brooder for 3 wk and then in rearing cages for 3 wk. This research was approved by The Ethics Committee of the Faculty of Veterinary Medicine, University of Selcuk (report no: 2009/73). RESEARCH NOTE Feeds Used and Feed Content During the first 4 wk, chick growing feed (24 to 28% crude protein, 2800 to 3000 kcal/kg metabolic energy) was used. Beginning with the 5th wk, laying quail feed (20% crude protein, 2800 kcal/kg metabolic energy) was used. Subjects were fed ad libitum, and the birds were always supplied with fresh water. Cages and Cabinets Twenty laying cages ( cm) (width depth height), multideck incubator brooders ( cm) (width depth height), a lower/upper deck battery ( cm), a fumigation room, and 5 incubators, each having a capacity of 768 quail eggs, were used. Eggs were gathered from standard cages, which were categorized according to groups, every morning at 07:00 and 08:00 and every afternoon at 16:00 and 17:00. All of the eggs were placed in a disinfection chamber and fumigated for 20 min by mixing 20 g potassium permanganate into 40 ml of 10% formalin solution per cubic meter of chamber. All eggs were then placed in the storage room at 15 to 16 C stable temperature and 65 to 70% relative humidity. Before the incubation period, eggs were gathered from breeding groups for a week (1 to 8 d) and stored. Experimental Design The general plan of the study is given in Table 1. Formation of the F 1 Generation 2403 From the first maternal flock, a total of 2250 hatching eggs were gathered, 750 per 3-wk period during weeks 8 to 10, 14 to 16, and 22 to 24. The eggs were used for the production of the parental (F 1 ) generation. The 750 hatching eggs were divided equally into 5 temperature Table 1. Experimental design 1 Grandparent (F 0 ) Parent (F 1 ) Offspring (F 2 ) Egg-gathering period Incubation temperature ( C) Egg number (n) Group Incubation temperature ( C) Egg number (n) 1st period (8 to 10 wk) 2nd period (16 to 18 wk) 3rd period (22 to 24 wk) Total eggs 2,250 2,250 1 Incubation humidity was adjusted to 65% for the first 14 d and to 75% between the 14th and 17th day.

3 2404 Yılmaz et al. Table 2. Incubation characteristics of hatching eggs and sex ratio and viability (%) of chicks (F 2, 3rd period) Group Item Significance Parental incubation temperature ( C) Incubation characteristic Hatching, all eggs (%) a ab ab b b * Hatching, fertile eggs (%) Fertility rate (%) a b ab b b * Early embryonic death ratio (%) Middle embryonic death ratio (%) Late embryonic death ratio (%) Total embryonic death ratio (%) Male ratio (%) a a b b b * Female ratio (%) b b a a a * Survival (to age 6 wk) (%) b a a a ab * a,b Differences among groups on the same line identified by different letters are significant (P < 0.05). *P < 0.05; = not significant. groups (36.7, 37.2, 37.7, 38.2, and 38.7 C). These incubation temperature groups were maintained separately thereafter. The quail chicks hatched from eggs of different temperature groups at different periods (8 to 10, 16 to 18, and 22 to 24 wk old) were raised to form the breeding members of the parent (F 1 ) generation. Formation of the F 2 Generation By placing 32 female and 8 male quail from the same temperature group into each breeding cage, 2250 hatching eggs were obtained from 160 females and 40 males for each period. A total of 480 females and 120 males were bred for 3 periods to produce the F 2 generation. From each cage, 150 eggs were gathered from each temperature group, marked, and placed into an incubator. For the production of the offspring (F 2 ) generation, the incubation temperature was set at 37.7 C; however, the eggs produced by different temperature groups during the previous incubation were marked and used for this incubation cycle. Quail hatched separately from different parental temperature groups were raised to the age of 5 to 6 wk. The sex of the individuals in the offspring (F 2 ) generation was assessed based on the results of tests involving chest feathers and cloaca, and was recorded according to the parental period and temperature group. Classification of Incubation Results The number of chicks hatched from the eggs in the parental (F 1 ) and offspring (F 2 ) generations, the number of embryo deaths associated with various periods, the number of infertile eggs, and the number of male and female chicks (4 to 5 wk old) represented the raw data for the study. Statistical Analysis Data on incubation and sex consisted of numerical counts and were therefore analyzed using the Chisquared test. The SPSS (2006) statistical package was used for the analysis of the data. RESULTS AND DISCUSSION Males were significantly more common (P < 0.05) during the embryonic period in parent quail eggs exposed to 36.7 C and 37.2 C (Table 2, Figure 1). The critical temperature is known for incubation in reptiles because both sexes hatch at nearly the same ratio (Doody et al., 2004; Doody et al., 2006; Lance, 2008). This temperature varies according to the species of reptile considered, and it is equal to 37.7 C in poultry. The present study found an increase in favor of females starting at the temperature of 37.7 C at 22 to 24 wk for the F 1 quail offspring (P < 0.05). In the offspring (F 2 ) generation, no statistical differences in the sex ratio were found for other periods. This result may be a consequence of the fact that the eggs in the parental (F 1 ) generation that were exposed to different temperatures were gathered from different grandparental (F 0 ) age groups. Offspring (F 2 ) were produced at a standard incubation temperature (37.7 C) from the eggs obtained from the groups consisting of these parents (F 1 ). However, parental (F 1 ) incubation temperatures had no statistically significant effect on the sex ratio (Table 2, Figure 1). This evidence suggests that grandparental (F 0 ) age can be a significant factor in sex determination as well (P < 0.05). Our research has found that temperature effects on eggs obtained from older-aged grandparents are reflected in the sex composition of the offspring (F 2 ) generation. These results are in agreement with the findings of Velando et al., (2002), Young and Badyaev (2004) and others that the sex composition of offspring in certain types of birds reflects maternal history. Our findings are consistent with the statement by Crews and Bull (2009) that specific chromosomal differences, together with temperature and male or female dominance, affect genes that influence sex composition. In addition, the internal organs of parents (F 1 ) reared for breeding may

4 RESEARCH NOTE 2405 Figure 1. Male and female ratios according to maternal incubation temperatures (F 2, 3rd period). Incubation temperature: top row, offspring (F 2 ) generation temperature was 37.7 C; bottom row, parent (F 1 ) generation incubation temperatures. Color version available. reflect the temperature applied during the embryonic period. Recent research on sex composition in poultry (Velando et al., 2002; Young and Badyaev, 2004; Hoshino et al., 2005; Takada et al., 2006; Endo et al., 2007; Ishimaru et al., 2008; Mattsson et al., 2008a; Mattsson et al., 2008b; Alekseevich et al., 2009; Brunström et al., 2009; Lee et al., 2009; Schoenmakers et al., 2009; Smith et al., 2009) has suggested the idea that sex composition may reflect the operation of different mechanisms from those traditionally invoked to explain the phenomenon. We also found that when the parents (F 1 ) had been incubated at 38.7 C as eggs, the feathers observed over most of their body were not of a mature type, and some regions of the body such as back of the neck, abdominal region and back of the body were not feathered in maturity. The research reported here indicates that it is possible to hatch chicks below or above the standard incubation temperature (37.7 C) if the age of the grandparents at egglaying is taken into account (F 0 ). However, because low and high temperatures can cause embryonic deaths, commercial firms should include a cost analysis when considering an application of this sort. In addition, further studies should be conducted to find ways of lowering the embryonic death ratio. Conclusions The effects on sex composition of differences in the temperatures applied to the quail eggs during incubation were apparent in the offspring (F 2 ) generation. In addition, the age of grandparents (F 0 ) might have indirect effects on the sex composition of the F 2 -generation chicks. This research further suggests that interdisciplinary studies on sex differentiation in poultry can improve the level of research on the topic. Furthermore, evaluation of the incubation results obtained at different temperature applications suggests opportunities for further detailed studies of the effects of incubation temperature. ACKNOWLEDGMENTS This research was supported by TÜBİTAK (The Scientific and Technological Research Council of Turkey), TOVAG (Agriculture, Forestry & Veterinary Research Grant Committee), Project no: 106 O 048 and Selcuk University Research Foundation as Project no: A portion of the data in this paper was previously presented as a poster presentation at the XIIIth European Poultry Conference in Tours, France, August 23 27, A portion of the study was published in abstract form in the conference proceedings (Yilmaz et al., 2010). REFERENCES Alekseevich, L. A., N. A. Lukina, N. S. Nikitin, A. A. Nekrasova, and A. F. Smirnov Problems of sex determination in birds exemplified by gallus gallus domesticus. Russ. J. Genet. 45: doi: /s Blumberg, M. S., S. J. Lewis, and G. Sokoloff Incubation temperature modulates post-hatching thermoregulatory behavior in the Madagascar ground gecko, Paroedura pictus. J. Exp. Biol. 205: Broderick, A. C., B. J. Godley, and G. C. Hays Metabolic heating and the prediction of sex ratios for Green Turtles (Chelonia mydas). Physiol. Biochem. Zool. 74:

5 2406 Yılmaz et al. Brunström, B., J. Axelsson, A. Mattsson, and K. Halldin Effects of estrogens on sex differentiation in Japanese quail and chicken. Gen. Comp. Endocrinol. 163: Conway, C. J., and T. E. Martin Effects of ambient temperature on avian incubation behavior. Behav. Ecol. 11: Crews, D., P. Coomber, R. Baldwin, N. Azad, and F. G. Lima Brain organization in a reptile lacking sex chromosomes: Effects of gonadectomy and exogenous testosterone. Horm. Behav. 30: Crews, D Sex determination: Where environment and genetics meet. Evol. Dev. 5: Crews, D., and J. J. Bull Mode and tempo in environmental sex determination in vertebrates. Semin. Cell Dev. Biol. doi:doi: /j.semcdb Dawson, A Natural and anthropogenic environmental oestrogens: The scientific basis for risk assessment Comparative reproductive physiology of nonmammalian species. Pure Appl. Chem. 70: Doody, J. S., A. Georges, and J. E. Young Determinants of reproductive success and offspring sex in a turtle with environmental sex determination. Biol. J. Linn. Soc. Lond. 81:1 16. Doody, J. S., E. Guarino, A. Georges, B. Corey, G. Murray, and M. Ewert Nest site choice compensates for climate effects on sex ratios in a lizard with environmental sex determination. Evol. Ecol. 20: doi: /s Endo, D., S. Murakami, Y. Akazome, and M. K. Park Sex difference in Ad4BP/SF-1 mrna expression in the chick-embryo brain before gonadal sexual differentiation. Zool. Sci. 24: doi: /zsj Ensminger, M. E Chapter 3: Incubation and brooding. Pages in Poultry Science. Interstate Publ. Inc., Danville, IL. Fleming, A., and D. Crews Estradiol and incubation temperature modulate regulation of steroidogenic factor 1 in the developing gonad of the Red-Eared Slider Turtle. Endocrinology 142: Gabriel, W. N., B. Blumberg, S. Sutton, A. R. Place, and V. A. Lance Alligator aromatase cdna sequence and its expression in embryos at male and female incubation temperature. J. Exp. Zool. 290: Göth, A., and D. T. Booth Temperature-dependent sex ratio in a bird. Biol. Lett. 1: Graves, J. A. M., and S. Shetty Sex from W to Z: Evolution of vertebrate sex chromosomes and sex determining genes. J. Exp. Zool. 290: Harvey, R Practical Incubation. Hancock House Publ., Blaine, WA. Hoshino, A., M. Koide, T. Ono, and Y. Sadao Sex-specific and left-right asymmetric expression pattern of Bmp7 in the gonad of normal and sex-reversed chicken embryos. Dev. Growth Differ. 47: Ishimaru, Y., T. Komatsu, M. Kasahara, Y. Katoh-Fukui, H. Ogawa, Y. Toyama, M. Maekawa, K. Toshimori, R. A. S. Chandraratna, K. Morohashi, and H. Yoshioka Mechanism of asymmetric ovarian development in chick embryos. Development 135: doi: /dev Juliana, J. R. S., R. M. Bowden, and F. J. Janzen The impact of behavioral and physiological maternal effects on offspring sex ratio in the common snapping turtle, Chelydra serpentina. Behav. Ecol. Sociobiol. 56: Kraak, S. B. M., and I. Pen Chapter 7: Sex determining mechanisms in vertebrates. Pages in Sex Ratios: Concepts and Research Methods. I. C. W. Hardy, ed. Cambridge Univ. Press, Cambridge, UK. Lance, V. A Is regulation of aromatase expression in reptiles the key to understanding temperature-dependent sex determination? J. Exp. Zool. 309A. doi: /jez.465. Lee, S. I., W. K. Lee, J. H. Shin, B. K. Han, S. Moon, S. Cho, T. Park, H. Kim, and J. Y. Han Sexually dimorphic gene expression in the chick brain before gonadal differentiation. Poult. Sci. 88: doi: /ps Mattsson, A., E. Mura, B. Brunsttröm, G. Panzica, and K. Halldin. 2008a. Selective activation of estrogen receptor alpha in Japanese quail embryos affects reproductive organ differentiation but not the male sexual behavior or the parvocellular vasotocin system. Gen. Comp. Endocrinol. 159: Mattsson, A., J. A. Olsson, and B. Brunström. 2008b. Selective estrogen receptor a activation disrupts sex organ differentiation and induces expression of vitellogenin II and very low-density apolipoprotein II in Japanese quail embryos. Reproduction 136: Miller, D., J. Summers, and S. Silber Environmental versus genetic sex determination: A possible factor in dinosaur extinction? Fertil. Steril. 81: Milnes, M. R., R. N. Roberts, and L. J. Guillette Jr Effects of incubation temperature and estrogen exposure on aromatase activity in the brain and gonads of embryonic alligators. Environ. Health Perspect. 110: O Neill, M., M. Binder, C. Smith, J. Andrews, K. Reed, M. Smith, C. Millar, D. Lambert, and A. Sinclair ASW: A gene with conserved avian W-linkage and female specific expression in chick embryonic gonad. Dev. Genes Evol. 210: Pace, H. C., and C. Brenner Feminizing chicks: A model for avian sex determination based on titration of Hint enzyme activity and the predicted structure of an Asw-Hint heterodimer. Genome Biol. 4:R18.6 Robbins, G. E. S Partridges & Francolins, Their Conservation, Breeding and Management. World Pheasant Assoc., Reading, UK. Schoenmakers, S., E. Wassenaar, J. W. Hoogerbrugge, J. S. E. Laven, J. A. Grootegoed, and W. M. Baarends Female meiotic sex chromosome inactivation in chicken. PLoS Genet. 5:e Smith, C.A., K.N. Roeszler, T. Ohnesorg, D.M. Cummins, P.G. Farlie, T.J. Doran, and A.H. Sinclair The avian Z-linked gene DMRT1 is required for male sex determination in the chicken. Nature 461: SPSS SPSS Release Statistical packet program, SPSS for Windows. SPSS Inc., Chicago, IL. Sundström, H Mutation and diversity in avian sex chromosomes, PhD Diss. Department of Evolutionary Biology, Uppsala University, Uppsala, Sweden. Takada, S., J. Ota, N. Kansaku, H. Yamashita, T. Izumi, M. Ishikawa, T. Wada, R. Kaneda, Y. Lim Choi, K. Koinuma, S. Fujiwara, H. Aoki, H. Kisanuki, Y. Yamashita, and H. Mano Nucleotide sequence and embryonic expression of quail and duck Sox9 genes. Gen. Comp. Endocrinol. 145: Van Krey, H. P Reproductive biology in relation to breeding and genetics. Pages in Poultry Breeding and Genetics. R. D. Crawford, ed. Elsevier Sci. Publ., Amsterdam, the Netherlands. Velando, A., J. Graves, and J. Ortega-Ruano Sex ratio in relation to timing of breeding, and laying sequence in a dimorphic seabird. Behav. Ecol. 144:9 16. Willingham, E., R. Baldwin, J. K. Skipper, and D. Crews Aromatase activity during embryogenesis in the brain and adrenal kidney gonad of the Red-Eared Slider Turtle, a species with temperature-dependent sex determination. Gen. Comp. Endocrinol. 119: Woodard, A. E., P. Vohra, and V. Denton Commercial and Ornamental Game Bird Breeders Handbook. Hancock House Publ., Blaine, WA. Yilmaz, A., C. Tepeli, M. Garip, and T. Caglayan The effects of incubation temperature on sex of chicks. World s Poult. Sci. J. 66(Suppl.):489. (Abstr.) Young, R. L., and A. V. Badyaev Evolution of sex-biased maternal effects in birds: I. Sex-specific resource allocation among simultaneously growing oocytes. J. Evol. Biol. 17: