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Incubation Relative Humidity Effects on Allantoic Fluid Volume and Hatchability1
Incubation Relative Humidity Effects on Allantoic Fluid Volume and Hatchability1 R. J. BUHR Department of Poultry Science, The University of Georgia, Athens, Georgia 30602-2772 ABSTRACT Four incubation trials were conducted to evaluate the effects of incubation RH on hatchability, allantoic fluid volume, and embryo weight. In Trial 1, White Leghorn eggs were incubated at a RH of 55 or 69% and a dry bulb temperature of 37.8 C and hatchability was determined. In Trial 2, hatchability and allantoic fluid volume were determined (Day 14 of incubation) for eggs incubated at 43, 55, or 69% RH. In Trial 3, allantoic fluid volumes and embryo weights were determined on Days 10, 12, 14, and 16 of incubation for eggs incubated at 40,55, or 70% RH. In Trial 4, hatchability, allantoic fluid volumes, and embryo weights were determined on Days 13,14, and 15 of incubation for eggs incubated at 40, 55, or 70% RH from hens 34 and 49 wk of age. There were no differences (P > .05) in hatchability between eggs incubated at 55 or 69% RH in Trial 1, or among eggs incubated at 43,55, or 69% RH in Trial 2. In Trial 4, hatchability was highest for eggs from 34-wk-old hens incubated at 55% RH. In Trial 2, percentage egg weight loss per day of incubation (.68, .55, and .40%) and projected 19-d weight loss (12.9, 10.45, and 7.6%) decreased significantly with increased incubation RH of 43, 55, and 69%, respectively. Allantoic fluid volume (as a percentage of initial egg weight) on Day 14 of incubation significantly increased with the increase in RH; 13.86, 15.45, and 17.93% for RH of 43,55, and 69%, respectively. In Trial 3, allantoic fluid volume peaked on Day 14 of incubation and volume increased or decreased with corresponding incubation RH; 14.05, 16.25, and 19.44% for RH of 40, 55, and 70%. In Trial 4, allantoic fluid volume was greatest on Day 13 of incubation and volume increased or decreased with corresponding incubation RH 13.38,16.91, and 18.68% for eggs from 34-wk-old hens and 11.12,13.77, and 16.59% for eggs from 49-wk-old hens. Eggs from 49-wk-old hens had lower allantoic fluid volumes, but higher initial egg weights due to greater percentage egg weight loss during incubation. Incubation egg weight loss in different RH appears to result from the corresponding depletion of the allantoic fluid reservoir. (Key words: eggs, incubation, relative humidity, allantoic fluid, hatchability) 1995 Poultry Science 74:874-884
INTRODUCTION The allantoic sac arises as a diverticulum of the chick embryonic hind gut into which the mesonephric kidney begins to secrete urine during the 5th d of incuba-
Received for publication September 14, 1994. Accepted for publication January 12, 1995. Supported by state and Hatch funds allocated to the Georgia Agricultural Experiment Stations of The University of Georgia.
tion (Fiske and Boyden, 1926). The allantoic sac begins filling with fluid at 2.5 d and attains a maximum volume of 8 to 10 mL on Days 12 to 14 of incubation, depending on incubation temperature (Fiske and Boyden, 1926; Romanoff et al., 1938; Davis et al, 1988). The allantois and the chorionic membranes fuse to form the chorioallantoic membrane, which expands to lie beneath the eggshell membrane to which it becomes attached during Day 11 and 12 of incubation. The allantois is
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INCUBATION RELATIVE HUMIDITY AND ALLANTOIC FLUID
primarily an organ of nitrogenous metabolite storage whereas the chorion functions as the embryonic respiratory organ and for the resorption of eggshell calcium. On Day 13 of incubation, allantoic fluid p H and sodium and chloride concentrations fall as allantoic fluid volume decrease, maintaining allantoic fluid hypotonic to blood, permitting water removal from the allantoic fluid down an osmotic gradient (Walker, 1943; Stewart and Terepka, 1969; Hoyt, 1979). The pituitary-adrenal axis is established in the chick embryo at approximately 14.5 d of incubation. Hypophysectomy of Day 2 embryos resulted in significant increases in embryo water content on Days 12.5 to 18 of incubation, indicating reduced ability of these embryos to maintain proper water balance (Thommes and McCarter, 1966). Woods et al. (1971) reported prolonged elevation (Days 15.5 to 17.5) of allantoic fluid volume and disrupted embryo water balance in hypophysectomized embryos due to deficiencies in neurohypophyseal hormones. These deficiencies were partially restored with pituitary grafts to the e m b r y o ' s chorioallantoic membrane or by adrenocorticotropin hormone administration. Hardin and Bell (1969) reported phenotypic correlations indicating that Day 13 allantoic fluid volume was unrelated to embryo weight (r = .19) and egg weight loss (r = .21), but highly correlated with initial egg weight (r = .64) and Day 13 egg weight (r = .66) for eggs incubated at a RH of 55%. However, after adjustment for variation in initial egg weight, there were genetic differences in the amount of allantoic fluid between White Leghorn and meat-type lines and their line crosses. Significant genetic differences in yield of influenza vaccine from allantoic fluid of chicken embryos have been reported (Bell et al, 1964). Of the variation in vaccine yield, 53% was explained by variation in egg size. However, maternal breed type remained a highly significant source of variation in vaccine yield after adjustment for initial egg weight. Romanoff and Hayward (1943) reported the progressive changes in the volume of allantoic fluid under normal (37.5 C) and extreme (34.5, 36.0, 38.5, and 39.5 C)
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incubation temperatures at a consistent RH of 60%. On Day 13 of incubation, allantoic fluid volume p e a k e d a n d represented 9 to 10% of initial egg weight (7.3 mL) when incubated at 37.5 C, whereas at temperature extremes volume was depressed, 6.7 mL for 38.5 or 36.0 C and 4.5 mL for 39.5 or 34.5 C. They reported that incubation temperature extremes resulted in the underdevelopment of the allantoic sac and an earlier peak with high temperature and later peak in volume with low temperature. Hoyt (1979) measured the relative extraembryonic water content of the yolk, amniotic fluid, and allantoic fluid after 17 d of incubation under different incubation RH, during which eggs lost 4.5 to 17.8% of their initial weight. As desiccation increased, allantoic fluid volume decreased six times more than the embryo fluid content, with minimal changes in water content of the amniotic fluid and the yolk. Hoyt concluded that the ability of the avian embryo to differentially partition water loss results in a more stable internal environment for embryonic development when eggs are subjected to a range of RH. Davis et al. (1988) measured allantoic fluid volume from Day 12 to 19 of incubation for eggs incubated in 10, 55, and 85% RH. Allantoic fluid volume declined for all eggs from a peak on Day 12 of 10.5 mL, at which time no differences in volume were detected. Eggs incubated at 10% RH had excessive weight loss and a more rapid decline in allantoic fluid volume, whereas eggs incubated at 85% RH had lower weight loss and a slower decline in allantoic fluid volume than control eggs. Eggs experiencing either high or low weight loss first differed significantly from control eggs in allantoic fluid volume on Day 16 of incubation. The literature contains few studies evaluating high and low incubation RH effects on allantoic fluid volume and hatchability continuously from the time of setting. The present studies were undertaken to identify the influence of incubation RH within the range of 40 to 70% RH on embryonic allantoic fluid volume, egg weight loss, and hatchability in modern commercial White Leghorn eggs that possess high hatchability.
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MATERIALS AND METHODS Trial 1 To assess the effects of incubation RH of 55 and 69% on embryonic mortality and hatchability, four weekly settings of hatching eggs collected from Hy-Line® White Leghorn hens at 35,36,37, and 38 wk of age were stored for less than 1 wk at 14.4 C and 60% RH. One hundred and eighty eggs per humidity treatment and setting were allowed to warm to room temperature (22 C) overnight (15 h) and set into Jamesway2 252B incubators operating at 37.8 C and at RH of 55 or 69%. Relative humidity was regulated by wet bulb contact thermometers (28.9 and 32.2 C, respectively). Incubators contained only eggs utilized for each trial. Relative humidity was calculated from wet and dry bulb temperatures measured with thermistor probes and remote temperature recorders (TempMentor™).3 Eggs were candled on the 7th, 14th, and 18th d of incubation, and on the 18th d eggs containing viable embryos were transferred into mass hatching baskets by RH treatment and placed into a single Jamesway 252B hatcher operating at 36.9 C. On Day 20, RH was increased to 69%. All candled eggs were opened to macroscopically distinguish between infertility and early embryonic mortality. Percentage fertility of eggs set, hatchability of eggs set, hatchability of fertile eggs, and hatchability of viable embryos transferred on the 18th d of incubation were determined. Hatchability of viable embryos at transfer was used as an indicator of late embryonic mortality immediately prior to hatching. On the day of hatch (Day 22) all unhatched eggs were broken out and embryos staged according to Hamburger and Hamilton (1951) to determine embryonic age. Trial 2 To assess the effects of incubation RH (43,55, and 69%) on allantoic fluid volume of Day 14 embryos, three settings were made of fertile hatching eggs collected from
the same flock of Hy-Line® White Leghorn hens at 37,43, and 56 wk of age. This range in RH was chosen to bracket recommendations for successful hatchability reported in the literature (Lundy, 1969) and was obtained with wet bulb contact thermometer (26.7, 28.9, and 32.2 C, respectively) RH controls. Eggs were collected, stored, and set in Jamesway 252B incubators as described in Trial 1. In the first setting 30 eggs per humidity treatment (55 and 69% RH) were weighed prior to setting and again on Days 7 and 14 of incubation. Egg weight loss was determined from Day 7 and 14 egg weights. On Day 14 of incubation 30 eggs per humidity treatment containing viable embryos were removed from the incubator, cooled to room temperature for 30 min, and opened for allantoic fluid determination. The shell over the air cell was removed with forceps, and the inner egg membrane moistened with a drop of water to permit visualization of the underlying blood vessels of the chorioallantoic membrane and to allow piercing of the membrane with minimum danger of hemorrhage. The egg was inverted and the allantoic fluid poured into a funnel supported by a graduated cylinder, from which allantoic fluid volume was determined to the nearest .1 mL (Hoyt, 1979). After adjustment for initial egg weight, correlations were determined between initial egg weight and allantoic fluid volume, and between Day 14 egg weight and allantoic fluid volume. The remaining 270 eggs (excluding eggs removed at candling) continued in each RH treatment until transfer into a single hatcher on Day 18 of incubation. In the second setting an additional low humidity treatment was added: 43% RH. Two hundred and seventy-four eggs from 43-wk-old hens were set for each incubation RH, 43, 55, and 69%. On Day 14 of incubation, 60 eggs were weighed and sampled for allantoic fluid, and the remaining eggs continued through hatching. The third setting replicated the second, using 237 eggs from 56-wk-old hens for each of the three incubation RH treatments. Trial 3
2
Jamesway Manufacturing Co., Fort Atkinson, WI 53538. 3 Ryan Instrument, Redmond, WA 98073-0599.
Trial 3 was designed to determine whether allantoic fluid volume increased
INCUBATION RELATIVE HUMIDITY AND ALLANTOIC FLUID
with elevated RH or whether the greater volume detected in Trial 2 on Day 14 of incubation was due to a slower rate of allantoic fluid loss. Two sequential settings were made in two newly installed NatureForm NMC-20004 incubators (37.8 C). Allantoic fluid volume was determined from 30 eggs on Days 10, 12, 14, and 16 of incubation. In the first setting, eggs from a flock of Hy-Line® White Leghorn hens at 27 wk of age were placed (without prewarming) into incubators operating at 40 and 55% RH, whereas in the second setting, eggs from hens at 30 wk of age were placed into incubators operated at 55 and 70% RH. One hundred and fifty eggs per humidity treatment were weighed prior to setting and on the 7th d of incubation, and 30 eggs containing viable embryos per humidity treatment were weighed on the day of sampling. Allantoic fluid volume was determined as described in Trial 2. Embryo weight was determined by removing the embryo from the egg, separating the embryo from the yolk sac and chorioallantoic membrane by cutting their attachments at the umbilicus, blotting the embryo with paper toweling, and weighing to the nearest .01 g. After adjustment for initial egg weight, correlations between initial egg weight and allantoic fluid volume or embryo weight were determined. In addition, correlations between sampling day egg weight and allantoic fluid volume or embryo weight were determined. Hatchability was not evaluated in this trial. Trial 4 Trial 4 was designed to determine whether allantoic fluid volume increased and decreased with elevated or reduced RH on Days 13, 14, and 15 of incubation and whether allantoic fluid varied with eggs of different weight from flocks of different ages. Eggs from a flock of Hy-Line® White Leghorn hens 34 wk old and from a separate flock at 49 wk of age were set concurrently (without prewarming) into three NatureForm incubators operating at 37.8 C, and RH of 40, 55, and 70%. The ability to run the three RH treatment
4
NatureForm Co., Jacksonville, FL 32202.
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concurrently was made possible by the installation of additional incubators. Eighty-eight eggs per humidity treatment and hens age were weighed prior to setting and egg weight equalized for each sampling day and RH treatment (34-wk-old = 57.34 g and 49-wk-old = 59.45 g). On the 7th d of incubation and on the day of sampling 25 eggs containing viable embryos per humidity treatment were weighed and from these weights egg weight loss was determined. Allantoic fluid volume was determined from 25 eggs on Days 13, 14, and 15 of incubation as described in Trial 2 and embryo weight was determined as described in Trial 3. After adjustment for initial egg weight, correlations between initial egg weight and allantoic fluid volume or embryo weight were determined, in addition to correlations between sampling day egg weight and allantoic fluid volume or embryo weight. An additional 247 eggs from 34-wk-old hens and 249 eggs from 49-wk-old hens were also set for each RH for determination of hatchability (Day 21). Statistical Analysis Fertility and hatchability data were analyzed using the General Linear Models (GLM) procedure for least squares analysis of disproportionate subclass frequency (SAS Institute, 1991). The percentage response was based on using the normal approximation for the binomial value, as the cell frequencies were large. Least squares means was used to separate means within significant main effects (P < .05). Sources of variation in the model included main effects: for Trials 1 and 2 were RH and setting, for Trial 3 were RH, setting, and sampling day, and for Trial 4 were RH, hens age, and sampling day. Interaction terms for main effects were tested for significance. Egg weight, egg weight loss during incubation, embryo weight, and allantoic fluid volume data were analyzed by analysis of variance in Trials 2,3, and 4. Sampling day significantly influenced allantoic fluid volume and embryo weight in Trials 3 and 4. The main effect setting was frequently significant within Trials 1, 2, and 3. Therefore, means are presented for each trial by setting and sampling day. In Trials 2,3, and 4 after adjustment for initial egg weight,
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correlations between initial egg weight and allantoic fluid volume or embryo weight were determined, in addition to correlations between sampling day egg weight and allantoic fluid volume or embryo weight.
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RESULTS AND DISCUSSION In Trial 1, elevated incubation RH at 69% did not result in differences (P > .05) in hatchability or distribution of embryonic mortality compared with control eggs incubated at 55% RH (Table 1). The average for the four settings was 97% fertility, 88.1% hatchability of eggs set, 90.8% hatchability of fertile eggs, and 94.7% hatchability of viable embryos at transfer. The main period of mortality for both RH treatments occurred following transfer into the hatcher. The lack of a difference between RH treatments in embryo mortality after transfer into the hatcher indicates that elevated incubation at 69% RH was not detrimental to embryo survival or ability to hatch. Incubation RH (69%) was calculated within 1% of the value for which the high RH contact thermometer was chosen (70%). For Trial 2, initial egg weight, percentage egg weight lost per day of incubation, and allantoic fluid volume expressed as a percentage of initial egg weight are presented in Table 2. Initial egg weight was heavier (P < .05) for eggs set at 69% RH than at 55% RH for the first setting, but was not different among RH treatments for the second and third settings. Initial egg weight increased significantly with hens' age over the three settings. Percentage egg weight loss per day of incubation was inversely related to incubation RH, with the greatest loss for eggs incubated at 43% RH (.68%) and the lowest loss for eggs incubated at 69% RH (.40%). Eggs incubated at 55% RH had intermediate percentage egg weight loss (.54%). These values projected as 19-d egg weight losses were 12.8,10.4, and 7.7% of initial egg weight for RH of 43, 55, and 69%, respectively. The low incubation RH (43%) was calculated to be 3% higher than the value for which the contact thermometer was chosen (40%). Allantoic fluid volume was positively influenced by incubation RH (P < .05).
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INCUBATION RELATIVE HUMIDITY AND ALLANTOIC FLUID
TABLE 2. Egg weight, percentage incubation egg weight loss, and percentage allantoic fluid volume for eggs incubated at relative humidities of 43, 55, and 69%, Trial 2 Relative humidity Variable setting
1
55%
Pooled
69%
SEM
54.02" 57.80 62.56
55.85a 58.46 61.30
.58 .42 .53
.68' .67'
.52" .55" .57"
.41" .39< .41 c
.01 .01 .01
13.06c 14.67=
15.77 14.82" 15.77"
17.11 18.13a 18.54"
.48 .36 .38
43%
'~1
Initial egg weight Setting 1 Setting 2 Setting 3 Percentage egg weight loss per day of incubation Setting 1 Setting 2 Setting 3 Allantoic fluid volume as a percentage of initial egg weight Setting 1 Setting 2 Setting 3
(SI 2
58.03 61.58
"^Means within a row with no common superscript differ significantly (P < .05). !Number of eggs in Setting 1 n = 30, Settings 2 and 3 n = 60, hens age 37, 43, and 56 wk. 2 There was no 43% RH treatment for Setting 1.
Values for allantoic fluid overlapped among settings and RH treatments due to the increase in initial egg weight with each successive setting. This overlap among RH treatments disappeared when allantoic fluid volume was expressed as a percentage of initial egg weight. Allantoic fluid volume on Day 14 of incubation expressed as a percentage of initial egg weight was 17.1 to 18.5% for eggs incubated at 69% RH, 14.8 to 15.8% for eggs incubated at 55% RH, and 13 to 14.7% for eggs incubated at 43% RH. The difference in allantoic fluid volume as a percentage of egg initial weight was significant in all settings except for eggs incubated at 55 and 69% RH in the first setting, in which egg weight differed significantly. These results are in agreement with those reported by Davis et ah (1988) that incubation RH can positively or negatively influence allantoic fluid by Day 14 of incubation. A similar trend with higher values was calculated for allantoic fluid volume as a percentage of Day 14 egg weight. After adjustment for initial egg weight, correlation coefficients (r) between initial egg weight or Day 14 egg weight
and allantoic fluid volume were significant and ranged from r = .30 to .70. No pattern associated with RH treatment was observed. In Trial 2, for all three settings, fertility (95.9%), hatchability of fertile eggs (91.7%), and hatchability of viable eggs at transfer (95%) were not significantly different among RH treatments. The results of Trials 1 and 2 indicate no adverse effects on hatchability with continuous incubation RH from 43 to 69% and the projected 19-d egg weight loss from 7.4 to 12.9%. Gildersleeve (1984) subjected broiler eggs to 55% RH or increasing RH profiles from 52 to 67% during incubation and reported no difference in 18-d egg weight loss although differences were present earlier. In addition, Gildersleeve reported no differences in fertility or hatchability with this programmed change in RH. Simkiss (1980) removed 6 mL of allantoic fluid from Day 13 embryos (equivalent to 100% increase in normal incubation water loss) and reported normal hatchability and equivalent percentage body composition but reduced chick weight by 6.6 g. In a separate experiment,
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TABLE 3. Egg weight, percentage egg weight loss, percentage allantoic fluid volume, and percentage embryo weight for eggs incubated at 40, 55, or 70% relative humidity1 and from which allantoic fluid volume was determined on Days 10, 12, 14, and 16 of incubation, Trial 3 Days of incubation Variable Initial egg weight, g 40% RH 55% RH Pooled SEM 55% RH 70% RH Pooled SEM Percentage egg weight loss per day of incubation, % 40% RH 55% RH Pooled SEM 55% RH 70% RH Pooled SEM Allantoic fluid volume as a percentage of initial egg weight, % 40% RH 55% RH Pooled SEM 55% RH 70% RH Pooled SEM Embryo weight as a percentage of initial egg weight, % 40% RH 55% RH Pooled SEM 55% RH 70% RH Pooled SEM
10
12
14
16
62.43 63.57 .76 63.99 63.52 .75
63.32 61.95 .90
63.36 63.14 .73 60.79b 63.54" .72
62.83 64.19 .78 65.03" 62.07b .77
62.15 62.91 .99
.73" .51b .02 .52" .36b .01
.70' .50" .02 .54* .33" .01
.67" .48 b .02 .56" .35b .01
.64" .50b .02 .49" .35 b .01
4.87b 6.38a .31 5.65" 7.34a .28
12.91 b 15.20" .46 14.75" 16.00" .44
14.05b 17.46" .53 15.04b 19.44" .45
8.67" 11.13" .45 12.52b 14.37" .44
3.83a 3.49b .05 3.79" 3.52b .06
7.64 7.34 .14 7.76" 7.23" .16
15.95" 14.63b .25 16.09" 14.60b .26
27.22 26.64 .36 26.42 26.68 .36
a b
- Means within days of incubation and setting with no common superscript differ significantly (P < .05). Relative humidity 40 and 55%, hens age 27 wk, n = 30; Relative humidity 55 and 70%, hens age 30 wk, n =
30.
Simkiss (1980) increased incubation water loss (an additional 8 g) by drilling holes in the eggshell of Day 13 embryos. No evidence of dehydration was detected. Simkiss (1980) concluded that the chick embryo has the regulatory ability to recycle the water in the egg, to grow to a size commensurate with the available water supply, to tolerate a wide range of eggshell porosity and incubation conditions. In Trial 3, initial egg weight did not differ between eggs in the two settings (mean 63.1 to 63 g) (Table 3). However, in the second setting eggs sampled on Days
14 and 16 of incubation differed significantly in initial egg weight for the 70 and 55% RH treatment. Percentage egg weight loss per day of incubation in Trial 3 was inversely related to incubation RH and did not differ among the four sampling days. Projected 19-d egg weight loss was 13.0, 9.4, 10.1, and 6.6% for incubation RH of 40, 55, 55, and 70%, respectively. Incubation RH was calculated to equal the set point values of the NatureForm incubators. Allantoic fluid volume was positively related to incubation RH and peaked on Day 14 of incubation (Table 3). Allantoic
INCUBATION RELATIVE HUMIDITY AND ALLANTOIC FLUID
fluid as a percentage of initial egg weight and sampling day egg weight increased with RH on Days 10, 12, 14, and 16 of incubation. Allantoic fluid volume and percentage egg weight loss per day of incubation varied significantly between the two 55% RH groups. Therefore the two settings were analyzed and are reported independently. These differences between the 55% RH groups occurred although eggs were collected from the same flock and set in the same incubator 3 wk apart, and reflects the inherent variability between settings and the importance of concurrent evaluation of RH treatments. After adjustment for initial egg weight, correlations between initial egg weight or egg weight on Day 10 and allantoic fluid volume were not significant for all RH treatments. Positive correlations (P < .05) were found for both initial egg weight or day of sampling egg weight and allantoic fluid volume on Day 12 (r = .39 to .59) and on Days 14 and 16 (r = .67 to .79) of incubation. The magnitude of the correlations appeared to be independent of RH treatment and were determined by the particular eggs within each treatment. Initial egg weight for eggs sampled on Day 14 was greater for eggs set in the 70% RH than the 55% RH incubator and corresponding allantoic fluid as a percentage of initial egg weight or Day 14 egg weight was also greater for the 70% RH treatment. However, on Day 16, although initial egg weight was lower for eggs set in the 70% RH than in the 55% RH, allantoic fluid measurements and calculated percentages were greater for eggs incubated at 70% RH, indicating the positive influence of RH on allantoic fluid volume. Embryo weights expressed as a percentage of initial egg weight were consistently higher in the low RH treatments on Day 10 through 15 of incubation. This finding is in contrast to that of Romanoff (1929), who reported that elevated incubation RH positively influenced embryonic growth rate; 80% RH accelerated embryo wet weight compared to embryos incubated at 40% RH. However, Romanoff's experiments utilized eggs from the same flock in succession and were not controlled for egg weight, and the water temperature of the
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humidifier was not reported. Townsley (1930) reported that, when RH was supplied via steam, as RH is increased operating incubation temperature must be decreased (and similarly when RH is low the incubation temperature should be increased) to optimize hatch time, hatchability, size of chicks, and chick livability. Penquite (1938) found no affect of RH (40, 62, or 80%) on embryo wet or dry weight and nitrogen content (Day 4 through 20) after the influence of egg weight was removed for eggs incubated in commercial incubators designed to maintain uniform conditions at all levels. The differences found in this study, heavier embryos incubated at lower incubation RH, may be due to greater fluctuation in incubation temperature due to increased frequency of humidity misting (room temperature spray) at the higher RH incubation treatments. Romanoff et al. (1938) and Penquite (1938) reported greater effects of incubation temperature on acceleration or depression of embryo weight during the 1st wk of incubation, and no detectable effects during the 2nd or 3rd wk. The greater effect of incubation temperature during the 1st wk than the 2nd or 3rd and the onset of albumen utilization by the embryo (Day 12.5) may explain the diminished difference in embryo weight at 16 d of incubation. In the present study, after adjustment for initial egg weight, correlations between initial egg weight and embryo weight were significant for only two sampling times, 40% RH on Day 10 (r = .52) and for the 55% RH on Day 16 (r = .37 and .40). In Trial 4, initial egg weight was significantly heavier for 49-wk-old hens (59.5 g) than for 34-wk-old hens (57.34 g) (Table 4). Egg weight did not differ within flocks for eggs placed in the three RH treatments or for the 3 d of allantoic fluid sampling. Percentage egg weight loss significantly increased with lower RH for both flocks and did not differ among the three sampling days. Percentage egg weight loss was higher for eggs from 49-wk-old hens than for eggs from 34-wk-old hens within the same RH treatment. Projected 19-d egg weight loss for eggs from 34-wk-old hens was 16.6, 12.7, and 9.1% incubation at RH of 40, 55,
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TABLE 4. Egg weight, percentage egg weight loss, percentage allantoic fluid volume, and percentage embryo weight for eggs incubated at 40, 55, or 70% relative humidity1 from which allantoic fluid volume was determined on Days 13, 14, and 15 of incubation, Trial 4 Days of incubation Variable Initial egg weight, g Hens age 34 wk 40% RH 55% RH 70% RH Pooled SEM Hens age 49 wk 40% RH 55% RH 70% RH Pooled SEM Percentage egg weight loss per day of incubation, % Hens age 34 wk 40% RH 55% RH 70% RH Pooled SEM Hens age 49 wk 40% RH 55% RH 70% RH Pooled SEM Allantoic fluid volume as a percentage of initial egg weight, % Hens age 34 wk 40% RH 55% RH 70% RH Pooled SEM Hens age 49 wk 40% RH 55% RH 70% RH Pooled SEM Embryo weight as a percentage of initial egg weight, % Hens age 34 wk 40% RH 55% RH 70% RH Pooled SEM Hens age 49 wk 40% RH 55% RH 70% RH Pooled SEM
13
14
15
56.36 57.66 57.86 .67
57.92 57.67 57.36 .63
57.35 56.33 57.59 .71
59.03 60.18 58.82 .55
59.97 59.27 59.93 .63
59.16 58.90 60.25 .63
.89' .64b .48' .02
.85" .69b .47' .02
.88" .68" .49' .02
.91" .72b .52' .03
.99* .69b .47' .04
.97' .73b .52' .02
13.38' 16.91b 18.68" .47
9.67' 12.74b 15.24" .49
5.22' 9.42b 12.79" .53
11.12' 13.77b 16.59" .44
9.00' 12.06b 14.52" .52
4.34' 7.27b 10.83" .41
13.45 12.94 12.74 .17
18.37" 17.71b 17.72b .21
25.34" 25.24"b 24.27b .23
13.32" 12.72" 12.47b .16
18.06" 17.61b 17.06b .20
24.40 24.64 23.76 .33
a-c Means for variables within days of incubation and hen age with no common superscript differ significantly (P < .05). x Number of eggs per treatment and day of sampling n = 25.
and 70% respectively, and was 18.2, 13.6, and 9.6% for eggs from 49-wk-old hens incubated at RH 40, 55, and 70% respectively.
Allantoic fluid volume was again positively related to incubation RH and was maximal on Day 13 of incubation (Table 4). Allantoic fluid volume was greater for
INCUBATION RELATIVE HUMIDITY AND ALLANTOIC FLUID
the 34-wk-old flock t h a n for the 49-wk-old flock, although initial egg weight was the opposite. This difference resulted from the greater egg weight loss of eggs from 49-wk-old hens than from 34-wk-old hens. Trial 4 utilized eggs from different flocks in order to collect allantoic fluid samples from different age hens that were incubated concurrently. Allantoic fluid volume as a percentage of initial egg weight and sampling egg weight increased with RH on all 3 d of collection. After adjustment for initial egg weight, correlations between initial egg weight or sampling day egg weight and allantoic fluid volume were significant (r = .4 to .83). As in Trial 3, the lowest humidity treatments had heavier embryos. Significant differences in embryo weight were detected among RH treatments for the 34-wk-old flock on Day 14 and 15 and for the 49-wk-old flock on Days 13 and 14. This difference is possibly due to a higher incubation temperature in the 40% RH incubator than in the 55 or 70% incubator, with less frequent additions of water from the mister nozzles. This concept is supported by the lower coefficient of variation for mean temperature in the 40% RH (.51%) incubator than in the 55 (.69%) or 70% RH (.73%) incubators. Correlations between initial egg weight or sampling egg weight and embryo weight were not significant. Fertility in Trial 4 was 98.9% for eggs from 34-wk-old hens and 99.1% for eggs from 49-wk-old hens and did not differ among RH treatments. Hatchability of viable embryos transferred was significantly greater for eggs from 34-wk-old hens incubated at 55% RH (98.7%) than either at 40 (93.3%) or 70% RH (92.2%). There was no difference in hatchability for eggs from 49-wk-old hens incubated at 40 (90.6%), 55 (93%), or 70% RH (89%). Lower hatchability for eggs from 34-wk-old hens incubated at 40 or 70% RH and for all eggs from 49-wk-old hens was due to an increased percentage of unhatched eggs containing live embryos that had pipped the eggshell. These results agree with those of Kirk et al. (1980), in which broiler hatching eggs incubated for 21 d and 7.5 h at increased RH had increased late embryo mortality and pipped eggs. The depres-
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sion in hatchability detected in Trial 4 but not in Trials 1 and 2 may be due to the difference in incubation length (22 vs 21 d) recommended by the incubator manufacturer. Therefore, additional investigation of the influence of incubation RH on incubation length, hatch time, and true hatchability is necessary. Results in the present study demonstrate that allantoic fluid volume is directly influenced by incubation RH and can be decreased or increased relative to an intermediate RH of 55%. Furthermore, although incubation RH ranged from 43 to 69% in Trials 1 and 2, there were no differences in hatchability or embryonic mortality. These findings indicate that the chicken embryo can maintain proper water balance by regulating allantoic fluid volume within this range of incubation RH. These results suggest that incubation RH and egg water loss during incubation may not be primary causes of elevated embryonic mortality. Coefficients of variation for daily rate of water loss from eggs of seven species of terns ranged from 4 to 46%, with an average of 28% (Rahn et al, 1976), indicating that in some species there is considerably more water loss variation than in others. The average coefficient of variability for eggshell conductance for 29 species of precocious and altricial birds was 25% (Ar et al, 1974) and 18% for 54 species of ducks and geese (Hoyt, 1979). Coefficients of variation for daily rate of water loss from eggs in Trial 2 ranged from 9.5 to 16.8%; in Trial 3 ranged from 9.5 to 23.6%; and in Trial 4 ranged from 10 to 37.7% for eggs from 34-wk-old hens a n d 6.8 to 22.9% for e g g s from 49-wk-old hens. Overall, the coefficients of variation for daily egg weight loss varied 1.8- to 3.8-fold. The results of this study agree with those of Hoyt (1979) that the avian embryo has the ability to regulate their state of hydration and maintain a fairly stable environment for development within this range of RH values. This study further supports the positive influence of egg weight and the negative influence of egg weight loss during incubation on allantoic fluid volume.
REFERENCES Ar, A., C. V. Paganelli, R. B. Reeves, D. G. Greene, and H. Rahn, 1974. The avian egg: water vapor
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conductance, shell thickness and functional pore area. Condor 76:153-158. Bell, A. E., H. W. McNary, and J. O. MacFarlane, 1964. Genetic variation in the vaccine yield from embryonating avian eggs. Poultry Sri. 43: 1258-1262. Davis, T. A., S. S. Shen, and R. A. Ackerman, 1988. Embryonic osmoregulation: Consequences of high and low water loss during incubation of the chicken egg. J. Exp. Zool. 245:144-156. Fiske, C. H., and E. A. Boyden, 1926. Nitrogen metabolism in the chick embryo. J. Biol. Chem. 70:535-556. Gildersleeve, R. P., 1984. The effect of humidity and broiler strain on egg weight losses during incubation. Poultry Sci. 63:2140-2144. Hamburger V., and H. L. Hamilton, 1951. A series of normal stages in the development of the chick embryo. J. Morphol. 88:49-92. Hardin, R. T., and A. E. Bell, 1969. Factors relating to genetic differences in the amount of chick allantoic fluid. Poultry Sci. 48:1655-1660. Hoyt, D. F., 1979. Osmoregulation by avian embryos: The allantois functions like a toad's bladder. Physiol. Zool. 52:354-362. Kirk, S., G. C. Emmans, R. McDonald, and D. Arnot, 1980. Factors affecting the hatchability of eggs from broiler breeders. Br. Poult. Sci. 21:37-53. Lundy, H., 1969. A review of the effects of temperature, humidity, turning and gaseous environment in the incubator on the hatchability of the hen's egg. Pages 143-178 in: The Fertility and Hatchability of the Hen's Egg. T. C. Carter and B. M. Freeman, ed. Oliver and Boyd, Edinburgh, Scotland. Penquite, R., 1938. Influence of temperature and humidity upon the growth of chick embryos in a mechanically ventilated incubator. Iowa Agric. Exp. Sta. Bull. 232. Ames, IA. Rahn, H., C. V. Paganelli, I. C. T. Nisbet, and G. C. Whittow, 1976. Regulation of incubation water
loss in eggs of seven species of terns. Physiol. Zool. 49:245-259. Romanoff, A. L., 1929. Effect of humidity on the growth, calcium metabolism, and mortality of the chick embryo. J. Exp. Zool. 54:343-348. Romanoff, A. L., and F. W. Hayward, 1943. Changes in volume and physical properties of allantoic and amniotic fluids under normal and extreme temperatures. Biol. Bull. 84:141-147. Romanoff, A. L., L. I. Smith, and R. A. Sullivan, 1938. Biochemistry and biophysics of the developing hen's egg. III. Influence of temperature. Cornell Univ. Agric. Exp. Sta. Mem. 216:1-42. SAS Institute, 1991. SAS/STAT® Guide for Personal Computers. Version 6 Edition. SAS Institute Inc., Cary, NC. Simkiss, K., 1980. Eggshell porosity and the water metabolism of the chick embryo. J. Zool. London 192:1-8. Stewart, M. E., and A. R. Terepka, 1969. Transport functions of the chick chorioallantoic membrane, 1. Normal histology and evidence for active electrolyte transport from the allantoic fluid in vivo. Exp. Cell Res. 58:93-106. Thommes, R. C, and C. F. McCarter, 1966. Adenohypophyseal control of water balance in the developing chick embryo. Am. Zool. 6: 517.(Abstr.) Townsley, T. S., 1930. The humidity factor in incubation. Pages 136-145 in: 4th World's Poultry Congress Proceedings. London, UK. Walker, P. A., 1943. Physical properties of the allantoic and amniotic fluids of the chick. II. Hydrogen ion concentration. J. Gen. Physiol. 26: 503-512. Woods, J. E., G. W. de Vries, and R. C. Thommes, 1971. Ontogenesis of the pituitary-adrenal axis in the chick embryo. Gen. Comp. Endocrinol. 17:407-415.
INTRODUCTION The allantoic sac arises as a diverticulum of the chick embryonic hind gut into which the mesonephric kidney begins to secrete urine during the 5th d of incuba-
Received for publication September 14, 1994. Accepted for publication January 12, 1995. Supported by state and Hatch funds allocated to the Georgia Agricultural Experiment Stations of The University of Georgia.
tion (Fiske and Boyden, 1926). The allantoic sac begins filling with fluid at 2.5 d and attains a maximum volume of 8 to 10 mL on Days 12 to 14 of incubation, depending on incubation temperature (Fiske and Boyden, 1926; Romanoff et al., 1938; Davis et al, 1988). The allantois and the chorionic membranes fuse to form the chorioallantoic membrane, which expands to lie beneath the eggshell membrane to which it becomes attached during Day 11 and 12 of incubation. The allantois is
874
INCUBATION RELATIVE HUMIDITY AND ALLANTOIC FLUID
primarily an organ of nitrogenous metabolite storage whereas the chorion functions as the embryonic respiratory organ and for the resorption of eggshell calcium. On Day 13 of incubation, allantoic fluid p H and sodium and chloride concentrations fall as allantoic fluid volume decrease, maintaining allantoic fluid hypotonic to blood, permitting water removal from the allantoic fluid down an osmotic gradient (Walker, 1943; Stewart and Terepka, 1969; Hoyt, 1979). The pituitary-adrenal axis is established in the chick embryo at approximately 14.5 d of incubation. Hypophysectomy of Day 2 embryos resulted in significant increases in embryo water content on Days 12.5 to 18 of incubation, indicating reduced ability of these embryos to maintain proper water balance (Thommes and McCarter, 1966). Woods et al. (1971) reported prolonged elevation (Days 15.5 to 17.5) of allantoic fluid volume and disrupted embryo water balance in hypophysectomized embryos due to deficiencies in neurohypophyseal hormones. These deficiencies were partially restored with pituitary grafts to the e m b r y o ' s chorioallantoic membrane or by adrenocorticotropin hormone administration. Hardin and Bell (1969) reported phenotypic correlations indicating that Day 13 allantoic fluid volume was unrelated to embryo weight (r = .19) and egg weight loss (r = .21), but highly correlated with initial egg weight (r = .64) and Day 13 egg weight (r = .66) for eggs incubated at a RH of 55%. However, after adjustment for variation in initial egg weight, there were genetic differences in the amount of allantoic fluid between White Leghorn and meat-type lines and their line crosses. Significant genetic differences in yield of influenza vaccine from allantoic fluid of chicken embryos have been reported (Bell et al, 1964). Of the variation in vaccine yield, 53% was explained by variation in egg size. However, maternal breed type remained a highly significant source of variation in vaccine yield after adjustment for initial egg weight. Romanoff and Hayward (1943) reported the progressive changes in the volume of allantoic fluid under normal (37.5 C) and extreme (34.5, 36.0, 38.5, and 39.5 C)
875
incubation temperatures at a consistent RH of 60%. On Day 13 of incubation, allantoic fluid volume p e a k e d a n d represented 9 to 10% of initial egg weight (7.3 mL) when incubated at 37.5 C, whereas at temperature extremes volume was depressed, 6.7 mL for 38.5 or 36.0 C and 4.5 mL for 39.5 or 34.5 C. They reported that incubation temperature extremes resulted in the underdevelopment of the allantoic sac and an earlier peak with high temperature and later peak in volume with low temperature. Hoyt (1979) measured the relative extraembryonic water content of the yolk, amniotic fluid, and allantoic fluid after 17 d of incubation under different incubation RH, during which eggs lost 4.5 to 17.8% of their initial weight. As desiccation increased, allantoic fluid volume decreased six times more than the embryo fluid content, with minimal changes in water content of the amniotic fluid and the yolk. Hoyt concluded that the ability of the avian embryo to differentially partition water loss results in a more stable internal environment for embryonic development when eggs are subjected to a range of RH. Davis et al. (1988) measured allantoic fluid volume from Day 12 to 19 of incubation for eggs incubated in 10, 55, and 85% RH. Allantoic fluid volume declined for all eggs from a peak on Day 12 of 10.5 mL, at which time no differences in volume were detected. Eggs incubated at 10% RH had excessive weight loss and a more rapid decline in allantoic fluid volume, whereas eggs incubated at 85% RH had lower weight loss and a slower decline in allantoic fluid volume than control eggs. Eggs experiencing either high or low weight loss first differed significantly from control eggs in allantoic fluid volume on Day 16 of incubation. The literature contains few studies evaluating high and low incubation RH effects on allantoic fluid volume and hatchability continuously from the time of setting. The present studies were undertaken to identify the influence of incubation RH within the range of 40 to 70% RH on embryonic allantoic fluid volume, egg weight loss, and hatchability in modern commercial White Leghorn eggs that possess high hatchability.
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MATERIALS AND METHODS Trial 1 To assess the effects of incubation RH of 55 and 69% on embryonic mortality and hatchability, four weekly settings of hatching eggs collected from Hy-Line® White Leghorn hens at 35,36,37, and 38 wk of age were stored for less than 1 wk at 14.4 C and 60% RH. One hundred and eighty eggs per humidity treatment and setting were allowed to warm to room temperature (22 C) overnight (15 h) and set into Jamesway2 252B incubators operating at 37.8 C and at RH of 55 or 69%. Relative humidity was regulated by wet bulb contact thermometers (28.9 and 32.2 C, respectively). Incubators contained only eggs utilized for each trial. Relative humidity was calculated from wet and dry bulb temperatures measured with thermistor probes and remote temperature recorders (TempMentor™).3 Eggs were candled on the 7th, 14th, and 18th d of incubation, and on the 18th d eggs containing viable embryos were transferred into mass hatching baskets by RH treatment and placed into a single Jamesway 252B hatcher operating at 36.9 C. On Day 20, RH was increased to 69%. All candled eggs were opened to macroscopically distinguish between infertility and early embryonic mortality. Percentage fertility of eggs set, hatchability of eggs set, hatchability of fertile eggs, and hatchability of viable embryos transferred on the 18th d of incubation were determined. Hatchability of viable embryos at transfer was used as an indicator of late embryonic mortality immediately prior to hatching. On the day of hatch (Day 22) all unhatched eggs were broken out and embryos staged according to Hamburger and Hamilton (1951) to determine embryonic age. Trial 2 To assess the effects of incubation RH (43,55, and 69%) on allantoic fluid volume of Day 14 embryos, three settings were made of fertile hatching eggs collected from
the same flock of Hy-Line® White Leghorn hens at 37,43, and 56 wk of age. This range in RH was chosen to bracket recommendations for successful hatchability reported in the literature (Lundy, 1969) and was obtained with wet bulb contact thermometer (26.7, 28.9, and 32.2 C, respectively) RH controls. Eggs were collected, stored, and set in Jamesway 252B incubators as described in Trial 1. In the first setting 30 eggs per humidity treatment (55 and 69% RH) were weighed prior to setting and again on Days 7 and 14 of incubation. Egg weight loss was determined from Day 7 and 14 egg weights. On Day 14 of incubation 30 eggs per humidity treatment containing viable embryos were removed from the incubator, cooled to room temperature for 30 min, and opened for allantoic fluid determination. The shell over the air cell was removed with forceps, and the inner egg membrane moistened with a drop of water to permit visualization of the underlying blood vessels of the chorioallantoic membrane and to allow piercing of the membrane with minimum danger of hemorrhage. The egg was inverted and the allantoic fluid poured into a funnel supported by a graduated cylinder, from which allantoic fluid volume was determined to the nearest .1 mL (Hoyt, 1979). After adjustment for initial egg weight, correlations were determined between initial egg weight and allantoic fluid volume, and between Day 14 egg weight and allantoic fluid volume. The remaining 270 eggs (excluding eggs removed at candling) continued in each RH treatment until transfer into a single hatcher on Day 18 of incubation. In the second setting an additional low humidity treatment was added: 43% RH. Two hundred and seventy-four eggs from 43-wk-old hens were set for each incubation RH, 43, 55, and 69%. On Day 14 of incubation, 60 eggs were weighed and sampled for allantoic fluid, and the remaining eggs continued through hatching. The third setting replicated the second, using 237 eggs from 56-wk-old hens for each of the three incubation RH treatments. Trial 3
2
Jamesway Manufacturing Co., Fort Atkinson, WI 53538. 3 Ryan Instrument, Redmond, WA 98073-0599.
Trial 3 was designed to determine whether allantoic fluid volume increased
INCUBATION RELATIVE HUMIDITY AND ALLANTOIC FLUID
with elevated RH or whether the greater volume detected in Trial 2 on Day 14 of incubation was due to a slower rate of allantoic fluid loss. Two sequential settings were made in two newly installed NatureForm NMC-20004 incubators (37.8 C). Allantoic fluid volume was determined from 30 eggs on Days 10, 12, 14, and 16 of incubation. In the first setting, eggs from a flock of Hy-Line® White Leghorn hens at 27 wk of age were placed (without prewarming) into incubators operating at 40 and 55% RH, whereas in the second setting, eggs from hens at 30 wk of age were placed into incubators operated at 55 and 70% RH. One hundred and fifty eggs per humidity treatment were weighed prior to setting and on the 7th d of incubation, and 30 eggs containing viable embryos per humidity treatment were weighed on the day of sampling. Allantoic fluid volume was determined as described in Trial 2. Embryo weight was determined by removing the embryo from the egg, separating the embryo from the yolk sac and chorioallantoic membrane by cutting their attachments at the umbilicus, blotting the embryo with paper toweling, and weighing to the nearest .01 g. After adjustment for initial egg weight, correlations between initial egg weight and allantoic fluid volume or embryo weight were determined. In addition, correlations between sampling day egg weight and allantoic fluid volume or embryo weight were determined. Hatchability was not evaluated in this trial. Trial 4 Trial 4 was designed to determine whether allantoic fluid volume increased and decreased with elevated or reduced RH on Days 13, 14, and 15 of incubation and whether allantoic fluid varied with eggs of different weight from flocks of different ages. Eggs from a flock of Hy-Line® White Leghorn hens 34 wk old and from a separate flock at 49 wk of age were set concurrently (without prewarming) into three NatureForm incubators operating at 37.8 C, and RH of 40, 55, and 70%. The ability to run the three RH treatment
4
NatureForm Co., Jacksonville, FL 32202.
877
concurrently was made possible by the installation of additional incubators. Eighty-eight eggs per humidity treatment and hens age were weighed prior to setting and egg weight equalized for each sampling day and RH treatment (34-wk-old = 57.34 g and 49-wk-old = 59.45 g). On the 7th d of incubation and on the day of sampling 25 eggs containing viable embryos per humidity treatment were weighed and from these weights egg weight loss was determined. Allantoic fluid volume was determined from 25 eggs on Days 13, 14, and 15 of incubation as described in Trial 2 and embryo weight was determined as described in Trial 3. After adjustment for initial egg weight, correlations between initial egg weight and allantoic fluid volume or embryo weight were determined, in addition to correlations between sampling day egg weight and allantoic fluid volume or embryo weight. An additional 247 eggs from 34-wk-old hens and 249 eggs from 49-wk-old hens were also set for each RH for determination of hatchability (Day 21). Statistical Analysis Fertility and hatchability data were analyzed using the General Linear Models (GLM) procedure for least squares analysis of disproportionate subclass frequency (SAS Institute, 1991). The percentage response was based on using the normal approximation for the binomial value, as the cell frequencies were large. Least squares means was used to separate means within significant main effects (P < .05). Sources of variation in the model included main effects: for Trials 1 and 2 were RH and setting, for Trial 3 were RH, setting, and sampling day, and for Trial 4 were RH, hens age, and sampling day. Interaction terms for main effects were tested for significance. Egg weight, egg weight loss during incubation, embryo weight, and allantoic fluid volume data were analyzed by analysis of variance in Trials 2,3, and 4. Sampling day significantly influenced allantoic fluid volume and embryo weight in Trials 3 and 4. The main effect setting was frequently significant within Trials 1, 2, and 3. Therefore, means are presented for each trial by setting and sampling day. In Trials 2,3, and 4 after adjustment for initial egg weight,
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correlations between initial egg weight and allantoic fluid volume or embryo weight were determined, in addition to correlations between sampling day egg weight and allantoic fluid volume or embryo weight.
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RESULTS AND DISCUSSION In Trial 1, elevated incubation RH at 69% did not result in differences (P > .05) in hatchability or distribution of embryonic mortality compared with control eggs incubated at 55% RH (Table 1). The average for the four settings was 97% fertility, 88.1% hatchability of eggs set, 90.8% hatchability of fertile eggs, and 94.7% hatchability of viable embryos at transfer. The main period of mortality for both RH treatments occurred following transfer into the hatcher. The lack of a difference between RH treatments in embryo mortality after transfer into the hatcher indicates that elevated incubation at 69% RH was not detrimental to embryo survival or ability to hatch. Incubation RH (69%) was calculated within 1% of the value for which the high RH contact thermometer was chosen (70%). For Trial 2, initial egg weight, percentage egg weight lost per day of incubation, and allantoic fluid volume expressed as a percentage of initial egg weight are presented in Table 2. Initial egg weight was heavier (P < .05) for eggs set at 69% RH than at 55% RH for the first setting, but was not different among RH treatments for the second and third settings. Initial egg weight increased significantly with hens' age over the three settings. Percentage egg weight loss per day of incubation was inversely related to incubation RH, with the greatest loss for eggs incubated at 43% RH (.68%) and the lowest loss for eggs incubated at 69% RH (.40%). Eggs incubated at 55% RH had intermediate percentage egg weight loss (.54%). These values projected as 19-d egg weight losses were 12.8,10.4, and 7.7% of initial egg weight for RH of 43, 55, and 69%, respectively. The low incubation RH (43%) was calculated to be 3% higher than the value for which the contact thermometer was chosen (40%). Allantoic fluid volume was positively influenced by incubation RH (P < .05).
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INCUBATION RELATIVE HUMIDITY AND ALLANTOIC FLUID
TABLE 2. Egg weight, percentage incubation egg weight loss, and percentage allantoic fluid volume for eggs incubated at relative humidities of 43, 55, and 69%, Trial 2 Relative humidity Variable setting
1
55%
Pooled
69%
SEM
54.02" 57.80 62.56
55.85a 58.46 61.30
.58 .42 .53
.68' .67'
.52" .55" .57"
.41" .39< .41 c
.01 .01 .01
13.06c 14.67=
15.77 14.82" 15.77"
17.11 18.13a 18.54"
.48 .36 .38
43%
'~1
Initial egg weight Setting 1 Setting 2 Setting 3 Percentage egg weight loss per day of incubation Setting 1 Setting 2 Setting 3 Allantoic fluid volume as a percentage of initial egg weight Setting 1 Setting 2 Setting 3
(SI 2
58.03 61.58
"^Means within a row with no common superscript differ significantly (P < .05). !Number of eggs in Setting 1 n = 30, Settings 2 and 3 n = 60, hens age 37, 43, and 56 wk. 2 There was no 43% RH treatment for Setting 1.
Values for allantoic fluid overlapped among settings and RH treatments due to the increase in initial egg weight with each successive setting. This overlap among RH treatments disappeared when allantoic fluid volume was expressed as a percentage of initial egg weight. Allantoic fluid volume on Day 14 of incubation expressed as a percentage of initial egg weight was 17.1 to 18.5% for eggs incubated at 69% RH, 14.8 to 15.8% for eggs incubated at 55% RH, and 13 to 14.7% for eggs incubated at 43% RH. The difference in allantoic fluid volume as a percentage of egg initial weight was significant in all settings except for eggs incubated at 55 and 69% RH in the first setting, in which egg weight differed significantly. These results are in agreement with those reported by Davis et ah (1988) that incubation RH can positively or negatively influence allantoic fluid by Day 14 of incubation. A similar trend with higher values was calculated for allantoic fluid volume as a percentage of Day 14 egg weight. After adjustment for initial egg weight, correlation coefficients (r) between initial egg weight or Day 14 egg weight
and allantoic fluid volume were significant and ranged from r = .30 to .70. No pattern associated with RH treatment was observed. In Trial 2, for all three settings, fertility (95.9%), hatchability of fertile eggs (91.7%), and hatchability of viable eggs at transfer (95%) were not significantly different among RH treatments. The results of Trials 1 and 2 indicate no adverse effects on hatchability with continuous incubation RH from 43 to 69% and the projected 19-d egg weight loss from 7.4 to 12.9%. Gildersleeve (1984) subjected broiler eggs to 55% RH or increasing RH profiles from 52 to 67% during incubation and reported no difference in 18-d egg weight loss although differences were present earlier. In addition, Gildersleeve reported no differences in fertility or hatchability with this programmed change in RH. Simkiss (1980) removed 6 mL of allantoic fluid from Day 13 embryos (equivalent to 100% increase in normal incubation water loss) and reported normal hatchability and equivalent percentage body composition but reduced chick weight by 6.6 g. In a separate experiment,
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TABLE 3. Egg weight, percentage egg weight loss, percentage allantoic fluid volume, and percentage embryo weight for eggs incubated at 40, 55, or 70% relative humidity1 and from which allantoic fluid volume was determined on Days 10, 12, 14, and 16 of incubation, Trial 3 Days of incubation Variable Initial egg weight, g 40% RH 55% RH Pooled SEM 55% RH 70% RH Pooled SEM Percentage egg weight loss per day of incubation, % 40% RH 55% RH Pooled SEM 55% RH 70% RH Pooled SEM Allantoic fluid volume as a percentage of initial egg weight, % 40% RH 55% RH Pooled SEM 55% RH 70% RH Pooled SEM Embryo weight as a percentage of initial egg weight, % 40% RH 55% RH Pooled SEM 55% RH 70% RH Pooled SEM
10
12
14
16
62.43 63.57 .76 63.99 63.52 .75
63.32 61.95 .90
63.36 63.14 .73 60.79b 63.54" .72
62.83 64.19 .78 65.03" 62.07b .77
62.15 62.91 .99
.73" .51b .02 .52" .36b .01
.70' .50" .02 .54* .33" .01
.67" .48 b .02 .56" .35b .01
.64" .50b .02 .49" .35 b .01
4.87b 6.38a .31 5.65" 7.34a .28
12.91 b 15.20" .46 14.75" 16.00" .44
14.05b 17.46" .53 15.04b 19.44" .45
8.67" 11.13" .45 12.52b 14.37" .44
3.83a 3.49b .05 3.79" 3.52b .06
7.64 7.34 .14 7.76" 7.23" .16
15.95" 14.63b .25 16.09" 14.60b .26
27.22 26.64 .36 26.42 26.68 .36
a b
- Means within days of incubation and setting with no common superscript differ significantly (P < .05). Relative humidity 40 and 55%, hens age 27 wk, n = 30; Relative humidity 55 and 70%, hens age 30 wk, n =
30.
Simkiss (1980) increased incubation water loss (an additional 8 g) by drilling holes in the eggshell of Day 13 embryos. No evidence of dehydration was detected. Simkiss (1980) concluded that the chick embryo has the regulatory ability to recycle the water in the egg, to grow to a size commensurate with the available water supply, to tolerate a wide range of eggshell porosity and incubation conditions. In Trial 3, initial egg weight did not differ between eggs in the two settings (mean 63.1 to 63 g) (Table 3). However, in the second setting eggs sampled on Days
14 and 16 of incubation differed significantly in initial egg weight for the 70 and 55% RH treatment. Percentage egg weight loss per day of incubation in Trial 3 was inversely related to incubation RH and did not differ among the four sampling days. Projected 19-d egg weight loss was 13.0, 9.4, 10.1, and 6.6% for incubation RH of 40, 55, 55, and 70%, respectively. Incubation RH was calculated to equal the set point values of the NatureForm incubators. Allantoic fluid volume was positively related to incubation RH and peaked on Day 14 of incubation (Table 3). Allantoic
INCUBATION RELATIVE HUMIDITY AND ALLANTOIC FLUID
fluid as a percentage of initial egg weight and sampling day egg weight increased with RH on Days 10, 12, 14, and 16 of incubation. Allantoic fluid volume and percentage egg weight loss per day of incubation varied significantly between the two 55% RH groups. Therefore the two settings were analyzed and are reported independently. These differences between the 55% RH groups occurred although eggs were collected from the same flock and set in the same incubator 3 wk apart, and reflects the inherent variability between settings and the importance of concurrent evaluation of RH treatments. After adjustment for initial egg weight, correlations between initial egg weight or egg weight on Day 10 and allantoic fluid volume were not significant for all RH treatments. Positive correlations (P < .05) were found for both initial egg weight or day of sampling egg weight and allantoic fluid volume on Day 12 (r = .39 to .59) and on Days 14 and 16 (r = .67 to .79) of incubation. The magnitude of the correlations appeared to be independent of RH treatment and were determined by the particular eggs within each treatment. Initial egg weight for eggs sampled on Day 14 was greater for eggs set in the 70% RH than the 55% RH incubator and corresponding allantoic fluid as a percentage of initial egg weight or Day 14 egg weight was also greater for the 70% RH treatment. However, on Day 16, although initial egg weight was lower for eggs set in the 70% RH than in the 55% RH, allantoic fluid measurements and calculated percentages were greater for eggs incubated at 70% RH, indicating the positive influence of RH on allantoic fluid volume. Embryo weights expressed as a percentage of initial egg weight were consistently higher in the low RH treatments on Day 10 through 15 of incubation. This finding is in contrast to that of Romanoff (1929), who reported that elevated incubation RH positively influenced embryonic growth rate; 80% RH accelerated embryo wet weight compared to embryos incubated at 40% RH. However, Romanoff's experiments utilized eggs from the same flock in succession and were not controlled for egg weight, and the water temperature of the
881
humidifier was not reported. Townsley (1930) reported that, when RH was supplied via steam, as RH is increased operating incubation temperature must be decreased (and similarly when RH is low the incubation temperature should be increased) to optimize hatch time, hatchability, size of chicks, and chick livability. Penquite (1938) found no affect of RH (40, 62, or 80%) on embryo wet or dry weight and nitrogen content (Day 4 through 20) after the influence of egg weight was removed for eggs incubated in commercial incubators designed to maintain uniform conditions at all levels. The differences found in this study, heavier embryos incubated at lower incubation RH, may be due to greater fluctuation in incubation temperature due to increased frequency of humidity misting (room temperature spray) at the higher RH incubation treatments. Romanoff et al. (1938) and Penquite (1938) reported greater effects of incubation temperature on acceleration or depression of embryo weight during the 1st wk of incubation, and no detectable effects during the 2nd or 3rd wk. The greater effect of incubation temperature during the 1st wk than the 2nd or 3rd and the onset of albumen utilization by the embryo (Day 12.5) may explain the diminished difference in embryo weight at 16 d of incubation. In the present study, after adjustment for initial egg weight, correlations between initial egg weight and embryo weight were significant for only two sampling times, 40% RH on Day 10 (r = .52) and for the 55% RH on Day 16 (r = .37 and .40). In Trial 4, initial egg weight was significantly heavier for 49-wk-old hens (59.5 g) than for 34-wk-old hens (57.34 g) (Table 4). Egg weight did not differ within flocks for eggs placed in the three RH treatments or for the 3 d of allantoic fluid sampling. Percentage egg weight loss significantly increased with lower RH for both flocks and did not differ among the three sampling days. Percentage egg weight loss was higher for eggs from 49-wk-old hens than for eggs from 34-wk-old hens within the same RH treatment. Projected 19-d egg weight loss for eggs from 34-wk-old hens was 16.6, 12.7, and 9.1% incubation at RH of 40, 55,
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TABLE 4. Egg weight, percentage egg weight loss, percentage allantoic fluid volume, and percentage embryo weight for eggs incubated at 40, 55, or 70% relative humidity1 from which allantoic fluid volume was determined on Days 13, 14, and 15 of incubation, Trial 4 Days of incubation Variable Initial egg weight, g Hens age 34 wk 40% RH 55% RH 70% RH Pooled SEM Hens age 49 wk 40% RH 55% RH 70% RH Pooled SEM Percentage egg weight loss per day of incubation, % Hens age 34 wk 40% RH 55% RH 70% RH Pooled SEM Hens age 49 wk 40% RH 55% RH 70% RH Pooled SEM Allantoic fluid volume as a percentage of initial egg weight, % Hens age 34 wk 40% RH 55% RH 70% RH Pooled SEM Hens age 49 wk 40% RH 55% RH 70% RH Pooled SEM Embryo weight as a percentage of initial egg weight, % Hens age 34 wk 40% RH 55% RH 70% RH Pooled SEM Hens age 49 wk 40% RH 55% RH 70% RH Pooled SEM
13
14
15
56.36 57.66 57.86 .67
57.92 57.67 57.36 .63
57.35 56.33 57.59 .71
59.03 60.18 58.82 .55
59.97 59.27 59.93 .63
59.16 58.90 60.25 .63
.89' .64b .48' .02
.85" .69b .47' .02
.88" .68" .49' .02
.91" .72b .52' .03
.99* .69b .47' .04
.97' .73b .52' .02
13.38' 16.91b 18.68" .47
9.67' 12.74b 15.24" .49
5.22' 9.42b 12.79" .53
11.12' 13.77b 16.59" .44
9.00' 12.06b 14.52" .52
4.34' 7.27b 10.83" .41
13.45 12.94 12.74 .17
18.37" 17.71b 17.72b .21
25.34" 25.24"b 24.27b .23
13.32" 12.72" 12.47b .16
18.06" 17.61b 17.06b .20
24.40 24.64 23.76 .33
a-c Means for variables within days of incubation and hen age with no common superscript differ significantly (P < .05). x Number of eggs per treatment and day of sampling n = 25.
and 70% respectively, and was 18.2, 13.6, and 9.6% for eggs from 49-wk-old hens incubated at RH 40, 55, and 70% respectively.
Allantoic fluid volume was again positively related to incubation RH and was maximal on Day 13 of incubation (Table 4). Allantoic fluid volume was greater for
INCUBATION RELATIVE HUMIDITY AND ALLANTOIC FLUID
the 34-wk-old flock t h a n for the 49-wk-old flock, although initial egg weight was the opposite. This difference resulted from the greater egg weight loss of eggs from 49-wk-old hens than from 34-wk-old hens. Trial 4 utilized eggs from different flocks in order to collect allantoic fluid samples from different age hens that were incubated concurrently. Allantoic fluid volume as a percentage of initial egg weight and sampling egg weight increased with RH on all 3 d of collection. After adjustment for initial egg weight, correlations between initial egg weight or sampling day egg weight and allantoic fluid volume were significant (r = .4 to .83). As in Trial 3, the lowest humidity treatments had heavier embryos. Significant differences in embryo weight were detected among RH treatments for the 34-wk-old flock on Day 14 and 15 and for the 49-wk-old flock on Days 13 and 14. This difference is possibly due to a higher incubation temperature in the 40% RH incubator than in the 55 or 70% incubator, with less frequent additions of water from the mister nozzles. This concept is supported by the lower coefficient of variation for mean temperature in the 40% RH (.51%) incubator than in the 55 (.69%) or 70% RH (.73%) incubators. Correlations between initial egg weight or sampling egg weight and embryo weight were not significant. Fertility in Trial 4 was 98.9% for eggs from 34-wk-old hens and 99.1% for eggs from 49-wk-old hens and did not differ among RH treatments. Hatchability of viable embryos transferred was significantly greater for eggs from 34-wk-old hens incubated at 55% RH (98.7%) than either at 40 (93.3%) or 70% RH (92.2%). There was no difference in hatchability for eggs from 49-wk-old hens incubated at 40 (90.6%), 55 (93%), or 70% RH (89%). Lower hatchability for eggs from 34-wk-old hens incubated at 40 or 70% RH and for all eggs from 49-wk-old hens was due to an increased percentage of unhatched eggs containing live embryos that had pipped the eggshell. These results agree with those of Kirk et al. (1980), in which broiler hatching eggs incubated for 21 d and 7.5 h at increased RH had increased late embryo mortality and pipped eggs. The depres-
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sion in hatchability detected in Trial 4 but not in Trials 1 and 2 may be due to the difference in incubation length (22 vs 21 d) recommended by the incubator manufacturer. Therefore, additional investigation of the influence of incubation RH on incubation length, hatch time, and true hatchability is necessary. Results in the present study demonstrate that allantoic fluid volume is directly influenced by incubation RH and can be decreased or increased relative to an intermediate RH of 55%. Furthermore, although incubation RH ranged from 43 to 69% in Trials 1 and 2, there were no differences in hatchability or embryonic mortality. These findings indicate that the chicken embryo can maintain proper water balance by regulating allantoic fluid volume within this range of incubation RH. These results suggest that incubation RH and egg water loss during incubation may not be primary causes of elevated embryonic mortality. Coefficients of variation for daily rate of water loss from eggs of seven species of terns ranged from 4 to 46%, with an average of 28% (Rahn et al, 1976), indicating that in some species there is considerably more water loss variation than in others. The average coefficient of variability for eggshell conductance for 29 species of precocious and altricial birds was 25% (Ar et al, 1974) and 18% for 54 species of ducks and geese (Hoyt, 1979). Coefficients of variation for daily rate of water loss from eggs in Trial 2 ranged from 9.5 to 16.8%; in Trial 3 ranged from 9.5 to 23.6%; and in Trial 4 ranged from 10 to 37.7% for eggs from 34-wk-old hens a n d 6.8 to 22.9% for e g g s from 49-wk-old hens. Overall, the coefficients of variation for daily egg weight loss varied 1.8- to 3.8-fold. The results of this study agree with those of Hoyt (1979) that the avian embryo has the ability to regulate their state of hydration and maintain a fairly stable environment for development within this range of RH values. This study further supports the positive influence of egg weight and the negative influence of egg weight loss during incubation on allantoic fluid volume.
REFERENCES Ar, A., C. V. Paganelli, R. B. Reeves, D. G. Greene, and H. Rahn, 1974. The avian egg: water vapor
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conductance, shell thickness and functional pore area. Condor 76:153-158. Bell, A. E., H. W. McNary, and J. O. MacFarlane, 1964. Genetic variation in the vaccine yield from embryonating avian eggs. Poultry Sri. 43: 1258-1262. Davis, T. A., S. S. Shen, and R. A. Ackerman, 1988. Embryonic osmoregulation: Consequences of high and low water loss during incubation of the chicken egg. J. Exp. Zool. 245:144-156. Fiske, C. H., and E. A. Boyden, 1926. Nitrogen metabolism in the chick embryo. J. Biol. Chem. 70:535-556. Gildersleeve, R. P., 1984. The effect of humidity and broiler strain on egg weight losses during incubation. Poultry Sci. 63:2140-2144. Hamburger V., and H. L. Hamilton, 1951. A series of normal stages in the development of the chick embryo. J. Morphol. 88:49-92. Hardin, R. T., and A. E. Bell, 1969. Factors relating to genetic differences in the amount of chick allantoic fluid. Poultry Sci. 48:1655-1660. Hoyt, D. F., 1979. Osmoregulation by avian embryos: The allantois functions like a toad's bladder. Physiol. Zool. 52:354-362. Kirk, S., G. C. Emmans, R. McDonald, and D. Arnot, 1980. Factors affecting the hatchability of eggs from broiler breeders. Br. Poult. Sci. 21:37-53. Lundy, H., 1969. A review of the effects of temperature, humidity, turning and gaseous environment in the incubator on the hatchability of the hen's egg. Pages 143-178 in: The Fertility and Hatchability of the Hen's Egg. T. C. Carter and B. M. Freeman, ed. Oliver and Boyd, Edinburgh, Scotland. Penquite, R., 1938. Influence of temperature and humidity upon the growth of chick embryos in a mechanically ventilated incubator. Iowa Agric. Exp. Sta. Bull. 232. Ames, IA. Rahn, H., C. V. Paganelli, I. C. T. Nisbet, and G. C. Whittow, 1976. Regulation of incubation water
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