J. Appl. Poult. Res.
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J APPL POULT RES 2007. 16:628-634. doi:10.3382/japr.2005-00088
© 2007 Poultry Science Association
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Research Reports

Differential Effects of Heat Stress in Three Strains of Laying Hens1

D. J. Franco-Jimenez*,2, S. E. Scheideler*, R. J. Kittok*, T. M. Brown-Brandl{dagger}, L. R. Robeson*, H. Taira* and M. M. Beck*,3,4

* Department of Animal Science, University of Nebraska, Lincoln 68583; and {dagger} USDA-US Meat Animal Research Center, Clay Center, NE 68933

Correspondence: 3 Corresponding author: mbeck{at}clemson.edu


   SUMMARY
TOP
 SUMMARY
DESCRIPTION OF PROBLEM
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS AND APPLICATIONS
 REFERENCES AND NOTES
 
Hy-Line Brown, W36, and W98 hens were housed for 2 wk at 22°C, exposed to 35°C heat stress (HS) for 2 wk, and had 2 wk of recovery at 22°C. Production parameters (egg production, feed intake, egg quality measures, and mortality), acid-base status (pH, pCO2, HCO3–, pO2), intestinal calcium uptake, and expression of hepatic heat shock protein-70 were measured. All production parameters except mortality were reduced by HS in all 3 strains. There were strain x temperature interactions for egg production, feed intake, shell thickness, specific gravity, and yolk weight, with the least severe reduction in W98 hens. The W36 hens were intermediate for egg production and feed intake, and the Brown and W36 hens were equivalent for shell thickness, specific gravity, and yolk weight. There were no interactions for egg, albumen, and shell weights, Haugh units, and intestinal calcium uptake, and all were reduced by HS. Blood pH and pCO2 were reduced by HS, and hepatic heat shock protein-70 was increased, with no differences among strains. Mortality rates during HS were 16, 8, and 4% for the Brown, W98, and W36 hens, respectively, but were not subjected to statistical analysis. The results suggest a mechanism in the W98 bird that appears to confer an advantage during HS with regard to egg production.

Key Words: heat stress • laying hen • strain • production • heat shock


   DESCRIPTION OF PROBLEM
TOP
 SUMMARY
 DESCRIPTION OF PROBLEM
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS AND APPLICATIONS
 REFERENCES AND NOTES
 
Heat stress (HS) is well known to reduce the reproductive performance of laying hens by interrupting egg production, an effect caused not only by a reduction in feed intake but also by a disruption of hormones responsible for ovulation and a decrease in responsiveness of granulosa cells to luteinizing hormone [1, 2]. In addition, HS negatively affects the strength, weight, thickness, and ash content of the eggshell [3, 4, 5]; these responses involve various aspects of calcium metabolism, including a reduction in free ionized calcium in the blood [6]. Several acid-base imbalances have been observed in birds exposed to HS, particularly respiratory alkalosis [7, 8], which reduces HCO3– in blood and limits the amount of CO3= available for eggshell formation [9]. Resistance to HS varies among strains and families of birds [10, 11, 12]. The physiological characteristics involved in the conveyance of thermotolerance are not clear, but HS proteins (HSP), especially HSP70, may be involved [13]. Management adjustments can also improve the resistance of laying hens to HS conditions, but usually these short-term adjustments are limited by cost and the trained personnel needed to perform the tasks. Genetic manipulation of breeders based on some component of thermotolerance might be an appropriate approach to improving egg production and survivability under HS conditions. However, a better understanding of the physiological processes that take place in the heat-stressed bird is needed in pursuing this strategy. The objective of this study was to characterize physiological responses in 3 strains of laying hens that appear to differ with regard to HS resistance.


   MATERIALS AND METHODS
TOP
 SUMMARY
 DESCRIPTION OF PROBLEM
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 CONCLUSIONS AND APPLICATIONS
 REFERENCES AND NOTES
 
Birds
Hy-Line laying hens (Brown, W36, and W98) [14] at 38 wk of age were provided water and a layer diet (2,947 kcal of ME/kg of feed, 3.8% Ca, 0.5% P, 17.0% protein) on an ad libitum basis, and maintained at 22°C in cages of 22 in. length x 24 in. height x 16.5 in. width, with 4 hens per cage (90.75 in.2 of space per bird). The 40 cages were randomly allocated within the house. For the actual experiments, 32 hens of each strain were randomly assigned to 24 cages (4 hens per cage and 8 replicate cages per strain) within an environmental chamber (11 ft width x 21 ft length x 8 ft height). The photo-period was 16L:8D. The hens were held for 2 wk at 22°C and 50% RH, exposed to HS (35°C and 50% RH) for 2 wk, and then allowed to recover for 2 wk at 22°C and 50% RH. All animals and experimental procedures were conducted with the approval of the University of Nebraska-Lincoln Institutional Animal Care and Use Committee. The parameters measured, sampling intervals, and analytical methods used in this study are summarized in Table 1Go.


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  		Table 1. Summary of measurements taken, sampling intervals, and methods used
 
Calcium uptake (CaT) was measured in vitro in duodenal slices incubated with 45Ca, according to methods described previously [15], and is expressed as the rate of uptake (nmol/g per min). Briefly, thin slices of duodenal tissue were incubated in transport buffer containing the 45Ca, and the reaction was stopped by mannitol at 4 or 9 min of incubation. The isotope was extracted in 2.5% trichloroacetic acid, centrifuged, and the supernatant was added to the scintillation cocktail. Counts per minute were determined by using a beta-counter, and nanomoles of calcium per gram of tissue were calculated. The HSP70 analysis [16] was carried out on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels in a dual vertical electrophoresis system [17] by using prestained molecular markers [18] and rat liver tissue as the positive control. Proteins were extracted from homogenized liver tissue, filtered, centrifuged, and the aqueous phase collected; protein concentration was determined by using the bicinchoninic acid protein assay [19]. Thirty micrograms of protein was loaded and gels were run at 150 and 200 V through the 5% stacking and 10% separating gels, respectively. Proteins were transferred from the gels to 45-µm nitrocellulose membranes [18] by electroblotting [17] at 14 V for 1 h. Membranes were blocked in dry milk-Tris-buffered saline and incubated with mouse monoclonal anti-HSP70 and anti-ß-actin. Immunoreactive proteins were visualized by using SuperSignal West Pico chemiluminescence [19]. Blood gases were measured in a blood gas analyzer [20]; blood samples collected from the brachial vein and held under anaerobic conditions were injected into the analyzer within minutes after sample collection. Ion-selective (pH) and modified pH electrodes within the machine measure pH and pCO2, respectively; pO2 is measured amperometrically by the creation of a current at the electrode surface [20].

Statistical Analysis
The data for production parameters (egg production, feed intake, yolk weight, shell thickness, and specific gravity) were analyzed as a completely randomized design with repeated measures in a 3 x 3 factorial experiment, with strain as one factor and temperature as the repeated factor, with a level of significance of 0.05. Mortality data were not analyzed statistically. The data for HSP70 and CaT were analyzed as a completely randomized design in a 3 x 2 factorial experiment with a level of significance of 0.10; because samples for these analyses were taken postmortem, this analysis did not include repeated measures. The data from acid-base measurements were analyzed as a completely randomized design with repeated measures in a 3 x 2 factorial experiment, with strain as one factor and temperature as the repeated factor, with a level of significance of 0.10. Differences among means were obtained based on Fisher’s protected LSD test. The method applied was based on the linear mixed model [21], where data were fit to a model that included the effects of strain, cage (strain), temperature, temperature x strain, and temperature x cage (strain). The effect of cage (strain) was used as the error term for testing the effect of strain. Cage (strain) was considered a random effect. The model for the design is:


Formula

where Yijk is the variable of interest for cage j assigned to strain i and temperature k, µ is the overall mean, {alpha}i is the strain effect, {omega}j is the cage (strain) effect (whole-plot error), ßj is the temperature effect, ({alpha}ß)ij is the strain x temperature interaction, and {varepsilon}ijk is the temperature x cage (strain) effect (split-plot error). To use this model, we must assume that {omega}j is the random effect of cage (strain) and {varepsilon}ijk is the random error associated with cage (strain) by temperature. The best covariance structure (compound symmetry) was chosen by using the value of Akaike’s information criterion [21].


   RESULTS AND DISCUSSION
TOP
 SUMMARY
 DESCRIPTION OF PROBLEM
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
CONCLUSIONS AND APPLICATIONS
 REFERENCES AND NOTES
 
Tables 2Go, 3Go, and 4Go show the results of this study. All production parameters were reduced by HS in all strains, with significant strain x temperature interactions. Feed intake (Table 2Go) was reduced (P < 0.05) during HS in all 3 strains of hens, and all 3 responded during the recovery period by consuming more feed than during the initial 2-wk period at 22°C. Feed intake was significantly reduced in all 3 strains (P < 0.05). Although feed intake was not statistically significant among strains at 35°C, the reduction was least in the W98 birds (27%), compared with 35 and 29% reductions in the Brown and W36 hens, respectively. Egg production (Table 2Go) was differentially affected by HS in the 3 strains. The effect was most pronounced in the Brown hens (31% reduction), least severe in the W98 hens (13%), and intermediate in the W36 hens (19.7%). In all 3 strains, egg production increased during the recovery period at 22°C, although not to pre-HS rates. Production in the W98 came closest, reaching a rate that was between the pre-HS and HS rates (P > 0.05). In Brown and W36 hens, the recovery rates remained significantly lower than during pre-HS (P < 0.05).


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  		Table 2. Production parameters of Hy-Line Brown, W98, and W36 laying hens as affected by heat stress1
 

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  		Table 3. Production parameters and intestinal calcium uptake of Hy-Line Brown, W98, and W36 laying hens as affected by heat stress1
 

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  		Table 4. Acid-base parameters in Hy-Line Brown, W98, and W36 laying hens as affected by heat stress1
 
These results, with the low mortality in the 2 white strains, are the primary explanation for the assertion that the W98 bird is physiologically more heat tolerant than the Brown or W36 hen. In this study, mortality in the W98 and W36 hens was 8 and 4%, respectively, compared with 16% in the Brown hens. The more severe effect on mortality of Brown hens was not unexpected, however, because they are larger birds with a heavier feather cover. It should be noted that the mortality rate between the W98 and the W36 birds has not been consistent in the literature; in some studies the W98 has had higher survivability during HS, and in other studies the W36 has had the advantage [22], but on average, they are essentially similar with regard to survivability of HS.

The other element in the evaluation of the W98 bird as the more heat-tolerant of the 2 white strains was based on the effects of HS on egg quality. Yolk weight, shell thickness, and specific gravity were negatively affected by HS in all strains, with the smallest effect on yolk weight occurring in the W98 birds (P < 0.05). There was only a 0.01-mm difference in reduction of shell thickness between the W98 (0.05 mm) and W36 (0.04 mm) hens, compared with a 0.07-mm reduction in the Brown strain. This means that not only did the W98 bird have fairly high livability during HS, but this strain was also able to sustain a higher level of egg production and egg equality than the other 2 strains. Another interpretation of these results is that feed intake in the W98 bird during HS remained high enough to sustain a higher level of egg production in spite of the stress, thus conferring an advantage with regard to egg production.

Egg weight, albumen weight, shell weight, and Haugh units were affected by strain and temperature, but without interactions (Table 3Go). Across all temperatures, the egg weight, albumen weight, and shell weight of the W98 bird were greater than those of the W36 bird (P < 0.05) and were similar to those of the considerably larger Brown hens (P > 0.05). Heat stress significantly reduced egg weight, albumen weight, and shell weight (P < 0.05), and neither egg weight nor albumen weight recovered during the 2 wk subsequent to HS (P > 0.05).

In considering possible physiological mechanisms that may contribute to the advantages of the W98 bird during HS, the results of the CaT and acid-base measurements in this study identify several potential candidates. Intestinal calcium uptake (Table 3Go) was measured before and during HS, but not during the recovery period. In all strains, HS reduced CaT, with by far the largest reduction in the Brown hens (52.5 vs. 30% reduction in the 2 white strains). Across all temperatures, the W98 hen had higher CaT than the Brown hen (P < 0.05) and similar, but slightly higher, CaT than the W36.

Acid-base status was disturbed by HS, as expected, with both strain and temperature main effects, but no interactions. When the strains were examined individually for pH and pCO2, the following effects were noted: HS caused an elevation in pH from 7.35 to 7.39, averaged across strains, which is not a physiologically significant elevation. When individual strain responses were examined (data not shown), the W98 hens showed the least elevation in blood pH in response to HS, with a 0.038-unit increase compared with 0.049 and 0.05 in the W36 and Brown hens, respectively. The failure to detect a significant increase in pH is likely explained by the compensation of the kidneys for HS-induced alkalosis by the time the blood sample was taken [23]. In contrast, the reduction in pCO2 (Table 4Go) in blood that typically occurs in response to hyperventilation in animals [23] was considerably less (13%) in the W98 birds than in the other 2 strains (W36, 24%; Browns, 17%; data not shown). Therefore, during HS the W98 birds had more CO2 in the blood available to react with H2O to produce a greater amount of HCO3– (P < 0.10; Table 4Go). During HS, reductions in CO2 (and thus also in HCO3–) interfere with the shell-forming reaction in the shell gland [24], resulting in reduced shell quality.

The HSP70 response to HS was as expected, with an overall increase across the 3 strains (Table 4Go and Figure 1Go) and with no interaction observed. Again, in looking at the individual strain responses (data not shown), the W98 hens and, interestingly, the Brown hens, responded with 33 and 36% increases, respectively, whereas in the W36 hens, the increase was only 5.5%. The function of HSP70 during stress is to prevent the synthesis and aggregation of abnormal proteins [25] and to restore (with several other proteins) the integrity of the progesterone receptor-binding domain [25, 26, 27].


Figure 1
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  		Figure 1. Western immunoblot showing hepatic heat shock protein-70 (HSP70; upper bands) expression in Hy-Line hens before (B) and after (A) heat stress. The lower bands represent the housekeeping gene ß-actin, which was used as a loaded control for each band. Rat liver tissue was used as a positive control (+C). The negative control is not shown because of the lack of bands.

 
The results of this study provide evidence that the W98 hen has a physiological advantage over the W36 and Brown hens with regard to greater stability of acid-base mechanisms during HS in spite of a slightly larger body size than the W36 hen. The slightly higher mortality seen in the W98 hen in this study would likely be offset in an economic model by the higher egg production and greater egg, yolk, albumen, and shell weights, as well as greater stability of the acid-base balance during HS. Whether the effects seen are related to the HSP70 response or to other unidentified cellular or molecular factors remains to be determined, but if such factors could be identified, the use of functional genomics to optimize genetic improvement of performance and survival during HS could become a viable option for breeding programs.


   CONCLUSIONS AND APPLICATIONS
TOP
 SUMMARY
 DESCRIPTION OF PROBLEM
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS AND APPLICATIONS
REFERENCES AND NOTES
 

  1. The Hy-Line W98 laying hen responded better to HS conditions with regard to egg production and some egg quality measures (egg, yolk, albumen, and shell weights) than did the Brown or W36 hens.
  2. At least part of the proposed explanation for this response is that the level of disruption of acid-base parameters (PCO2, HCO3–, and possibly pH) was not as severe in the W98 strain.
  3. The possible involvement of the HSP70 protein in the performance of W98 hens during HS should be investigated. Cellular or molecular mechanisms, or both, involved in the acid-base response should be investigated for possible inclusion in functional genomics programs of primary breeders.


   ACKNOWLEDGMENTS
 
The authors thank N. Savery for manuscript preparation.


   FOOTNOTES
 
1 Published as journal series 15053, Agricultural Research Division, University of Nebraska-Lincoln, NE 68583. Supported by USDA Regional Research Project NE-1022. Back

2 Current address: California State Polytechnic University-Pomona, Animal and Veterinary Sciences, Pomona, CA 91768. Back

4 Current address: Department of Animal and Veterinary Sciences, Clemson University, Clemson, SC 29634. Back


   REFERENCES AND NOTES
TOP
 SUMMARY
 DESCRIPTION OF PROBLEM
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS AND APPLICATIONS
 REFERENCES AND NOTES
 

  1. Donoghue, D., B. F. Krueger, B. M. Hargis, A. M. Miller, and M. E. El Halawani. 1989. Thermal stress reduces serum luteinizing hormone and bioassayable hypothalamic content of luteinizing hormone releasing hormone in the hen. Biol. Reprod. 41:419–424.[Abstract]
  2. Novero, R. P., M. M. Beck, E. W. Gleaves, A. L. Johnson, and J. A. Deshazer. 1991. Plasma progesterone, luteinizing hormone concentrations and granulose cell responsiveness in heat-stressed hens. Poult. Sci. 70:2335–2339.[ISI][Medline]
  3. Miller, P. C., and M. L. Sunde. 1975. The effects of precise constant and cyclic environmentals on shell quality and other performance factors with Leghorn pullets. Poult. Sci. 54:36–46.[ISI]
  4. DeAndrade, A. N., J. C. Rogler, W. R. Featherston, and C. W. Alliston. 1976. Influence of constant elevated temperature and diet on egg production and shell quality. Poult. Sci. 55:685–693.[ISI]
  5. DeAndrade, A. N., J. C. Rogler, W. R. Featherston, and C. W. Alliston. 1977. Interrelationships between diet and elevated temperature (cyclic or constant) on egg production and shell quality. Poult. Sci. 56:1178–1188.[ISI]
  6. Odom, T. W., P. C. Harrison, and W. G. Bottje. 1986. Effects of thermal-induced respiratory alkalosis on blood ionized calcium levels in the domestic hen. Poult. Sci. 65:570–573.[ISI][Medline]
  7. Teeter, R. G., M. O. Smith, F. N. Owens, S. C. Arp, S. Sangiah, and J. E. Breazile. 1985. Chronic heat stress and respiratory alkalosis: Occurrence and treatment in broiler chicks. Poult. Sci. 64:1060–1064.[ISI][Medline]
  8. Teeter, R. G., and M. O. Smith. 1986. High chronic ambient temperature stress effects on broiler acid-base balance and their response to supplemental ammonium chloride, potassium chloride, and potassium carbonate. Poult. Sci. 65:1777–1781.[ISI][Medline]
  9. Mongin, P. E. 1968. Role of acid-base balance in the physiology of egg-shell formation. World’s Poult. Sci. J. 24:200–230.[CrossRef][ISI]
  10. Fox, T. W. 1951. Studies on heat tolerance in domestic fowl. Poult. Sci. 30:477–483.[ISI]
  11. KheirEldin, M. A., and C. S. Shaffner. 1954. Familial differences in resistance to high environmental temperatures in chicks. Poult. Sci. 33:1064. (Abstr.)
  12. Fox, T. W. 1980. The effects of thiouracil and thyroxine on resistance to heat shock. Poult. Sci. 59:2391–2396.[ISI][Medline]
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  14. Hy-Line International, West Des Moines, IA.
  15. Al-Batshan, H. A., S. E. Scheideler, B. L. Black, J. D. Garlich, and K. E. Anderson. 1994. Duodenal calcium uptake, femur ash, and eggshell quality decline with age and increase following molt. Poult. Sci. 73:1590–1596.[ISI][Medline]
  16. Givisiez, P. E. N., J. A. Ferro, M. I. T. Ferro, S. N. Kronka, E. Decuypere, and M. Macari. 1999. Hepatic concentration of heat shock protein 70 kD (HSP70) in broilers subjected to different thermal treatments. Br. Poult. Sci. 40:292–296.[CrossRef][ISI][Medline]
  17. Owl Separation Systems, Portsmouth, NH.
  18. Bio-Rad Laboratories, Hercules, CA.
  19. Pierce, Rockford, IL.
  20. Stat Profile 3, NOVA Biomedical, Waltham, PA.
  21. SAS Institute. 2001. SAS Users Guide. Version 8.2. SAS Institute, Cary, NC.
  22. Franco, D. J. 2004. Effect of heat stress on production, physiological, and metabolic parameters in three varieties of laying hens. PhD Diss. Univ. Nebraska, Lincoln.
  23. Hayes, J. A. Respiratory alkalosis. http://www.emedici-ne.com/med/topic2009.htm. Accessed Nov. 2, 2005.
  24. Eastin, W. C., Jr., and E. Spaziani. 1978. On the mechanism of calcium secretion in the avian shell gland (uterus). Biol. Reprod. 19:505–518.[Abstract]
  25. Lee, Y. J. 1992. Differential relocalization of each member of Hsp70 and Hsp28 families during chronic heating. J. Therm. Biol. 17:161–168.[CrossRef][ISI]
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  27. Hernandez, P. M., A. Chadli, and D. Toft. 2002. Hsp40 binding is the first step in the Hsp90 chaperoning pathway for the progesterone receptor. J. Biol. Chem. 277:11873–11881.[Abstract/Free Full Text]
  28. Butcher, G. D., and R. D. Miles. 2004. Egg specific gravity—Designing a monitoring program. http://www.afn.org/~poultry/flkman5.htm Accessed March 06, 2004.
  29. Haugh, R. R. 1937. The Haugh unit for measuring egg quality. US Egg Poult. Mag. 43:552–555, 572–573.




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