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Poult Sci 2007. 86:538-544
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PHYSIOLOGY, ENDOCRINOLOGY, AND REPRODUCTION

Physiological Changes to Transient Exposure to Heat Stress Observed in Laying Hens

D. J. Franco-Jimenez*,1 and M. M. Beck*,2

* Department of Animal Science, University of Nebraska-Lincoln, 68583

2 Corresponding author: mbeck1{at}unl.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Hy-Line W-36, W-98, and Brown hens lay approximately the same number of eggs/hen housed to 80 wk; however, little is known about differences in performance during heat stress (HS). Two experiments were performed. The first experiment evaluated intestinal calcium uptake (CaT), heat shock protein-70 (HSP70) liver expression, and endocrine status in the 3 strains under heat stress in response to 1 h of transient exposure to high temperature before onset of 18 h of HS. The second experiment evaluated the differences between W-36 and W-98 in acid-base status observed at 2 different ambient temperatures. The HSP70 and CaT data were analyzed as a completely randomized design (CRD) using a 3 x 2 factorial with strain as a 1 factor and preexposed and control treatments as the other. Estrogen and progesterone data were analyzed as a CRD using repeated measures in a 3 x 2 x 2 factorial with strain as a the first factor, preexposure and control treatments as the second factor, and phase of blood collection as the third factor. The data of the second experiment were analyzed as a CRD using repeated measures in a 2 x 2 x 2 factorial with strain, temperature, and phase of blood collection as the factors. The method applied in both experiments was based on the mixed model (SAS). The results show a strain effect, with the higher CaT in the W-36. The results indicated that transient exposure to HS did not induce changes in HSP70 liver expression. In the second experiment, the blood gas values did not differ between strains, except for the partial pressure of CO2, in which the values at 22°C are higher for the W-36. At 38°C, there was an increase in blood pH and a reduction in HCO3– in both strains. The results indicate that endocrine, acid-base status, and Ca homeostasis represent important factors to be considered in assessing genetic differences for thermotolerance.

Key Words: laying hen • heat stress • reproductive hormone • heat shock protein • acid-base status


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In summertime, high environmental temperatures can be hazardous to laying hens, not only because of mortality, but also because of the reduction in the number and quality of the eggs produced during heat stress (HS). Whereas studies on alleviation of HS have focused on costly management adjustments, genetic improvement of heat tolerance may provide a low-cost solution, particularly attractive to developing countries with hot climates. During HS, the blood flow to unfeathered extremities (comb and leg) and other tissues such as tongue, larynx, and trachea increases the loss of heat by radiation, conduction, and convection (Richards, 1971; Van Kampen, 1971; Nolan et al., 1978). Birds exhibit a variety of panting patterns to lose heat as water vapor (Frankel et al., 1962; Richards, 1970; Kassim and Sykes, 1982). The increase in respiration rate leads to a reduction in blood partial pressure of CO2 (PCO2), HCO3–, and an increase in blood pH, resulting in respiratory alkalosis (Bottje and Harrison, 1985; Teeter et al., 1985). The higher blood pH reduces the amount of ionized Ca in the blood (Odom et al., 1986), which is the form of Ca utilized by the shell gland. Also, in laying hens, blood HCO3– plays an important role in formation of the CaCO3 required for eggshell formation. Genetic variation in response to HS has been shown to exist among breeds (Fox, 1951, 1980), sire and dam families (KheirEldin and Shaffner, 1954), mortality rates, and a bird’s ability to control increases in body temperature (Lee et al., 1945). Management manipulations such as heat conditioning (Davis et al., 1991; Yahav et al., 1997), changes in diet composition, and feed restriction (McCormick et al., 1979; Bollengier-Lee et al., 1999) have been implemented to reduce the effect of HS, but usually these adjustments are not easy to apply and tend to require trained personnel. Heat shock proteins (HSP) have been suggested to play a role in cellular protection under high ambient temperature, with a proposed relationship between the development of thermotolerance and HSP synthesis, especially HSP-70 (HSP70; Lindquist and Craig, 1988). It has been suggested that preexposure to HS might improve the productive performance and reduce the mortality of the birds when they are reexposed to HS later on (Arjona et al., 1988, 1990). During HS, HSP70 locates in the nucleus, binds to the RNA synthesis machinery, and prevents synthesis and aggregation of abnormal protein (Lee, 1992). There is evidence that shows that avian progesterone receptor hormone-binding domain is very unstable and rapidly loses its activity at elevated temperatures (Smith, 1993). However, the hormone-binding ability can be restored after a transient loss using an in vitro 5 protein system containing HSP-40, HSP70, HSP organizing protein, HSP-90, and adenosine triphosphate (Hernandez et al., 2002). The objective of the first experiment was to determine whether pre-exposure to HS can induce changes in hepatic HSP70 expression, reproductive hormone levels in blood, and intestinal Ca uptake in the 3 varieties of laying hens. The objective of the second experiment was to determine whether there are differences between Hy-Line W-36 and W-98 in acid-base status observed at 2 different ambient temperatures.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
General
All animal and experimental procedures were conducted with approval of the University of Nebraska-Lincoln Institutional Animal Care and Use Committee. Laying hens were provided water and a layer diet (2,947 ME Kcal/kg of feed, 3.8% Ca, 0.5% P, 17.0% protein) ad libitum and maintained at 22°C in cages of 55 x 41.25 x 60 cm (22 x 16.5 x 24 in), with 4 hens of each strain per cage [562 cm2 (90.75 in2) of space per bird]. The photoperiod consisted of 16L:8D. There were approximately 40 cages the 3 strains were randomly allocated to in the hen house.

Experimental Protocol
Experiment 1.
Twelve hens of each strain (W-98, W-36, and Brown) of approximately 60 wk of age were randomly chosen and randomly assigned to 1 of the 2 treatments (preexposed to HS and nonpreexposed to HS). The hens from the preexposed group (6 hens per strain) were exposed to 35°C for 1 h, whereas the rest of the hens remained at 22°C. After 5 d at 22°C, all 36 birds were exposed to 35°C for 18 h. Blood samples were collected for estrogen and progesterone determination using RIA, and all the birds were euthanized by cervical dislocation to collect liver and duodenum samples. Intestinal Ca uptake was determined using the protocol described by Al-Batshan et al. (1994), whereas hepatic expression of HSP70 was obtained using western immunoblotting.

Sampling Protocol
Blood samples were collected throughout the brachial vein using 5-mL sterilized syringes with a 22-gauge x 1 inch (2.54 cm) needle. Blood was transferred into 15 x 85 mm heparinized tubes. Immediately, plasma was separated from the red blood cells by centrifugation at 2,000 x g. Saturated Na citrate was added to the plasma samples (20 µL/mL) to prevent further clotting (Novero et al., 1991). The samples were stored at –20°C until they were assayed for estrogen (E2) and progesterone (P4). Hens were euthanized by cervical dislocation shortly before oviposition (as determined by abdominal palpation) at approximately the same time each day, and immediately, a 3-cm segment from the midduodenal loop was cut into 6 thin slices (1.5 cm x 2 mm wide) and in vitro Ca transport assay was then conducted as described by Al-Batshan et al. (1994), with a slight modification of tissue incubation time.

Hormone Analysis
P4 RIA.
Progesterone was assayed by RIA validated for the chicken at the Animal Science Physiology Laboratory, University of Nebraska-Lincoln. The methods of the assay to determine plasma progesterone concentrations have been described by Roberson et al. (1989). For validation of the assay with chicken plasma, recovery of added mass (7.8 and 15.6 pg) from 10 µL of plasma from 4 independent samples averaged 115 ± 4.7%. Assay determination of 10, 12.5, and 15 µL of sample from each of 9 independent samples were highly correlated (10 and 12.5 µL, r = 0.986; 10 and 15 µL, r = 0.960; and 12.5 and 15 µL, r = 0.983). The intraassay and interassay CV were 3.8 and 6%, respectively.

E2 RIA.
Estrogen was assayed by RIA validated for the chicken at the Animal Science Physiology Laboratory, University of Nebraska-Lincoln. Radioimmunoassay for E2 was validated as follows for chicken plasma at the Animal Sciences Physiology Laboratory, University of Nebraska. Duplicate aliquots (6.6 µL) of sample were extracted twice with 2 mL of diethyl ether, and extract residues were subjected to E2 RIA, as described by Kojima et al. (1992). The assay utilized an antiserum to E2 at a dilution of 1:1,600.000 (Lilly lot 022367) provided by N. R. Mason (Lilly Research Laboratories, Indianapolis, IN). Pooled avian plasma samples (n = 4) were assayed at 100, 50, 25, and 12.5 µL. Four pools of avian plasma were used to determine recovery of added E2 (0.2, 1.6, and 12.8 pg). Recovery ranged from 76.1 to 111.8%, averaging 87.85 ± 10.65%. Parallelism was determined by using the ALLFIT program (DeLean et al., 1978). Slopes of the dilutions of plasma and the standard curve were not different, as determined by the ALLFIT program (P = 0.26). The intraassay and interassay CV were 6.4 and 8.2%, respectively.

Ca Uptake.
Six thin slices of duodenum tissue (approximately 1.5 cm x 2 mm wide) were taken from the loop and incubated in disposable beakers containing 2.0 mL of Ca transport buffer (CaTB): 140 mM NaCl, 4.8 mM KCl, 1.2 mM KH2PO4, 1.2 mM MgSO4, and 25 mM N-2 hydroxy-ethylpiperazine-N'-2-ethanesulfonic acid at pH 7.40 (Sigma-Aldrich, St. Louis, MO) for 10 min at 37°C (5 mM glucose and 0.5 mM CaCl2 were added the same day of assay). After incubation in CaTB, the assay was begun by transferring the tissues to identical beakers containing CaTB and 45Ca (25,000 cpm/100 µL). The tissues were incubated for 4 (3 tissue slices per bird) and 9 (3 tissue slices per bird) min at 37°C in a shaking water bath. The reaction was terminated by transferring the slices at 10-s intervals to beakers containing 4 mL of 300 mM mannitol. The 45Ca was extracted from the tissue in 2 mL of 2.5% trichloroacetic acid for 60 min at 37°C in a shaking water bath. The tissue samples were weighed after extraction and recorded, and the supernatant was poured off into 15 x 85 mm test tubes and centrifuged for 5 min at 500 x g. One milliliter of the supernatant was then transferred into a 20-mL scintillation vial; 6 mL of EcoLite (ICN Pharmaceuticals Inc., Costa Mesa, CA) scintillation cocktail was added; and the radioactivity of 45Ca was counted in a ß-counter (Packard C1900 liquid scintillation analyzer, Packard Instrument Co., Meriden, CT). Data were calculated as the rate of Ca uptake by duodenal tissue and were expressed and analyzed as a rate (nmol/g per min) or as total Ca (nmol/g). Calcium uptake at 9 min (Ca9min) is calculated as follows: 66 nM Ca x counts per minute of sample (from 9-min incubation period) ÷ total counts per minute of isotope buffer x 2 ÷ milligrams of tissue. Rate of Ca uptake was calculated by subtracting the calculated Ca uptake at the 4-min incubation period (Ca4min) from the calculated Ca uptake at the 9 min incubation period and dividing by 5: (Ca9min – Ca4min)/5.

Tissue Homogenization and Preparation for Western Blot Analysis
All procedures were performed at 4°C. Liver tissues were individually homogenized in a volume (6 mL/g of tissue) of extraction buffer containing 50 mM Tris-HCl (pH 8.0), 500 mM KCl, 2 mM dithiothreitol, 1 mM EDTA, and 0.05% protease inhibitor cocktail (Sigma-Aldrich) in a 15-mL polypropylene tube using a Polytron homogenizer (Brinkmann Instruments Inc., Westbury, NY). The homogenate was filtered through cheesecloth into a microcentrifuge tube and centrifuged at 16,000 x g for 15 min at 4°C. The aqueous phase between the upper fat layer and the pellet was collected into a fresh microcentrifuge tube and recentrifuged at 3,000 x g for 15 min at 4°C. The resulting supernatant was collected into a fresh microcentrifuge tube and stored at –20°C before being used in western blot analysis. Protein concentrations were calculated using the BCA Protein Assay Kit (Pierce Biotechnology, Rockford, IL) with BSA as the standard. Concentrations of protein ranged from 8.75 to 34.8 µg/µL.

Western Blot Analysis.
Prestained molecular weight markers (Bio-Rad Laboratories, Hercules, CA) were included, and rat liver tissue was used as the positive control. Thirty micrograms of total protein were loaded as described by Givisiez et al. (1999). Proteins were separated by SDS-PAGE by using 10% gels with 5% stacking gels in a dual vertical gel electrophoresis system (Owl Separation Systems Inc., Portsmouth, NH). Gels were run at 150 V until the dye band reached the end of the stacking gel and then at 200 V until the dye band reached the end of the gel. Proteins were transferred from the polyacrylamide gels to 45-µm nitrocellulose membranes (Bio-Rad Laboratories) by electroblotting in a Semidry blotting apparatus (Owl Separation Systems Inc.) at a constant setting of 14 V for 1 h. Membranes were blocked with 3% dry milk in Tris-buffered saline with 0.05% Tween-20 (TBST, pH 7.5) for 1 h. After washing with TBST, membranes were agitated for 16 to 20 h at 4°C in 10 µL/10 mL of mouse monoclonal anti-HSP70 (Sigma-Aldrich) and 10 µL/10 mL of mouse monoclonal anti ß-actin (Sigma-Aldrich) in 3% milk TBST. Membranes were then washed and treated with goat anti-mouse IgG peroxidase conjugated (Santa Cruz Biotechnology Inc., Santa Cruz, CA), diluted 1:5000 in antibody buffer, and agitated for 1 h. The membrane was washed 2 times for 15 min with TBST, followed by 2 additional washing with Tris-buffered saline for 15 min each. Immunoreactive proteins were visualized using SuperSignal West Pico chemiluminescence (Pierce Biotechnology). Western blots were electronically scanned with HP Scanjet 6200C (Hewlett-Packard Co., Palo Alto, CA) and saved as 840 x 430 pixel TIF file images. These digital images were then analyzed using Scion Image (Scion Corp., Frederick, MD), subtracting the background density and dividing the value for each specific HSP70 band by the value of the respective ß-actin band with a constant measurement area of 60 x 32 pixels. The digital numbers obtained were the relative density values integrated for the intensity and the size of each band.

Experiment 2.
The 2 varieties of laying hens (W-36 and W-98) were maintained in the hen house. At approximately 40 wk of age, 10 hens from each line (20 total) were transferred (5 at a time) to a temperature-controlled chamber. They were held in the chamber for 1 h first at 22°C and then at 38°C for an additional hour. Venous blood samples (brachial vein) were collected in heparinized sample tubes before and at the end of exposure to 22°C and again before and at the end of exposure to 38°C. The blood was analyzed immediately in a blood gas analyzer (Stat Profile 3, Nova Biomedical, Waltham, MA) and pH, partial pressure of oxygen, PCO2, and HCO3– were obtained.

Statistical Analysis
Experiment 1.
The western blot and Ca transport data were analyzed as a CRD using a factorial 3 x 2, with strain as 1 factor and preexposed and control treatments as the other. The data from estrogen and progesterone were analyzed as a CRD using repeated measures in a factorial 3 x 2 x 2, with strain as the first factor, preexposure and control treatments as the second factor, and phase of blood collection (previous to 18 h of HS or after the 18 h of HS) as the third factor. The method applied for this repeated measures analysis was based on the mixed model (PROC MIXED; SAS Institute, 2001), and data were fitted to a model that included the effects of treatment, strain, phase, treatment x strain, treatment x phase, strain x phase, phase x treatment, and phase x treatment x strain. The model for the design is as follows:


Formula

where Yijkl = the variable of interest for the j hen assigned to strain i, treatment k, and phase l; µ = the overall mean; {alpha}j = the strain effect; ßk = the treatment effect; ({alpha} ß)jk = the strain x treatment interaction; Sl = the phase effect; ({alpha}Sjl) = the strain x phase interaction; (ßSkl) = the treatment x phase interaction; ({alpha} ßSjkl) = the strain x treatment x phase interaction; and Eijkl = the error term. To use this model, we must assume that Eijkl, is the random error associated with the j hen of i strain in the k treatment at l phase. The best covariance structure was chosen using the value of the Akaike’s information criterion (Littell et al., 1999). The differences between means were determined by Fisher’s protected least significant difference with a level of significance of {alpha} = 0.1.

Experiment 2.
The data were analyzed as a CRD using repeated measures in a factorial 2 x 2 x 2 with strain as a first factor, temperature as another, and phase of blood collection (before and after exposure) as the last factor. The method applied was based on the mixed model of SAS (SAS Institute, 2001), in which data were fitted to a model that included the effects of strain, temperature, phase, strain x temperature, strain x phase, temperature x phase, and strain x temperature x phase. The model for the design is as follows:


Formula

where Yijkl = the variable of interest for the j hen assigned to strain i, temperature k, and phase l; µ = the overall mean; {alpha}j = the strain effect; ßk = the temperature effect; ({alpha} ß)jk = the strain x temperature interaction; Sl = the phase effect; ({alpha}Sjl) = the strain x phase interaction; (ßSkl) = the temperature x phase interaction; ({alpha} ßSjkl) = the strain x temperature x phase interaction; and Eijkl = the error term. To use this model, we must assume that Eijkl, is the random error associated with the j hen of i strain in the k temperature at l phase. The best covariance structure was chosen using the value of the Akaike’s information criterion (Littell et al., 1999). The differences between means were determined by Fisher’s protected least significant difference with a level of significance of {alpha} = 0.1.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Experiment 1
The rate of duodenal Ca uptake (Table 1Go) was higher in W-36 hens than in Browns; W-98 was intermediate and not statistically different from either of the 2 other strains. Hepatic expression of HSP70 was not increased by preexposure to 1 h of HS in either W-36 or W-98 hens; Brown hens that were preexposed to 1 h of HS had a lower relative density in the western blot analysis (Figure 1Go) than any other group. There was a treatment effect with an increase in hepatic HSP70 expression in the nonpreexposed group (Table 2Go). There was strain x phase interaction for estrogen (Table 3Go), with only the Brown hens showing a reduction after 18 h of exposure to HS. Progesterone levels were not affected by HS (data not shown).


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  		Table 1. Intestinal Ca uptake
 

Figure 1
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  		Figure 1. Western blot analysis of heat shock protein-70 (HSP70) expression in liver tissue. The protein band in the upper panel represents immunoblot of 70 kDa heat shock protein for the specific strains and treatments of liver samples from birds after 18 h of heat stress exposure. The bands are represented as follows: W-98 C = W-98 strain not preexposed to heat stress; W-98 PHS = W-98 strain preexposed to heat stress; W-36 C = W-36 strain not preexposed to heat stress; W-36 PHS = W-36 strain preexposed to heat stress; B C = Brown strain not preexposed to heat stress; B PHS = Brown strain preexposed to heat stress; +Cont = positive control obtained from rat liver. The lower panel band represents immunoblot 43 kDa of ß-actin protein used as a loading control. The negative control is not shown because of an absence of bands.

 

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  		Table 2. Relative heat shock protein-70 (HSP70) liver expression
 

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  		Table 3. Estrogen levels (strain x phase effect)
 
Experiment 2
During the 1-h chamber exposure to 22°C, the W-98 and W-36 hens exhibited similar blood pH, PCO2, and HCO3– values. However, blood pH levels decreased over time in both lines when pre- and post-22°C exposure samples were compared (Table 4Go). A similar temperature x phase interaction is shown for bicarbonate levels, but in this case, there was an increase from its initial value at 22°C and a reduction from its initial value at 38°C after 1 h of exposure in both strains (Table 4Go). There was a strain x temperature effect for PCO2, with a lower value for the W-98 at 22°C, but not differences observed at 38°C between the strains. Blood oxygen levels showed a strain x phase interaction with a reduction from its higher initial value for the W-36 strain and no effect for the W-98 strain.


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  		Table 4. Changes in blood acid-base status
 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In Experiment 1, the results showed an important difference in intestinal Ca uptake after HS exposure, particularly in the W-36 over the Brown hens, a finding that is consistent with earlier studies (Franco and Beck, 2003; Franco, 2004; Franco et al., 2004; Franco-Jimenez and Beck, 2005). Also, it has been reported that the W-36 strain is more efficient, using lower levels of dietary Ca than the W-98 strain in thermoneutral conditions (Scheideler et al., 2004). The ability of duodenal cells to continue taking up Ca during HS episodes could be 1 characteristic of hens that breeders might use in their selection. In addition, we were unable to show a beneficial effect of a 1-h exposure to HS before a longer episode, with regard to thermotolerance associated with induction of liver HSP70. This finding is consistent with some earlier studies (Yahav et al., 1997; Givisiez et al., 1999) and is in contrast to another study (Lindquist and Craig, 1988). It appears, from a synthesis of these papers, that heat conditioning by preexposure may not be associated with increased hepatic HSP70, unless the second exposure is a higher temperature or for a longer time. There is considerable disagreement in the literature about the actual role of HSP70 in the development of thermotolerance, and it is possible that the mechanism involving HSP70 is different from its response to single episodes of HS. Heat stress has been associated with HSP70 induction to provide protection against the subsequent cellular injuries to cells and tissues (Lindquist and Craig, 1988). Thermotolerance is usually associated with HSP70 induction, and heat conditioning resulted in an increase in the expression of HSP70 (Lindquist and Craig, 1988). In any case, it is evident from the other findings of previous studies (Franco and Beck, 2003; Franco, 2004; Franco et al., 2004) that the Brown hens generally respond less well to hot conditions. Estrogen levels show a treatment x phase interaction, and the reduction in plasma estrogen in the Brown hens after 18 h of HS exposure is consistent with the lower rate of Ca uptake observed in this strain and is further evidence for their reduced adaptability to HS. They may be the only birds that were actually stressed in this experiment. Neither E2 nor P4 were affected in W-98 and W-36 hens. Although it might appear contradictory, the lower levels of HSP70 in the preexposed HS group could be associated with poor adaptation. However, if we consider that the expression of HSP70 is related to the degree of stress to which an individual has been subjected, then the preexposed HS group suffered less stress after 18 h of HS exposure than the control group. This lower response in the preexposed HS group might be due to HS acclimation and could be indicative of acquired thermotolerance. There is also the possibility that transient preexposure to HS might reduce the hepatic expression of HSP70 during the subsequent episode.

In Experiment 2, the results reinforce the theory that at higher ambient temperatures, the hens are susceptible to respiratory alkalosis due to the panting process (Bottje and Harrison 1985; Teeter et al., 1985), which reduces the amount of CO2 and the amount of HCO3– in blood, both required by the shell gland to support shell formation (Etches, 1996). The 2 strains had the same response in terms of changes in blood pH, HCO3–, and PCO2 when exposed to high ambient temperatures (38°C). However, the ability of the W-98 hens to maintain shell thickness and the specific gravity of eggs laid during HS shown in earlier studies (Franco and Beck, 2003; Franco, 2004; France et al., 2004) may indicate that this strain somehow handles the unbalance in acid-base status in a different way to support shell formation. Oxygen blood levels are not affected by 1 h of exposure to high ambient temperatures (38°C). The drop in pH and the increment on HCO3– for both strains at 22°C is not clear. One possibility might be a depression in respiratory rate (not measured), with accompanying retention of CO2, which later on may be traduced in a higher production of HCO3– from carbonic acid dissociation.

Based on the results and on interpretations from earlier studies in the literature, it can be proposed that the Brown hens are more susceptible to HS, whereas the W-36 and W-98 hens seem to adapt or respond better to HS. In addition, W-98 birds may apparently manage the acid-base unbalance better at high ambient temperatures to sustain a better shell quality, observed in previous studies. Heat shock proteins have been suggested to be involved in cellular protection in adverse situations, and these proteins may improve thermotolerance of the bird. We were unable to induce HSP70 with 1 h of preexposure to HS before a longer episode, which may indicate that the time of exposure was not long enough or certain adaptation had already occurred in those preexposed birds, reducing the amount of HSP70 needed for protection when the bird was reexposed to HS. In either case, this confirms the finding of Givisiez et al. (1999), who suggested that HSP70 levels are modulated by additional factors and are only 1 of the other factors involved in cellular protection. More research is needed to confirm these hypotheses and to elucidate whether HSP70 may be used as a good indicator of thermotolerance.


   ACKNOWLEDGMENTS
 
We thank N. Savery for manuscript preparation; L. Robeson, K. Hansen, H. Taira, F. Madison, and M. Burns for animal care and technical assistance; A. Cupp for allowing us to get rat liver tissue; and Hy-Line International (West Des Moines, IA) for providing us the initial set of birds.


   FOOTNOTES
 
1 Present address: Department of Animal and Veterinary Science, California State Polytechnic University, Pomona. Back

Received for publication July 24, 2006. Accepted for publication August 31, 2006.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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Franco-Jimenez, D. J., and M. M. Beck. 2005. Intestinal calcium uptake, shell quality and reproductive hormones levels of three laying hens varieties after prolonged egg production. Int. J. Poult. Sci. 4:518–522.

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