Thyroxine and duodenal development in chicken embryos LINDAM . BREWER A N D T. W. BETZ
Can. J. Zool. Downloaded from www.nrcresearchpress.com by University of Auckland on 12/05/14 For personal use only.
Department ofBiology, Crrrleton University, Otrnwt~,O n f . ,Canoda KIS 5B6 Received July 17. 1978
BREWER,L. M . , and T . W. BETZ. 1979. Thyroxine and duodenal development in chicken embryos. Can. J . Zool. 57: 416-423. To test whether thyroxine (T,) can stimulate duodenal development in chickens, it was administered to 16.5-day-old embryos (strain Hyline 934F ofGullus gallus) (approximately stage 42). Treatment time was closer to the prehatching phosphatase increase, and the single dosage (5 pg) larger than before. Control groups received the following: (1) D-T, for possible physicochemical variables, (2) windowed shells and saline vehicle, (3) windowed shells or (4) no treatment. Subsequent accumulation of duodenal alkaline phosphatase activity (EC 3.1.3.1) and protein content were monitored as developmental parameters (n = 8-43). Yolk sac retraction, distl-ibution of developmental stages, and mortality were used as indices of maturation and survival. L-Thyroxine tl-eatment increased duodenal alkaline phosphatase significantly ( p < 0.01) within 48 h, 24 h sooner than normal. This precocious increase was transient inembryos treated at stage 41. Duodenal protein content was elevated significantly in stage 43 embryos treated with L - T ~but , it was not different from control values at later stages. Pooling of time data did not greatly alter duodenal parameters. Yolk sac retraction and shell pipping began sooner in embryos treated with L-T4 than control embryos, however, distribution of developmental stages was unaltered. Increased mortality was found 72 h after L-T4 treatment. After D-T, treatment no parameter was significantly different. The parameters responding after treatment may be stimulated by thyroxine during development. BREWER,L. M., et T. W. BETZ. 1979. Thyroxine and duodenal development in chicken embryos. Can. J . Zool. 57: 416-423. L'administration de thyroxine (T,) a des embryons de poulets de 16.5 jours (souche Hyline 934F de Gallus gallus, au stade 42 environ) a permis de deceler son effet sur le developpement du duodenum. Comparativement aux travaux anterieurs, le traitement comportait une dose unique plus importante (5pg) et a ete effectue b un moment plus rapproche d e I'augmentation d e phosphatase qui precede I'eclosion. Les groupes-temoins ont reGu les traitements suivants: (I) D-T4 pour deceler les variables physicochimiques possibles, (2) des coquilles percees et une solution saline en guise de solvant, (3) des coquilles percees et (4) aucun traitement. L'accumulation subsequente de phosphatase alcaline dans le duodenum (EC 3.1.3.1) et le contenu en proteines ont servi de parametres (n = 8-43). Le retrait du sac vitellin, la distribution des stades de developpement et la mortalite ont servi d'indices de maturation et de survie. Le traitement a la L-thyroxine augmente la phosphatase alcaline du duodenum d e f a ~ o nsignificative @ < 0.01) en moins de 48h, soit 24h plus t8t qu'il n'est normal. Cette augmentation precoce est tempordire chez les embryons traites au stade 41. Le contenu en proteines du duodenum est augmente significativement chezdes embryons traites a la L - T au ~ stade 43, mais les valeurs ne diffierent pas de celles qu'on enregistre chez les temoins a des stades plus avances. Le groupement des donnees par rapport au temps ne modifie pas beaucoup les parametres du duodenum. Le retrait du sac vitellinet le bris de lacoquille commencent plus t8t chezles embryons traites a l a ~ - que T ~ chez les embryons-temoins; cependant, la distribution des stades de developpement reste la mkme. Soixante-douze heures apres le traitement la L - T ~la, mortalite est augmentee. Les parametl-es ne subissent toutefois pas de changement significatif a la suite d'un traitement a la D-T4. Les parametres qui sont affectes par le traitement peuvent i t r e stimules par la thyroxine au cours du developpement. [Traduit par le journal]
Introduction The terminal phase of duodenal development in chicken embryos is marked by dramatic enzymic and structu~tlchanges. During the 5 days prior to hatching alkaline phosphatase (EC 3.1.3.1) accumulation increases sharply (Moog 1950). paralleling develaprnent of the microvillar surface of epithelial cells (Overten and Shoup 1964). It is accepted that the embryonic ndenohypophysis stimulates this phase of duodenal development
(Hinni and Watterson 1963; Bellware and Betz 1970; Hart and Betz 19720, 1972b). The role of the thyroid glands i s as yet unclear. Thiourea treatment inhibits the sharp increase in alkaline phosphatase. and this effect can be reversed by thyroxine trearment (Moog 1961). Precocious increase in alkaline phosphatase activity can be induced by cortisone (Moog and Richardson 1955). Moog (1961) found that thyroxine did not produce this effect. while others have indicated that it can elevate phos-
0008-4301/79/0204 16-08$01 .OO/O @I979 National Research Council of Canada/Conseil national de I-echerchesdu Canada
Can. J. Zool. Downloaded from www.nrcresearchpress.com by University of Auckland on 12/05/14 For personal use only.
BREWER AND BET2
phatase (Betz and Mallon 1970; Mallon 1972). Moog (1961)suggested that thyroxine is permissive in the action of corticoids as cortisone treatment did not alleviate the effects of thiourea. Whether thiourea acts on duodenum solely via decreased thyroid gland function is not known. Chicken embryos made mostly adenohypophyseoprivic by partial decapitation have lower than normal circulating thyroxine levels beginning at day 1 1 (Daugeras-Bernard et al. 1976; Thommes et al. 1977). Their duodenal differentiation fails to progress beyond the 17.5-day level (Hinni and Watterson 1963; Bellware and Betz 1970; Hart and Betz 1972a). Stocum (1966)compared the duodenal effects of thiourea and partial decapitation and suggested that thyroxine levels must be maintained during development. Thyroxine treatment repaired the intestinal effects of hypophysectomy in neonatal rats (Yeh and Moog 1975). This effect has been implicated in chicken embryos, which could be different from neonatal rats, but has not been demonstrated because of toxicity (Mallon 1972). Recently thyroxine accelerated goblet cell development and alkaline phosphatase activity when added to chicken embryo duodenum in vitro, suggesting a direct effect (Black and Moog 1977, 1978; Brewer and Betz 1978). Accumulated evidence suggests that thyroxine plays a major role in intestinal development in other species (Chan et al. 1973; Koldovsky et al. 1974, 1975; Yeh and Moog 1975; Henning 1978; Kaltenbach et al. 1978). Its participation in duodenal development in chicken embryos is as yet unclear. This study was undertaken to reinvestigate this problem. L-Thyroxine was given prior to the prehatching increases in alkaline phosphatase specific activity and circulating T,. The dosage used was larger and the embryos were older at the time of treatment than previous1 y (Moog 196 1 ; Hinni and Watterson 1963; Mallon 1972). As well, staged embryos were used for greater accuracy and precision. D-T,, apparently without effect in chickens (Reineke and Turner 1945), was used as a physicochemical control, which has not been reported in previous experimental designs. Materials and Methods Fertilized white leghorn Gullus gullus (Hyline 934F) eggs were incubated at 38 f 0.5"C and 60 5% relative humidity in a forced-draft incubator for 34 to 40 h. Egg shells were windowed using a cone impregnated with diamond dust and mounted on a drill press, sealed with tape, and reincubated (see Betz 1975). On the 16th day of incubation (approximately 392 h), 0.1 mL of solution was placed on the chorioallantoic membrane. Treatments consisted of single applications of 5.7pg L-T4 ij,3',5,5'-tetraiodo-L-thyronine pentahydrate sodium salt,
+
417
Sigma), 5.7 pg D-T4 (3,3',5,5'-tetraiodo-D-thyroninepentahydrate sodium salt, Sigma), or saline vehicle. Thyroxine was dissolved in alkaline 0.9% saline and the pH reduced to 7.4. Additional groups were untreated with windowed shells or intact. At the time of treatment (1100 hours Eastern Standard Time) eggs were randomized to help avoid possible positional effects in the incubator. Only embryos with healthy appearing chorioallantoic membranes were treated. Normal intact eggs were candled at treatment time to determine embryonic vitality. Mortality was determined from the time of treatment until sampling, before which they were undisturbed. Twenty-four, 48, and 72 h after treatment embryos were decapitated, weighed, and staged according to Hamburger and Hamilton's stages of normal development (1951). Instances of yolk sac retraction, shell pipping, and the distribution of developmental stages were noted. Duodena were removed, stripped of pancreas and mesentery, blotted, weighed, frozen using a dry ice - acetone bath, then lyophilized 48h (Virtis unitrap) to uniform weight, and stored at -15°C. Prior to biochemical analysis duodena were ground separately in 1 mL glass-distilled water (0°C) using a ground glass homogenizer mounted on a Con-Torque stirrer. Homogenates were stored overnight (O°C), then their volumes brought to 2 m L with glass-distilled water. Each 2-mL sample was divided: 0.5 mL each was used for protein and alkaline phosphatase determination. and the remaining 1 mL was stored. The sample for protein determination was boiled 15 min in 10% (wlv) trichloroacetic acid (TCA) then spun IOmin at 700 x g (Sorvall GLC-I centrifuge). The pellet was resuspended in 10% TCA, stored overnight (0°C). then spun IOmin at 700 x g. The final pellet was dissolved in IOmL 0.5 N NaOH at 60°C. Protein determination was done on duplicate I-mL samples of this solution using the technique of Lowry el 01. (1951), and optical densities (560 nm) were measured on a Hitachi Perkin-Elmer 139 spectrophotometer against a bovine serum albumin standard curve (fraction V, essentially fatty acid free, Sigma). Determination of alkaline phosphatase using PhosphastrateQ alkaline substrate (phenolphthalein monophosphate dicyclohexylamine) according to the modification of Manning e l (11. (1967) of the Babson ef (11. technique (1966) was done using 0. I-mL aliquots of homogenate diluted with distilled water (20 to 200 dilution factor) at 37.5"C for 20 to 60min. Absorbance (550nrn) was measured against a phenolphthalein standard curve and for comparison an alkaline phosphatase standard from Escherichia coli (EC 3.1.3.1, type 111-R, Sigma) was used. The specific activity of alkaline phosphatase was determined as micrograms phenolphthalein released per hour per milligram protein. Control determinations of the specific activity of alkaline phosphatase and total protein using 25 identical samples obtained from a hornogenate of 10 duodena showed a maximum variation of 6%. Values for means plus or minus standard errors were calculated and means for staged and time-pooled results were compared using Duncan's new multiple range test (Duncan 1955) at p = 5%. Only groups receiving a treatment were pooled according to time, i.e., results of all stages were pooled as to time after treatment. Normal and windowed groups were not pooled; however, appropriate stages were compared with time-pooled groups (e.g., 43 normal with 24-h pool). Distributions of stages were compared using tables of binomial confidence limits at p = 5%.
Results Stages of Development At the time of treatment, stage 42 was the most common (Fig. 1). Twenty-four, 48, and 72h later, stages 43, 44, and 45 respectively were most common. At 72 h, some stage 45 embryos in all groups,
CAN. J. ZOOL. VOL. 57. 1979
,,$
I
Time 0-165 Days
Can. J. Zool. Downloaded from www.nrcresearchpress.com by University of Auckland on 12/05/14 For personal use only.
WINDOWED
,
48 h
- 1 8 v 2 Days
P
STAGE FIG.1 . Distribution of developmental stagesofchicken embryos as percentage composition. At time0 single dosesof L - T ~ , D-T,, or saline were applied and at 24.48, and 72 h samples were taken. Embryos pipping the shell are 45 PIP.
except those with windowed shells only, had begun to pip their shells. Stage distributions in all groups were not significantly different. Alkuline Phosphatase Mean duodenal alkaline-phosphatase-specific activity increased significantly from stage 42 to 45 in normal (unwindowed) embryos (Table 1). Twenty-four hours after any treatment, no significant differences in phosphatase activity were seen among any stage 43 embryos (Table 1, Fig. 2). At 48 h, the phosphatase activity of only embryos treated with L-T4 was significantly higher than in any other stage 44 group. Stage 43 and 45 phosphatase activities were also significantly elevated at this time. At 72 h, phosphataseactivity as a result of L-T4 treatment remained greatest among the stage 45 groups, although no longer significantly. Stage
44 embryos that had been treated 72 h previously with L-T, had activities not significantly different from stage 44 controls at 48 h. For comparison, sample results from all stages were pooled. Twenty-four hours after treatment, the phosphatase activity of the proup rreated with l-T4 was significantly higher than those of salinctreated and sfage 43 normal (unpooled) groups (Table 2). The group treated with L-T, remained significantly greater than all others at 48 h. and at 72 h no significant differences were found.
Total Duodenal Protein Content Total duodenal protein content of normal embryos increased significantly from stages 42 to 45 (Table 1). After 24 h, the protein contents of stage 43 embryos treated with L-T4 were significantly greater than those of all other groups, except those
419
BREWER A N D BETZ
TABLE 1. Mean alkaline phosphatase specific activity and total protein content of duodenal homogenates Treatment
Time after treatment
Stage
24 h
43
48 h
43
Can. J. Zool. Downloaded from www.nrcresearchpress.com by University of Auckland on 12/05/14 For personal use only.
44
Normal
Windowed
(26)‘' 1047.6k97.3b 2.8k0.2'
(20) 1500.2f 172.2 2.8k0.2
(8) 1185.8k246.9 2.3k0.2
-
-
-
-
(43) 1707.3k143.3 4.4k0.2
(14) 1526.1k251.6 3.8k0.2
(16) 1277.5k143.0 3.8k0.3
-
-
-
-
-
(20) 3240.8k488.3 5.0k0.3
45
44
45
L-T4
D-T4 (10) 1507.7k351.0 3.1k0.4
(16) 1584.6k233.1 3.6k0.3
(8) 3362.951313.4 3.8k0.4 (19) 1454.7k275.8 4.3k0.2
(20) 3444.8k343.2 4.3k0.2
-
(18) 6012.051103.2 4.0k0.2
-
-
-
-
-
(10) 1697.6k207.1 3.8k0.2
(9) 3839.8k133.8 4.9k0.3
(14) 2626.9k380.1 4.8k0.3
-
72 h
Saline
-
(1 5) 3737.8k918.9 5.0k0.4
NOTE:At $me of treatment, stage 42 normal specific activity and total protein were 855.9 ? 62.8 and 2.4 f 0.1, respectively (n "Sample sues are In parentheses. bSpecific activities are in micrograms per hour per milligram of total duodenal protein plus o r minus standard error.
Can. J. Zool. Downloaded from www.nrcresearchpress.com by University of Auckland on 12/05/14 For personal use only.
Department ofBiology, Crrrleton University, Otrnwt~,O n f . ,Canoda KIS 5B6 Received July 17. 1978
BREWER,L. M . , and T . W. BETZ. 1979. Thyroxine and duodenal development in chicken embryos. Can. J . Zool. 57: 416-423. To test whether thyroxine (T,) can stimulate duodenal development in chickens, it was administered to 16.5-day-old embryos (strain Hyline 934F ofGullus gallus) (approximately stage 42). Treatment time was closer to the prehatching phosphatase increase, and the single dosage (5 pg) larger than before. Control groups received the following: (1) D-T, for possible physicochemical variables, (2) windowed shells and saline vehicle, (3) windowed shells or (4) no treatment. Subsequent accumulation of duodenal alkaline phosphatase activity (EC 3.1.3.1) and protein content were monitored as developmental parameters (n = 8-43). Yolk sac retraction, distl-ibution of developmental stages, and mortality were used as indices of maturation and survival. L-Thyroxine tl-eatment increased duodenal alkaline phosphatase significantly ( p < 0.01) within 48 h, 24 h sooner than normal. This precocious increase was transient inembryos treated at stage 41. Duodenal protein content was elevated significantly in stage 43 embryos treated with L - T ~but , it was not different from control values at later stages. Pooling of time data did not greatly alter duodenal parameters. Yolk sac retraction and shell pipping began sooner in embryos treated with L-T4 than control embryos, however, distribution of developmental stages was unaltered. Increased mortality was found 72 h after L-T4 treatment. After D-T, treatment no parameter was significantly different. The parameters responding after treatment may be stimulated by thyroxine during development. BREWER,L. M., et T. W. BETZ. 1979. Thyroxine and duodenal development in chicken embryos. Can. J . Zool. 57: 416-423. L'administration de thyroxine (T,) a des embryons de poulets de 16.5 jours (souche Hyline 934F de Gallus gallus, au stade 42 environ) a permis de deceler son effet sur le developpement du duodenum. Comparativement aux travaux anterieurs, le traitement comportait une dose unique plus importante (5pg) et a ete effectue b un moment plus rapproche d e I'augmentation d e phosphatase qui precede I'eclosion. Les groupes-temoins ont reGu les traitements suivants: (I) D-T4 pour deceler les variables physicochimiques possibles, (2) des coquilles percees et une solution saline en guise de solvant, (3) des coquilles percees et (4) aucun traitement. L'accumulation subsequente de phosphatase alcaline dans le duodenum (EC 3.1.3.1) et le contenu en proteines ont servi de parametres (n = 8-43). Le retrait du sac vitellin, la distribution des stades de developpement et la mortalite ont servi d'indices de maturation et de survie. Le traitement a la L-thyroxine augmente la phosphatase alcaline du duodenum d e f a ~ o nsignificative @ < 0.01) en moins de 48h, soit 24h plus t8t qu'il n'est normal. Cette augmentation precoce est tempordire chez les embryons traites au stade 41. Le contenu en proteines du duodenum est augmente significativement chezdes embryons traites a la L - T au ~ stade 43, mais les valeurs ne diffierent pas de celles qu'on enregistre chez les temoins a des stades plus avances. Le groupement des donnees par rapport au temps ne modifie pas beaucoup les parametres du duodenum. Le retrait du sac vitellinet le bris de lacoquille commencent plus t8t chezles embryons traites a l a ~ - que T ~ chez les embryons-temoins; cependant, la distribution des stades de developpement reste la mkme. Soixante-douze heures apres le traitement la L - T ~la, mortalite est augmentee. Les parametl-es ne subissent toutefois pas de changement significatif a la suite d'un traitement a la D-T4. Les parametres qui sont affectes par le traitement peuvent i t r e stimules par la thyroxine au cours du developpement. [Traduit par le journal]
Introduction The terminal phase of duodenal development in chicken embryos is marked by dramatic enzymic and structu~tlchanges. During the 5 days prior to hatching alkaline phosphatase (EC 3.1.3.1) accumulation increases sharply (Moog 1950). paralleling develaprnent of the microvillar surface of epithelial cells (Overten and Shoup 1964). It is accepted that the embryonic ndenohypophysis stimulates this phase of duodenal development
(Hinni and Watterson 1963; Bellware and Betz 1970; Hart and Betz 19720, 1972b). The role of the thyroid glands i s as yet unclear. Thiourea treatment inhibits the sharp increase in alkaline phosphatase. and this effect can be reversed by thyroxine trearment (Moog 1961). Precocious increase in alkaline phosphatase activity can be induced by cortisone (Moog and Richardson 1955). Moog (1961) found that thyroxine did not produce this effect. while others have indicated that it can elevate phos-
0008-4301/79/0204 16-08$01 .OO/O @I979 National Research Council of Canada/Conseil national de I-echerchesdu Canada
Can. J. Zool. Downloaded from www.nrcresearchpress.com by University of Auckland on 12/05/14 For personal use only.
BREWER AND BET2
phatase (Betz and Mallon 1970; Mallon 1972). Moog (1961)suggested that thyroxine is permissive in the action of corticoids as cortisone treatment did not alleviate the effects of thiourea. Whether thiourea acts on duodenum solely via decreased thyroid gland function is not known. Chicken embryos made mostly adenohypophyseoprivic by partial decapitation have lower than normal circulating thyroxine levels beginning at day 1 1 (Daugeras-Bernard et al. 1976; Thommes et al. 1977). Their duodenal differentiation fails to progress beyond the 17.5-day level (Hinni and Watterson 1963; Bellware and Betz 1970; Hart and Betz 1972a). Stocum (1966)compared the duodenal effects of thiourea and partial decapitation and suggested that thyroxine levels must be maintained during development. Thyroxine treatment repaired the intestinal effects of hypophysectomy in neonatal rats (Yeh and Moog 1975). This effect has been implicated in chicken embryos, which could be different from neonatal rats, but has not been demonstrated because of toxicity (Mallon 1972). Recently thyroxine accelerated goblet cell development and alkaline phosphatase activity when added to chicken embryo duodenum in vitro, suggesting a direct effect (Black and Moog 1977, 1978; Brewer and Betz 1978). Accumulated evidence suggests that thyroxine plays a major role in intestinal development in other species (Chan et al. 1973; Koldovsky et al. 1974, 1975; Yeh and Moog 1975; Henning 1978; Kaltenbach et al. 1978). Its participation in duodenal development in chicken embryos is as yet unclear. This study was undertaken to reinvestigate this problem. L-Thyroxine was given prior to the prehatching increases in alkaline phosphatase specific activity and circulating T,. The dosage used was larger and the embryos were older at the time of treatment than previous1 y (Moog 196 1 ; Hinni and Watterson 1963; Mallon 1972). As well, staged embryos were used for greater accuracy and precision. D-T,, apparently without effect in chickens (Reineke and Turner 1945), was used as a physicochemical control, which has not been reported in previous experimental designs. Materials and Methods Fertilized white leghorn Gullus gullus (Hyline 934F) eggs were incubated at 38 f 0.5"C and 60 5% relative humidity in a forced-draft incubator for 34 to 40 h. Egg shells were windowed using a cone impregnated with diamond dust and mounted on a drill press, sealed with tape, and reincubated (see Betz 1975). On the 16th day of incubation (approximately 392 h), 0.1 mL of solution was placed on the chorioallantoic membrane. Treatments consisted of single applications of 5.7pg L-T4 ij,3',5,5'-tetraiodo-L-thyronine pentahydrate sodium salt,
+
417
Sigma), 5.7 pg D-T4 (3,3',5,5'-tetraiodo-D-thyroninepentahydrate sodium salt, Sigma), or saline vehicle. Thyroxine was dissolved in alkaline 0.9% saline and the pH reduced to 7.4. Additional groups were untreated with windowed shells or intact. At the time of treatment (1100 hours Eastern Standard Time) eggs were randomized to help avoid possible positional effects in the incubator. Only embryos with healthy appearing chorioallantoic membranes were treated. Normal intact eggs were candled at treatment time to determine embryonic vitality. Mortality was determined from the time of treatment until sampling, before which they were undisturbed. Twenty-four, 48, and 72 h after treatment embryos were decapitated, weighed, and staged according to Hamburger and Hamilton's stages of normal development (1951). Instances of yolk sac retraction, shell pipping, and the distribution of developmental stages were noted. Duodena were removed, stripped of pancreas and mesentery, blotted, weighed, frozen using a dry ice - acetone bath, then lyophilized 48h (Virtis unitrap) to uniform weight, and stored at -15°C. Prior to biochemical analysis duodena were ground separately in 1 mL glass-distilled water (0°C) using a ground glass homogenizer mounted on a Con-Torque stirrer. Homogenates were stored overnight (O°C), then their volumes brought to 2 m L with glass-distilled water. Each 2-mL sample was divided: 0.5 mL each was used for protein and alkaline phosphatase determination. and the remaining 1 mL was stored. The sample for protein determination was boiled 15 min in 10% (wlv) trichloroacetic acid (TCA) then spun IOmin at 700 x g (Sorvall GLC-I centrifuge). The pellet was resuspended in 10% TCA, stored overnight (0°C). then spun IOmin at 700 x g. The final pellet was dissolved in IOmL 0.5 N NaOH at 60°C. Protein determination was done on duplicate I-mL samples of this solution using the technique of Lowry el 01. (1951), and optical densities (560 nm) were measured on a Hitachi Perkin-Elmer 139 spectrophotometer against a bovine serum albumin standard curve (fraction V, essentially fatty acid free, Sigma). Determination of alkaline phosphatase using PhosphastrateQ alkaline substrate (phenolphthalein monophosphate dicyclohexylamine) according to the modification of Manning e l (11. (1967) of the Babson ef (11. technique (1966) was done using 0. I-mL aliquots of homogenate diluted with distilled water (20 to 200 dilution factor) at 37.5"C for 20 to 60min. Absorbance (550nrn) was measured against a phenolphthalein standard curve and for comparison an alkaline phosphatase standard from Escherichia coli (EC 3.1.3.1, type 111-R, Sigma) was used. The specific activity of alkaline phosphatase was determined as micrograms phenolphthalein released per hour per milligram protein. Control determinations of the specific activity of alkaline phosphatase and total protein using 25 identical samples obtained from a hornogenate of 10 duodena showed a maximum variation of 6%. Values for means plus or minus standard errors were calculated and means for staged and time-pooled results were compared using Duncan's new multiple range test (Duncan 1955) at p = 5%. Only groups receiving a treatment were pooled according to time, i.e., results of all stages were pooled as to time after treatment. Normal and windowed groups were not pooled; however, appropriate stages were compared with time-pooled groups (e.g., 43 normal with 24-h pool). Distributions of stages were compared using tables of binomial confidence limits at p = 5%.
Results Stages of Development At the time of treatment, stage 42 was the most common (Fig. 1). Twenty-four, 48, and 72h later, stages 43, 44, and 45 respectively were most common. At 72 h, some stage 45 embryos in all groups,
CAN. J. ZOOL. VOL. 57. 1979
,,$
I
Time 0-165 Days
Can. J. Zool. Downloaded from www.nrcresearchpress.com by University of Auckland on 12/05/14 For personal use only.
WINDOWED
,
48 h
- 1 8 v 2 Days
P
STAGE FIG.1 . Distribution of developmental stagesofchicken embryos as percentage composition. At time0 single dosesof L - T ~ , D-T,, or saline were applied and at 24.48, and 72 h samples were taken. Embryos pipping the shell are 45 PIP.
except those with windowed shells only, had begun to pip their shells. Stage distributions in all groups were not significantly different. Alkuline Phosphatase Mean duodenal alkaline-phosphatase-specific activity increased significantly from stage 42 to 45 in normal (unwindowed) embryos (Table 1). Twenty-four hours after any treatment, no significant differences in phosphatase activity were seen among any stage 43 embryos (Table 1, Fig. 2). At 48 h, the phosphatase activity of only embryos treated with L-T4 was significantly higher than in any other stage 44 group. Stage 43 and 45 phosphatase activities were also significantly elevated at this time. At 72 h, phosphataseactivity as a result of L-T4 treatment remained greatest among the stage 45 groups, although no longer significantly. Stage
44 embryos that had been treated 72 h previously with L-T, had activities not significantly different from stage 44 controls at 48 h. For comparison, sample results from all stages were pooled. Twenty-four hours after treatment, the phosphatase activity of the proup rreated with l-T4 was significantly higher than those of salinctreated and sfage 43 normal (unpooled) groups (Table 2). The group treated with L-T, remained significantly greater than all others at 48 h. and at 72 h no significant differences were found.
Total Duodenal Protein Content Total duodenal protein content of normal embryos increased significantly from stages 42 to 45 (Table 1). After 24 h, the protein contents of stage 43 embryos treated with L-T4 were significantly greater than those of all other groups, except those
419
BREWER A N D BETZ
TABLE 1. Mean alkaline phosphatase specific activity and total protein content of duodenal homogenates Treatment
Time after treatment
Stage
24 h
43
48 h
43
Can. J. Zool. Downloaded from www.nrcresearchpress.com by University of Auckland on 12/05/14 For personal use only.
44
Normal
Windowed
(26)‘' 1047.6k97.3b 2.8k0.2'
(20) 1500.2f 172.2 2.8k0.2
(8) 1185.8k246.9 2.3k0.2
-
-
-
-
(43) 1707.3k143.3 4.4k0.2
(14) 1526.1k251.6 3.8k0.2
(16) 1277.5k143.0 3.8k0.3
-
-
-
-
-
(20) 3240.8k488.3 5.0k0.3
45
44
45
L-T4
D-T4 (10) 1507.7k351.0 3.1k0.4
(16) 1584.6k233.1 3.6k0.3
(8) 3362.951313.4 3.8k0.4 (19) 1454.7k275.8 4.3k0.2
(20) 3444.8k343.2 4.3k0.2
-
(18) 6012.051103.2 4.0k0.2
-
-
-
-
-
(10) 1697.6k207.1 3.8k0.2
(9) 3839.8k133.8 4.9k0.3
(14) 2626.9k380.1 4.8k0.3
-
72 h
Saline
-
(1 5) 3737.8k918.9 5.0k0.4
NOTE:At $me of treatment, stage 42 normal specific activity and total protein were 855.9 ? 62.8 and 2.4 f 0.1, respectively (n "Sample sues are In parentheses. bSpecific activities are in micrograms per hour per milligram of total duodenal protein plus o r minus standard error.
