COMBINING ABILITY OF QUAIL LINES 1849 Hayman, B. I., 1957. Interaction, heterosis and diallel crosses. Genetics, 42: 336-355. Kempthorne, O., 1956. The theory of the diallel cross. Genetics, 41:451-459. Sprague, G. F., and L. A. Tatum, 1942. General versus specific combining ability in single crosses of corn. J. Amer. Soc. Agron. 34: 923-932. Wearden, S., D. Tindell and J. V. Craig, 1965. Use of a full diallel cross to estimate general and specific combining ability in chickens. Poultry Sci. 65: 1043-1053. Yao, T. S., 1961. Genetic variations in the progenies of the diallel crosses of inbred lines of chicken. Poultry Sci. 40: 1048-1062. Photoacceleration of Embryonic Development in Depigmented Japanese Quail Eggs MARILYN A. COLEMAN ' AND ROGER A. MCNABB Department of Biology, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061 INTRODUCTION ENVIRONMENTAL factors, such as light and temperature influence the rate of development of the chick embryo. Eggs incubated under white light hatch approximately one day earlier than those incubated under colored light (Shutze et al., 1962). Siegel et al. (1969) found that during the first week of incubation light had a greater effect than did light exposure during the second or third week, and that the acceleration of development was apparent by 10 hours of incubation. Kallen and Rudenberg (1964) observed abnormal development during the first and second days of incubation of embryos irradiated through a window in the shell. Abnormal embryogenesis and delayed hatching oc- 1. Current address: Poultry Science Dept., Auburn University, Auburn Alabama 36830 (Received for publication January 13, 1975) ABSTRACT Pigmented and depigmented Japanese quail eggs were incubated either in the presence of a light source for one week or in the dark. Acceleration of embryonic development was apparent by the first day of incubation in the depigmented eggs exposed to light. Embryonic weights of eggs exposed to light were significantly (P <.05) greater than eggs incubated in the dark by the fourth day of incubation. Air cell temperatures of depigmented eggs were significantly (P <.05) lower than pigmented eggs by the fourth day of incubation when both groups of eggs were exposed to a light source. Thus photoacceleration appeared to be a real effect, and great enough to overcome the lower egg temperature at which these embryos developed. Embryos developing in lighted depigmented eggs can decrease the hatching time of other groups incubated with them. POULTRY SCIENCE 54: 1849-1855, 1975 curred when incubation was conducted under either continuous or intermittent light (Tamimie and Fox, 1967). Isakson et al. (1970) showed that, 1) there were developmental differences in White Rock embryos incubated in darkness and under various intensities of incandescent light, 2) exposure to light interfered with normal morphogenesis in vitro, and 3) temperatures within culture dishes were not affected by irradiation. These observations suggest that the phenomenon observed in vitro is due to light energy rather than heat energy. Walter and Voitle (1972) showed that the stimulation of embryonic development in broilers is directly related to the length of time of irradiation. They found no differences in embryo weights until 12 days of incubation. Chick weights at hatching and at 4 and 8 weeks of age were not significantly different. They found that embryos exposed to light
1850 M. A. COLEMAN AND R. A. MCNABB hatched at 18 days of incubation, with no apparent deleterious effects on subsequent performance. Cooper (1972) found that hatchability of turkey eggs was improved by irradiation during the hatching period. He also reported that more frequent abnormalities and mortalities were associated with irradiation during incubation. Vince (1966) demonstrated that quail tended to hatch at the same time, if incubated together. Coordination of hatching times has been shown to be affected by vibration (Vince, 1966) and noises associated with lung ventilation (Vince et al., 1970). Vince et al. (1970) showed also that lung ventilation noises affected coordinated hatching in Japanese quail, but not in Bobwhite quail; however, pipping vibrations affected both species. This study was conducted to determine the role of eggshell pigments in screening Japanese quail embryos from external light. The effect of photoacceleration on synchrony of hatching also was determined. METHODS AND MATERIALS Eggs from Japanese quail (Coturnix coturnix japonica) were acquired from the Poultry Science Department of Virginia Polytechnic Institute and State University. All the eggs within a replicate were laid within a 4-hour span and were collected and used the same day. Eggs were paired according to size and pigmentation (both background and spot pattern, see Fig. 1). Eggs without a mate were not used in this study. One egg of each pair was depigmented totally by rubbing with tepid tap water in a teflon sponge. Eggs were never immersed. Control eggs (pigmented) were treated in the same manner on the pointed end of the egg only. Thus, control eggs were subject to the effects of washing, but the depigmented area was not exposed to light. All eggs were sprayed with a disinfectant and placed in open exposed 'o light FIG. 1. Method of selection of eggs used in this study. egg trays, pointed end downward. All eggs were incubated in the same incubator during the first 12 days of development, but two incubators were used during the last days of development for the hatching experiment. Eggs were placed in the different incubators at random. The temperature and relative humidity were the same in all three incubators (39 C, 90% R. H., respectively). Experiment 1. la) To investigate the role of egg shell pigments in the light, paired pigmented and depigmented eggs were placed in the presence of a lighted 60 watt, internally frosted, incandescent bulb placed 18 cm. from the top of the eggs. The bulb was mounted in an aluminized reflector to reduce stray light. No egg was further than 22 cm. from the surface of the light bulb, so that light intensities at the surfaces of the eggs ranged from 140-80 foot-candles (0.8-0.6 x 10 5 ergs/cm. 2 -sec).
PHOTOACCELERATION OF EMBRYO DEVELOPMENT 1851 At each 24-hour interval throughout the first 6 days of incubation pairs of eggs were opened and embryonic development compared using anatomical parameters such as somite number according to a modified Lilly technique (Hamilton, 1952). Representative drawings were made at each interval for each group. There were 6 replicates of each treatment and the experiment involved 250 eggs. lb) In this experiment additional controls were used to determine whether the effect demonstrated in la) was because of the light or because of the pigmentation removal. Pairs of pigmented and depigmented eggs were placed either in the dark or under a light as in la). At each 24-hour interval pairs of eggs from the light and dark were opened and the embryos compared. The embryos were weighed to the nearest.01 mg. on a Mettler balance. Embryonic age was determined by anatomical measurements according to a modified Lilly technique, as above. Experiment 2. Six pairs of eggs were pretreated as previously described in Experiment la). A36ga. copper-constantan thermocouple was inserted and cemented in the air cell of each egg. Air cell temperatures were measured at 12 intervals each day throughout the first 6 days of development in the light. The ages of the embryos were determined by anatomical observation on the last day. Experiment 3. One thousand eggs were selected and pretreated as described in Experiment lb) above (500 pigmented and 500 depigmented). All eggs were exposed to the two conditions of Experiment lb) for 6 days. For the remaining 12 days of the incubation period, all eggs were incubated in the dark. For these remaining 12 days, groups of eggs were placed in close proximity to determine whether photoaccelerated chicks could coordinate hatching time of other embryos. The groups were arranged as follows: 1) all eggs incubated in the dark (pigmented and depigmented), 2) all eggs incubated in the light (pigmented and depigmented), 3) all depigmented eggs (both light exposed and dark), 4) all pigmented (both light exposed and dark) and 5) a group that contained all four groups of eggs in the experiment. Eggs were checked at 6-hour intervals during the day for signs of pipping. Hatching time was determined to be when the chick first left the egg. Any chick that hatched between 6 p.m. and 6 a.m. was considered hatched at 6 a.m. If any chicks were not out of the eggs within 36 hours after the first chicks in that group hatched they were considered not hatched. Preliminary studies showed that if the chick had not shed the egg shell within 36 hours after the clutch started hatching then it would not live very long after "hatching." Data was analyzed either by a student's t test or by analysis of variance and a Duncan's multiple range test (Steele and Torrie, 1960). RESULTS Experiment I. a) Anatomical differences were detectable within the first 24-hour period of development. Embryos from depigmented eggs developed faster than those from pigmented eggs, although both were given the same amount of light. Figure 2 is a drawing of representative embryos at each 24-hour period throughout the first 5 days of development. There was no increase in abnormalities in the depigmented eggs. b) Embryos in depigmented eggs exposed to light developed significantly faster by anatomical criteria than those in pigmented eggs in the light, but no such differences (P <. 10) were observed between embryos of pigmented and depigmented eggs in the dark. Embryos in pigmented eggs exposed to the light did not develop significantly faster than those of pigmented or depigmented eggs maintained in darkness. The age in hours of
1852 M. A. COLEMAN AND R. A. MCNABB 5 O Q UJ 5 O DAYS OF H INCUBATION FIG. 2. Embryos from pigmented (top row) and depigmented (bottom row) eggs incubated in the presence of a light. At each day drawn, the embryos from depigmented eggs were more advanced in embryonic development, as shown by the following characteristics for each day: Day 1 cephalic development more noticeable, size of the primitive streak decreased Day 2 curviflexure, somite number, posterior neuropore closed Day 3 development of the head region, somite number, pharyngeal pouch advancements, and limb buds Day 4 fusion of the somites, development of the limb buds, pharyngeal pouch advancements Day 5 digitation of the limb buds, beak. the 4 groups of embryos throughout the first week of development are shown in Fig. 3. Although the embryos in depigmented eggs exposed to light developed significantly faster by the first 24-hour observation, differences in weight between those and the embryos in pigmented eggs in the light were not significant (P <. 10) until the 4th day of incubation. Even though the pigmented eggs in the light were not significantly more developed than those in the dark, there was a significant difference (P <.05) in weight by Day 4 of incubation. The weights of the four groups of embryos throughout the first week of incubation are shown in Fig. 4. Experiment 2. Fig. 5 demonstrates that although embryos in depigmented eggs developed at a faster rate than embryos in pigmented eggs, they had a significantly (P <.05) lower air cell temperature by Day 4 than pigmented eggs exposed to light. Embryos from depigmented eggs were 174 ± 2 hours old, whereas embryos from pigmented eggs were only ± 2 hours old at the end of the experiment. Experiment 3. Groups containing depigmented eggs exposed to the light hatched earlier than any other groups (Table 1). When all the eggs were hatched together the eggs hatched in 16 days, which is still earlier than normal for Japanese quail. Depigmented eggs exposed to light hatched at 15 days in any
PHOTOACCELERATION OF EMBRYO DEVELOPMENT 1853 200 3 o x 100 5? 50 T" 1 1 PIGMENTED O «light dark DEPIGMENTED A A light dark * /./ / g 4r '' jr / // i i i - 1 2 3 4 5 DAYS OF INCUBATION A? J^ 1 1 1 FIG. 3. Developmental age (in hours) of the embryos from pigmented and depigmented eggs exposed to the light and in the dark. Embryos from depigmented light exposed eggs are significantly (P <.05) more developed by day 3 of incubation. Standard errors are included within the symbols. two-egg group. Hatching of pigmented eggs exposed to light was accelerated when adjacent to depigmented eggs exposed to the light, but did not themselves speed up the pigmented eggs in the dark. Since the different groups hatched from 15 days to 18 days depending upon their grouping, it shows that pigmented and depigmented eggs exposed to light, or incubated in the dark, show the coordinated hatching demonstrated by Vince (1966) and Vince et al. (1970). DISCUSSION As Figure 3 demonstrates, light accelerated embryonic development of Japanese quail only after depigmentation. Light stimulated growth as shown in Figure 4, but not differentiation, in the pigmented eggs. Most previously studied eggs showed photoacceleration without changing shell background ' < on O.3 PIGMENTED O - - light dark DEPIGMENTED A A light dark 2 3 4 5 DAYS OF INCUBATION FIG. 4. Weight of embryos in grams from pigmented and depigmented eggs exposed to the light and in the dark. Embryos from light exposed eggs were significantly heavier (P <.05) by day 4 of incubation, than were embryos from eggs incubated in the dark. Standard errors are included within the symbols. pigmentation (Walter and Voitle, 1972) or spots (Cooper, 1972). Anatomical differences were apparent within 24 hours of incubation, but significant weight differences were not noted until 4 days of incubation. This compares favorably with results of Siegel et al. (1969) who were able to see differences within 10 hours of incubation in the light, but could not measure them until somite formation. Walter and Voitle (1972) found no differences in embryo weights until 12 days of incubation. This is probably due to the low lighting intensity which they used [only 10-30 footcandles, which is far below the intensity of 104 footcandles found by Isakson et al. (1970) to be most effective].
1854 M. A. COLEMAN AND R. A. MCNABB 40.0 O 39.5 < 39.0 38.5 38.0 37.5 Total no. eggs 400 A PIGMENTED O DEPIGMENTED 1 2 3 4 DAYS OF INCUBATION FIG. 5. Air-cell temperatures of pigmented and depigmented eggs incubated in the presence of a light. Air-cell temperature of depigmented eggs was significantly (P <.05) lower by 4 days of incubation. Below 104 footcandles illumination did not give the maximum "Light Effect" while at light intensities above this threshold embryonic abnormalities and mortalities were prevalent. No increase in embryonic mortality resulting from irradiation was found, in contrast to the works of Tamimie and Fox (1964) and Cooper (1972). The intensities used by these authors were far above the range used by Isakson et al. (1970). Light acts as energy per se, since in the light, pale depigmented eggs were significantly cooler (P <.05) than their pigmented, less reflective counterparts. Isakson et al. (1970) and Siegel et al. (1969) found that there was no differences between air cell temperatures in the light and dark. Depigmented and pigmented eggs incubated in the light showed normal coordinated hatching. It appears that the accelerating factors in the groupings are the embryos in the depigmented, lighted eggs, since these always hatch earlier no matter how they are grouped. Because Japanese quail are peculiar in their response to light, it would appear that further biophysical studies on the egg shells themselves would reveal the wavelengths of light that are active in the acceleration of embryonic development in the avian embryos. Since embryos from pigmented eggs are larger (Fig. 4) but not more developed (Fig. 3) in the lighted conditions, it would appear that there are two active wavelengths of light one affects growth, and the other differentiation. Pigmentation, which has been customarily TABLE 1. Changes in Japanese quail hatching times with different treatments Treatments All eggs hatched together Eggs from dark treatment hatched together Eggs from light treatment hatched together All pigmented eggs hatched together Type of eggs included Pigmented (L) Depigmented (L)' Pigmented (D) Depigmented (D) 2 Pigmented (D) Depigmented (D) Pigmented (L) Depigmented (L) Pigmented (L) Pigmented (D) All depigmented eggs hatched Depigmented (L) together Depigmented (D) 1 Eggs exposed to light during the first six days of incubation. 2 Eggs incubated in darkness throughout incubation. Average hatching time (days) 16 18 15 17 15
PHOTOACCELERATION OF EMBRYO DEVELOPMENT 1855 thought to serve as a camouflage, actually may serve as a light screen. Animals which are subjected to high intensity light (such as desert animals) are protected internally by a black peritoneal lining. Egg shell pigmentation may serve the same purpose. Quail in normal populations have a large deposit of egg shell pigments, but from their wide usage as laboratory animals a strain of quail has been developed which has unpigmented eggs. These unpigmented quail eggs offer yet another area for study of photoacceleration. ACKNOWLEDGMENTS We would like to thank P. B. Siegel of the Poultry Science Dept., V.P.I. & S.U. for the supply of quail eggs, Clete Sellers for drawings of the embryos, Ann Dumper, and Carol Luckner for technical assistance. REFERENCES Cooper, J. B., 1972. Effect of light during incubation on hatchability of turkey eggs. Poultry Sci. 51: 1105-1109. Hamilton, H. C, 1952. Lillie's Development of the Chick. 3rd Ed., Henry Holt and Co., New York pp. 78-91. Isakson, S. T., B. J. Huffman and P. B. Siegel, 1970. Intensities of incandescent light and the develop- F.D.A.-A.P.H.I.S. NOTES The Food and Drug Administration and the Animal and Plant Health Inspection Service have signed a "Memorandum of Understanding" calling for fuller cooperation between the two agencies in monitoring animal feeds and animal products for residues. The two agencies will exchange information more readily and generally cooperate more extensively. C.B.E. NOTES NEWS AND NOTES The newly installed Board of Directors of the Council of Biology Editors for 1975-1976 are: Chairment of chick embryos in ovo and in vitro. Comp. Biochem. Physiol. 34: 229-235. Kallen, J. K., and S. I. Rudenberg, 1964. Teratogenic effects of electric light on early chick embryos. Acta. Morphol. Neer Scand. 6: 95-99. Lauber, J. K., and J. V. Shutze, 1964. Accelerated growth of embryo chicks under the influence of light. Growth, 28: 179-190. Shutze, J. V., J. K. Lauber, M. Kato and W. Wilson, 1962. Influence of incandescent light and colored light on chick embryos during incubation. Nature, 96: 594-595. Siegel, P. B., S. T. Isakson, F. N. Coleman and B. J. Huffman, 1969. Photoacceleration of development in chick embryos. Comp. Biochem. Physiol. 28: 753-758. Steel, R. G., and J. H. Torrie, 1960. Principles and Procedures of Statistics, McGraw-Hill, New York, pp. 67-128. Tamimie, H. S., and M. W. Fox, 1967. Effect of continuous and intermittent light exposure on embryonic development of chicken eggs. Comp. Biochem. Physiol. 20: 793-799. Tyler, C, 1969. Avian egg shell: Their structure and characteristics. Int. Rev. Gen. Exp. Zool. 4: 81-124. Vince, M. A., 1966. Artificial acceleration of hatching in quail embryos. Anim. Behav. 14: 389-394. Vince, M. A., J. Green and S. Chinn, 1970. Changes in the timing of lung ventilation and late foetal development in quail. Comp. Biochem. Physiol. 39: 769-785. Walter, J. H., and R. A. Voitle, 1972. Effects of photoperiod during incubation on embryonic and post embryonic development of broilers. Poultry Sci. 51: 1122-1126. man Margaret Broadbent, Chairman-Elect Eli Chernin, Past-Chairman Karl F. Heumann, Secretary David Frost, Treasurer Sheila McGough, and Directors Lois DeBakey, Stephen Geiger and Erwin Neter. N.F.P.A. NOTES The National Fire Protection Association has announced that official approval has been given by the American National Standards Institute to the 1975 edition of the National Electrical Code. The National Electrical Code now bears the dual (Continued on page 1874)