Embryonic developmental plasticity of the chick: increased CO(2) during early stages of incubation changes the developmental trajectories during prenatal and postnatal growth

This study investigated the effect of non-ventilation of the incubator during the first 10 days of incubation on carbon dioxide (CO(2)) concentrations in the incubator and its effects on the embryonic and post-hatch development of the chicken (Gallus gallus). Two different incubation conditions were created, one incubator was kept at standard conditions, with adequate ventilation (V) and a second incubator was non-ventilated (NV) during the first ten days of incubation, allowing the CO(2) to rise. After the first 10 days, both incubations were continued under standard conditions. The experiment was repeated twice with different ages of the breeders (45 and 60 wks) which resulted in different CO(2) levels at ED10 (1.5 and 1%). The CO(2) concentration in the V incubators remained below 0.1% in these first 10 days. The eggs of the NV incubation showed higher pCO(2) levels in the air cell from ED10 until ED14 compared to the eggs of the V group. The NV embryos had significantly higher absolute and relative (to egg weight) body weights from ED10 until ED18, pointing to an accelerated embryonic growth. At internal pipping, the NV chick embryos had higher plasma corticosterone and T(3) levels and higher pCO(2) in the air cell. Chicks incubated under NV conditions hatched 10 h earlier in the first and 15 h earlier in the second experiment and the spread of hatch was narrower. During the post-hatch period, the NV chickens had a higher body weight compared to the V chickens. From these results, it is clear that higher levels of CO(2) during the first ten days of incubation have persistent (epigenetic) effects during the incubation and early post-hatch period.     

Statistical review of the henhouse experiments: the effects of a pulsed magnetic field on chick embryos.

This paper analyzes data from a study conducted by the United States Office of Naval Research on the effects of pulsed magnetic fields on chick embryos. The experiment involved incubation of eggs under carefully controlled conditions in six different laboratories. The original analysis included inappropriate statistical methodology for analyzing the experimental results. Since the conclusions from this study rest so heavily on the results of statistical analyses, choosing the proper methodology is imperative. The major aim of this paper then is to introduce more appropriate analytic tools and illustrate their use in the present context. Qualitatively our results agree with those of the original analysis; our findings about interactions between effects, however, makes interpretation of these effects more subtle. We apply linear logistic modeling to counts of damaged embryos, using as covariates factors corresponding to exposure, laboratory, incubator, run, and measurements of background radiation. This facilitates estimation of the size of the effects. The effects of laboratory, incubator, and run are explored both as fixed and random effects. We find statistically significant exposure and laboratory effects, in accordance with the original study. However, we also find that the inter-laboratory variation in exposure effect is at least as large as the exposure effect itself. The presence of such effects fundamentally alters the interpretation of the fitted model, as is graphically presented.     

The effect of pulsed and sinusoidal magnetic fields on the morphology of developing chick embryos

Several investigators have reported robust, statistically significant results that indicate that weak (∼ 1 μT) magnetic fields (MFs) increase the rate of morphological abnormalities in chick embryos. However, other investigators have reported that weak MFs do not appear to affect embryo morphology at all. We present the results of experiments conducted over five years in five distinct campaigns spanning several months each. In four of the campaigns, exposure was to a pulsed magnetic field (PMF); and in the final campaign, exposure was to a 60 Hz sinusoidal magnetic field (MF). A total of over 2500 White Leghorn chick embryos were examined. When the results of the campaigns were analyzed separately, a range of responses was observed. Four campaigns (three PMF campaigns and one 60 Hz campaign) exhibited statistically significant increases (P ≥ 0.01), ranging from 2-fold to 7-fold, in the abnormality rate in MF-exposed embryos. In the remaining PMF campaign, there was only a slight (roughly 50%), statistically insignificant (P = 0.2) increase in the abnormality rate due to MF exposure. When the morphological abnormality rate of all of the PMF-exposed embryos was compared to that of all of the corresponding control embryos, a statistically significant (P ≥ .001) result was obtained, indicating that PMF exposure approximately doubled the abnormality rate. Likewise, when the abnormality rate of the sinusoid-exposed embryos was compared to the corresponding control embryos, the abnormality rate was increased (approximately tripled). This robust result indicates that weak EMFs can induce morphological abnormalities in developing chick embryos. We have attempted to analyze some of the confounding factors that may have contributed to the lack of response in one of the campaigns. The genetic composition of the breeding stock was altered by the breeder before the start of the nonresponding campaign. We hypothesize that the genetic composition of the breeding stock determines the susceptibility of any given flock to EMF-induced abnormalities and therefore could represent a confounding factor in studies of EMF-induced bioeffects in chick embryos. Bioelectromagnetics 18:431–438, 1997. © 1997 Wiley-Liss, Inc.     

Naltrexone pretreatment blocks microwave-induced changes in central cholinergic receptors

Repeated exposure of rats to pulsed, circularly polarized microwaves (2,450-MHz, 2-microseconds pulses at 500 pps, power density 1 mW/cm2, at an averaged, whole-body SAR of 0.6 W/kg) induced biphasic changes in the concentration of muscarinic cholinergic receptors in the central nervous system. An increase in receptor concentration occurred in the hippocampus of rats subjected to ten 45-min sessions of microwave exposure, whereas a decrease in concentration was observed in the frontal cortex and hippocampus of rats exposed to ten 20-min sessions. These findings, which confirm earlier work in the authors' laboratory, were extended to include pretreatment of rats with the narcotic antagonist naltrexone (1 mg/kg, IP) before each session of exposure. The drug treatment blocked the microwave-induced changes in cholinergic receptors in the brain. These data further support the authors' hypothesis that endogenous opioids play a role in the effects of microwaves on central cholinergic systems.     

Influence of cortisol on developmental rhythms during embryogenesis in a tropical damselfish

Newly-spawned teleost eggs can vary widely in their maternal endowment of a variety of hormones, including cortisol. Field and laboratory experiments have shown that initial egg cortisol concentrations directly influence the size at hatching of the benthic spawning damselfish, Pomacentrus amboinensis. The present study examines the mechanism by which cortisol influences larval size at hatching by investigating the growth and developmental rhythms throughout embryogenesis. Newly spawned eggs of P. amboinensis were collected from natural benthic nests, and half of each clutch was incubated in a moderate level of cortisol (2.7 x 10(-6) M, equivalent to a concentration of 0.79 pg/egg). Cortisol was found to have no affect on the rate of cell-pulsations up to epiboly (18 hr post-fertilization), with cells pulsing at a mean rate of 56-60 pulses/min. Cortisol had an affect on the relative growth rate from the start of gastrulation to knot formation. Growth in the cortisol-supplemented embryos was pulsed, with periods of fast growth punctuated by long periods of stasis. Overall growth rates during this period were lower in the cortisol-supplemented embryos despite their higher growth during active periods. Pulse rates of somite cells and contraction rhythms of myotomes and the heart were twice as high in cortisol-supplemented embryos than controls. Despite this, cortisol-supplemented eggs developed at the same rate as controls and hatched at the same time. This study suggests that the maternal endowment of cortisol to eggs plays a vital role in determining the embryonic rhythms by which embryos grow and may be directly influencing metabolism.     

Inhomogeneous background magnetic field in biological incubators is a potential confounder for experimental variability and reproducibility

This report shows that the background magnetic field in biological incubators can vary by orders of magnitude within and between incubators. These variations can be observed within the same incubator in locations that are centimeters apart from each other as well as between incubators that are identical and located in the same laboratory. Additionally, the values measured were frequently outside the range of magnitudes found naturally on the Earth's surface or ordinary habitation spaces. Exposure to such altered magnetic field environments has been experimentally shown to be sufficient to cause numerous effects in cell cultures. Examples of the effects reported span from differential generation of free radicals and heat shock proteins to differences in cellular proliferation, differentiation, and death. Although the effects are not well established and the molecular mechanism of action is currently under debate, these observations alone support the notion that the inhomogeneity of the background magnetic field in incubators is a potential confounding source of the variability and reproducibility for studies performed on cell cultures. In this regard, it is recommended that special measures be adopted to control the background magnetic fields in incubators when investigating the biological effects of exposure to magnetic fields of comparable characteristics as the ones measured in this study, or when studying small biological effects in general. Bioelectromagnetics 34:337–348, 2013. © 2012 Wiley Periodicals, Inc.     

Effect of a 12 HZ and of a 460 HZ pulsed magnetic field on the weight of AKR mice

AKR mice were exposed to a 6 mT, 12 Hz or 460 Hz pulsed magnetic field (PMF) 30 minutes twice a week. The exposure took placein utero and/or during the life span for four consecutive generations. The adult mice exposed to the 460 Hz PMF only after the birth time were lighter than the controls; for the two frequencies the decrease in weight with the ageing was less pronounced than in the controls. When the exposure took placein utero the exposed new-born mice were heavier than the controls. The difference in weight progressively disappeared when the mice were exposed to the 12 Hz PMF, persisted when the mice were exposed to the 460 Hz PMF.     

Development of chicken embryos exposed to an intermittent horizontal sinusoidal 50 Hz magnetic field

The effects of intermittent exposure (2 h on/22 h off) to a 200 μT horizontal, sinusoidally oscillating (50 Hz) magnetic field were studied in 210 fertilized chicken eggs. Two hundred ten control eggs (sham-exposed) were incubated in the same chamber as the experimental eggs. Chick embryos were examined for developmental anomalies and maturity stage after 48 h of incubation. Immunohistochemical analysis of extracellular membrane components (laminin, fibronectin, and type IV collagen) were conducted on day 7 and histological examinations for malformations of brain, liver, and heart, on days 7, 12, and 18 of incubation. Furthermore, egg fertility and egg weights were evaluated on days 2, 7, 12, and 18. The investigation also measured the body weight of chickens for 90 days from hatching and included histological analysis of body organs. Each variable was investigated blind. Statistical comparison between exposed and sham-exposed values did not show significant differences in any of the variables investigated. Thus, it appears that the exposure of embryos to an intermittent 200 μT magnetic field at 50 Hz does not cause developmental anomalies, changes in maturity stage, alterations in distribution of extracellular membrane components, or malformations in the brain, liver, or heart. Moreover, there were no differences in body weight, morphology, or histology of central nervous system, liver, heart, or testis in 90-day-old chickens hatched from exposed in comparison to sham-exposed eggs. © 1996 Wiley-Liss, Inc.     

Gas exchange and hatchability of chicken eggs incubated at simulated high altitude

Chicken eggs laid at sea level were incubated at sea level (control conditions), at a simulated altitude of 5.5 km without any further measures (natural conditions), and at a simulated altitude of 5.7 km at optimal incubator gas composition (optimal conditions). Under optimal conditions the incubator relative humidity was 70% throughout incubation, the gas mixture supplied to the incubator contained 45% O2-55% N2, and the ventilation rate was reduced to 6% of control in order to maintain the normal air-space gas tensions and to compensate for the increased eggshell conductance at altitude. The embryos that developed under control conditions showed a normal CO2 production with 94% hatchability of fertile eggs. Under natural conditions at altitude all embryos died within a few days. Optimal conditions resulted in an almost normal gas exchange and in an improvement of hatchability from 0 to 81% of fertile eggs.     

Chick embryo development can be irreversibly altered by early exposure to weak extremely-low-frequency magnetic fields

Several reports have shown that weak, extremely-low-frequency (ELF), pulsed magnetic fields (PMFs) can adversely affect the early embryonic development of the chick. In this study, freshly fertilized chicken eggs were exposed during the first 48 h of postlaying incubation to PMFs with 100 Hz repetition rate, 1.0 μT peak-to-peak amplitude, and 500 μs pulse duration. Two different pulse waveforms were used, having rise and fall times of 85 μs (PMF-A) or 2.1 μs (PMF-B). It has been reported that, with 2 day exposure, these fields significantly increase the proportion of developmental abnormalities. In the present study, following exposure, the eggs were allowed to incubate for an additional 9 days in the absence of the PMFs. The embryos were taken out of the eggs and studied blind. Each of the two PMF-exposed groups showed an excess in the percentage of developmental anomalies compared with the respective sham-exposed samples. This excess of anomalies was not significant for the PMF-A-treated embryos (P = 0.173), whereas it was significant for the PMF-B-exposed group (P = 0.007), which showed a particularly high rate of early embryonic death. These results reveal that PMFs can induce irreversible developmental alterations and confirm that the pulse waveform can be a determinant factor in the embryonic response to ELF magnetic fields. The data also validate previous work based on the study of PMFs' effects at day 2 of embryonic development under field exposure. © 1994 Wiley-Liss, Inc.     

TEMPERATURE VARIABILITY WITHIN BACTERIOLOGICAL INCUBATORS

SUMMARY: Temperature variability within four bacteriological incubators regulated for 30° has been studied using readings taken from the normal thermometers and from others placed on the incubator shelves.
During 5-day incubation periods, differences between readings from individual thermometers and between thermometers on different shelves in the same incubator rarely exceeded 0·5° and 1° respectively. The mean temperature recorded on the top shelf was significantly higher ( P <0·05) than that on the bottom shelf for all incubators, and than that indicated by the normal thermometers for two incubators.
Colony counts for pasteurized milk were not materially affected by within incubator temperature differences in this experiment nor, at three of the four centres, by removal from the incubators for brief periods for counting after intermediate incubation times.     

Carbon-activated gas filtration during in vitro culture increased pregnancy rate following transfer of in vitro-produced bovine embryos

Many environmental conditions for in vitro embryo production (IVP) systems for cattle have been relatively standardised, e.g. media composition, temperature, pH, water quality, and atmospheric composition. However, little attention has been paid to the quality of ambient laboratory air and the gas environment in incubators. Although a few studies have examined the effects of chemical air contamination on IVP of human embryos, there are no published accounts for domestic animal embryos. Therefore, this study investigated the effects of an intra-incubator carbon-activated air filtration system (CODA) during in vitro culture (IVC) on embryonic development and subsequent pregnancy rate of bovine embryos. Immature cumulus-oocyte-complexes (COCs) were obtained twice-weekly by ultrasonic-guided transvaginal oocyte aspiration. The COCs were matured in TCM199/FCS/LH/FSH, fertilized with frozen-thawed Percoll-separated semen, and subsequently cultured for 7 day in SOFaaBSA. Day 7 embryos were transferred either fresh or frozen/thawed. The experimental design was a 2 x 2 factorial; presumptive zygotes were placed either in a conventional CO(2)-O(2)-N(2) incubator (Control group) or in an identical CO(2)-O(2)-N(2) incubator with a CODA intra-incubator air purification unit (CODA group) for IVC. The embryo production rate at Day 7 was not affected by the CODA air purification unit (23.4 and 24.7% morulae and blastocysts per oocyte for control and CODA, respectively) nor was there any significant effect on embryo stage or quality. However, the pregnancy rate was improved (P=0.043) for both fresh (46.3% versus 41.0%) and frozen/thawed embryos (40.8% versus 35.6%). In conclusion, atmospheric purification by the CODA intra-incubator air purification unit significantly increased pregnancy rate following transfer of in vitro-produced bovine embryos.     

Electrofusion of two-cell bovine embryos for the production of tetraploid blastocysts in vitro

Tetraploid bovine blastocysts were produced experimentally by electrofusion of in vitro matured and fertilized, zona-enclosed two-cell embryos (33-35 hr after initiation of sperm-egg incubation) using three fusion protocols. Field strengths of 1.0, 1.4, and 2.4 kV/cm were tested and the rate of fusion, subsequent cleavage, and blastocyst development were measured for each. High rates of fusion (76.5% +/- 2.8%), cleavage (72.5% +/- 7.4%) and blastocyst development (56.1% +/- 6.4%) were achieved with the application of 1. 4 kV/cm as a single 100-microseconds pulse. Embryos were scored 30 and 60 min after stimulation for fusion. No time effect for fusion, cleavage, or blastocyst development was observed. Chromosome preparations of day 7 blastocysts revealed 12.5% of fused embryos were tetraploid. This is a significant increase from that found in nonfused embryos where spontaneous tetraploidy did not occur. An electrical stimulus of 1.0 kV/cm applied as two 50-microseconds pulses produced significantly less one-cell embryos (64.2% +/- 3.0%) compared to 1.4 kV/cm while cleavage (79.9% +/- 3.4) and blastocyst development (44.6% +/- 4.0%) were not different from that for unexposed control embryos (89.5% +/- 2.3% and 57.2% +/- 3.2%, respectively). Embryos fused at 2.4 kV/cm applied as a single 30-microseconds pulse (69.7% +/- 5.7%) showed significantly lower cleavage (72.1% +/- 3.7%) and blastocyst rates (40.2% +/- 4.6%) compared to the unexposed control.     

Biological effects of low-frequency pulsed magnetic fields on the embryonic central nervous system development. A histological and histochemical study

Numerous experiments have yielded contradictory results on the harmful action of magnetic fields on embryonic development. Pulsed magnetic fields appear to be able to delay normal development of embryos. In the present study, fertilized Gallus domesticus eggs were exposed during incubation to pulsed magnetic fields (harmonic signals of 10 μT for 1 second with silences of 0.5 seconds) of 50 or 100 Hz frequency. Embryos extracted at 45 h of exposure to fields of 50 Hz or 100 Hz frequency had significantly (p<0.05) fewer somite pairs compared with controls of the same age. At 15 days of incubation, only embryos exposed to a 10 μT- 50 Hz field had a significantly (p<0.05) higher somatic weight. At 21 days of incubation, a significantly lower somatic weight (p<0.01) and development stage (p<0.05) was found in embryos exposed to a 10 μT-100 Hz field than in controls, while a lower development stage (p<0.05) alone was observed in those exposed to a 10 μT-50 Hz field. In addition, animals showed higher expression of the neural marker NSE (neural specific enolase) after 21 days of development as determined by immunohistochemistry, with very low expression of glycosaminoglycans identified by alcyan blue staining. These results suggest that pulsed magnetic fields may be able to hinder normal embryonic development in vivo and to alter normal neural function, at least at the intensities and frequencies analyzed in the present study.     

Directed and enhanced neurite growth with pulsed magnetic field stimulation

Pulsed magnetic field (PMF) stimulation was applied to mammalian neurons in vitro to influence axonal growth and to determine whether induced current would direct and enhance neurite growth in the direction of the current. Two coils were constructed from individual sheets of copper folded into a square coil. Each coil was placed in a separate water-jacketed incubator. One was energized by a waveform generator driving a power amplifier, the other was not energized. Whole dorsal root ganglia (DRG) explant cultures from 15-day Sprague-Dawley rat embryos were established in supplemented media plus nerve growth factor (NGF) at concentrations of 0-100 ng/mL on a collagen-laminin substrate. Dishes were placed at the center of the top and bottom of both coils, so that the DRG were adjacent to the current flowing in the coil. After an initial 12 h allowing DRG attachment to the substrate floor, one coil was energized for 18 h, followed by a postexposure period of 18 h. Total incubation time was 48 h for all DRG cultures. At termination, DRG were histochemically stained for visualization and quantitative analysis of neurite outgrowth. Direction and length of neurite outgrowth were recorded with respect to direction of the current. PMF exposed DRG exhibited asymmetrical growth parallel to the current direction with concomitant enhancement of neurite length. DRG cultures not PMF exposed had a characteristic radial pattern of neurite outgrowth. These results suggest that PMF may offer a noninvasive mechanism to direct and promote nerve regeneration.     

Radiological workers sensitivity to 50 Hz pulsed magnetic fields: preliminary results

In the present study the effect of extremely low frequency pulsed magnetic fields (PMF) was evaluated in lymphocyte cultures from 12 subjects occupationally exposed to low doses of ionising radiations. The PMF signal characteristics were repetition frequency 50 Hz, triangular shape, rise time about 1.2 ms and peak intensity 2.5 mT. The cytokinesis-block technique was employed to evaluate genotoxicity and cytotoxicity in terms of micronucleus frequency and cell proliferation, respectively. When PMF-exposed cultures were compared with their respective controls, a slight but statistically significant increase was detected in both the biological parameters investigated ( p<0.05). The results obtained suggest a possible role of specific employments involving exposure to ionising radiation, in the risk associated with electromagnetic field exposure.     

A novel approach for in vitro production of bovine embryos: use of the Oxoid atmosphere generating system

The importance of the incubator type is often overlooked when protocols for in vitro production of embryos are evaluated. In this study the ability of a standard CO2 Heraeus incubator and the Oxoid CO2Gen atmosphere-generating system to support bovine in vitro oocyte maturation, fertilization and embryo development is described for the first time. The Oxoid CO2Gen gas generating system, originally designed for the growth of bacteria, is based on the chemical reaction of ascorbic acid and air. When the sachet with ascorbic acid is placed in the confined volume of the airtight AnaeroJar, an atmosphere of 6% CO2 in 15% O2 is created, which is comparable to the 5% CO2 and 20% O2 used for standard in vitro production of bovine embryos. In the first set of experiments oocyte in vitro maturation (IVM), fertilization (IVF) and embryo culture (IVC) were allocated to one or the other of the culture systems. In the second set of experiments IVM and IVF took place in the Heraeus incubator, while IVC was allocated either to the Heraeus or to the AnaeroJar. During experiments the AnaeroJar was placed in the Heraeus incubator to ensure identical incubation temperatures of 38.8 degrees C. A standard protocol was used for production of embryos: 23 h of IVM in TCM-199, 20 h of IVF with frozen-thawed washed spermatozoa in TALP medium and 7 days of IVC (8 days after insemination) in B2 medium with bovine oviduct epithelial cells. In the first set of experiments, based on a total of 766 inseminated oocytes, the Day 8 blastocyst rates were the same in the Heraeus incubator and the AnaeroJar: 30% vs. 30% with oviduct cell coculture, and 21% vs. 18% without coculture. In the second set of experiments, based on 1963 inseminated oocytes, the average blastocyst rates were 27% vs. 32% from the Heraeus incubator and the AnaeroJar. In 2 of 6 replicates blastocyst rates were lower in the Heraeus incubator than in the jar; in the remaining replicates they were alike. No differences were noted in blastocyst kinetics or morphology. In conclusion, the Oxoid gas generating system seems to be a cheap, convenient and stable alternative to expensive CO2 incubators, not only for the growth of bacteria, but also for in vitro production of bovine embryos.     

Thermoelectrically Cooled Temperature-Gradient Apparatus for Comparative Cell and Virus Temperature Studies

Establishment of a near-linear temperature gradient in an incubator has been accomplished by the application of heat to one terminus of a conducting body, normally a metal bar, and the removal of heat from the other terminus of the conducting body. Such incubators have been complex and unwieldy because of the need for mechanical refrigeration. We have described a simplified temperature gradient incubator which uses thermoelectric module cooling coupled with electric heating. Along the gradient, 20 stations in two parallel rows of 10, each accommodating a 30-ml plastic cell culture flask, were continually monitored by an electronic thermometer, and the temperatures were recorded. By manipulation of two simple potentiometer controls, any temperature gradient between 0 and 50 C could be obtained. Minor deviations which occurred between theoretically perfect and obtained temperature gradients were reproducible and readily measured. The gradient incubator was particularly applicable to (i) simultaneously studying a given biological activity over the entire temperature range supporting the growth of a given cell, virus, or microorganism, or (ii) precisely defining the upper or lower temperature limits of a biological system by 10-point determinations. Preliminary experiments have demonstrated the usefulness of the apparatus in characterizing the temperature limits for growth in vitro of cells of reptilian cell lines. The gradient incubator was also successfully utilized for the characterization of the effect of temperature on the efficiency of plating of amphibian viruses and possible temperature variants of those viruses.     

Influence of environmental oxygen on development and hatching of aquatic eggs of the australian frog, Crinia georgiana

The effect of oxygen partial pressure (Po(2)) on development and hatching was investigated in aquatic embryos of the myobatrachid frog, Crinia georgiana, in the field and in the laboratory. Eggs from 29 field nests experienced widely variable Po(2) but similar temperatures. Mean Po(2) in different nests ranged between 2.9 and 19.3 kPa (grand mean 12.9 kPa), and mean temperature ranged between 11.9 degrees and 16.8 degrees C (grand mean 13.7 degrees C). There was no detectable effect of Po(2) or temperature on development rate or hatching time in the field, except in one nest at 2.9 kPa where the embryos died, presumably in association with hypoxia. Laboratory eggs were incubated at 15 degrees C at a range of Po(2) between 2 and 25 kPa. Between 5 and 25 kPa, there was almost no effect of Po(2) on development rate to stage 26, but the embryos hatched progressively earlier-at earlier stages and lower gut-free body mass-at lower Po(2). At 2 kPa, development was severely delayed, growth of the embryo slowed, and morphological anomalies appeared. A high tolerance to low Po(2) may be an adaptation to embryonic development in the potentially hypoxic, aquatic environment.     

Expression of heat shock protein in broiler embryo tissues after acute cold or heat stress

This study evaluated the expression of heat shock protein 70 kD (hsp70) in broiler chicken embryos subjected to cold (Experiment I) or high incubation temperature (Experiment II). In each experiment, fertile eggs were distributed in three incubators kept at 37.8 degrees C. At day 13 (D13), D16, and D19 of incubation, the embryos were subjected to acute cold (32 degrees C) or heat (40 degrees C) for 4-6 hr. Immediately after cold or heat exposure, samples from the liver, heart, breast muscle, brain, and lungs of 40 embryos were taken per age and treatment (control or stressed embryos). A tissue pool from 10 embryos was used as 1 replication. The levels of hsp70 in each tissue sample was quantified by Western blot analysis. The data were analyzed in a 3 x 2 factorial arrangement of treatments with four replications. hsp70 was detected in all embryo tissues, and the brain contained 2- to 5-times more hsp70 protein compared to the other tissues in either cold or heat stressed embryos. hsp70 increases were observed in the heart and breast muscle of cold stressed embryos at D16 and D19, respectively. Heat stressed embryos showed an increase of hsp70 in the heart at D13 and D19, and in the lung at D19 of incubation. Younger embryos had higher hsp70 synthesis than older embryos, irrespective of the type of thermal stressor. The results indicate that the expression of hsp70 in broiler chicken embryos is affected by cold and heat distress, and is tissue- and age-dependent.