Research Note: Phenotypic trends for the multigenerational advanced intercross of the Virginia body weight lines of chickens.

Random samples from generation S41 of the Virginia high and low 8-week body weight lines formed the base population for producing a multigenerational reciprocal intercross population. Although genetic mapping from this intercross has been reported, lacking are phenotypic trends across multiple generations. Here, we provide phenotypic information for the parental base population, the F1 reciprocal cross, and subsequent segregating recombinant generations F2 to F17. Heterosis for the selected trait in the F1 was negative for both reciprocal crosses. Phenotypic correlations for the selected trait in the recombinant generations were essentially nil for both males and females as was percent sexual dimorphism and coefficients of variation.

Low-coverage sequencing in a deep intercross of the Virginia body weight lines provides insight to the polygenic genetic architecture of growth: novel loci revealed by increased power and improved genome-coverage.

Genetic dissection of highly polygenic traits is a challenge, in part due to the power necessary to confidently identify loci with minor effects. Experimental crosses are valuable resources for mapping such traits. Traditionally, genome-wide analyses of experimental crosses have targeted major loci using data from a single generation (often the F2) with individuals from later generations being generated for replication and fine-mapping. Here, we aim to confidently identify minor-effect loci contributing to the highly polygenic basis of the long-term, bi-directional selection responses for 56-d body weight in the Virginia body weight chicken lines. To achieve this, a strategy was developed to make use of data from all generations (F2-F18) of the advanced intercross line, developed by crossing the low and high selected lines after 40 generations of selection. A cost-efficient low-coverage sequencing based approach was used to obtain high-confidence genotypes in 1Mb bins across 99.3% of the chicken genome for >3,300 intercross individuals. In total, 12 genome-wide significant, and 30 additional suggestive QTL reaching a 10% FDR threshold, were mapped for 56-d body weight. Only 2 of these QTL reached genome-wide significance in earlier analyses of the F2 generation. The minor-effect QTL mapped here were generally due to an overall increase in power by integrating data across generations, with contributions from increased genome-coverage and improved marker information content. The 12 significant QTL explain >37% of the difference between the parental lines, three times more than 2 previously reported significant QTL. The 42 significant and suggestive QTL together explain >80%. Making integrated use of all available samples from multiple generations in experimental crosses are economically feasible using the low-cost, sequencing-based genotyping strategies outlined here. Our empirical results illustrate the value of this strategy for mapping novel minor-effect loci contributing to complex traits to provide a more confident, comprehensive view of the individual loci that form the genetic basis of the highly polygenic, long-term selection responses for 56-d body weight in the Virginia body weight chicken lines.

Population dynamics of a long-term selection experiment in White Plymouth Rock chickens selected for low or high body weight.

The population dynamics of 2 lines of chickens from a long-term (59 generations) selection experiment were assessed based on pedigree data. These lines were propagated from phenotypic selection for low and high 8-wk BW in White Plymouth Rock chickens. Our objective was to determine whether the 2 lines maintained similar population structures over the selection horizon to allow meaningful comparisons of their performance data. A complete pedigree of 31,909 individuals, consisting of 102 founders, 1,064 from the parental generation, and 16,245 low weight (LWS) and 14,498 high weight (HWS) select chickens, was available. Inbreeding (F) and average relatedness (AR) coefficients were computed. Average F per generation and AR coefficients were 1.3 (SD 0.8) % and 0.53 (SD 0.001) for LWS, and 1.5 (SD 1.1) % and 0.66 (SD 0.001) for HWS. Mean F for the entire pedigree was 0.26 (0.16) and 0.33 (0.19), and maximum F was 0.64 and 0.63, in LWS and HWS, respectively. Based on Wright's fixation index, at generation 59, substantial genetic differences were established between lines. The effective population size was 39 in LWS and 33 in HWS. The effective number of founders was 17 and 15, effective number of ancestors were 12 and 8, and genome equivalents were 2.5 and 1.9 in LWS and HWS, respectively. About 30 founders explained the marginal contribution to both lines. By generation 59, only 7 male and 6 female founders contributed to both lines. Moderately high levels of inbreeding and low effective population sizes were inevitable, as this was a closed population. However, effects on the fitness of the population were expected to be less substantial because founders were a combination of 7 lines. The effective numbers of founders and ancestors were relatively low compared to the actual number of founders, as few ancestors contributed to descendants. Based on these evaluations, it can be inferred that LWS and HWS had similar population structures. Comparisons of selection responses in the 2 lines therefore should be reliable.

A genomic inference of the White Plymouth Rock genealogy.

Crossing of populations has been, and still is, a central component in domestication and breed and variety formation. It is a way for breeders to utilize heterosis and to introduce new genetic variation into existing plant and livestock populations. During the mid-19th century, several chicken breeds that had been introduced to America from Europe and Asia became the founders for those formed in the USA. Historical records about the genealogy of these populations are often unclear and inconsistent. Here, we used genomics in an attempt to describe the ancestry of the White Plymouth Rock (WPR) chicken. In total, 150 chickens from the WPR and 8 other stocks that historical records suggested contributed to its formation were whole-genome re-sequenced. The admixture analyses of the autosomal and sex chromosomes showed that the WPR was likely founded as a cross between a paternal lineage that was primarily Dominique, and a maternal lineage where Black Java and Cochin contributed in essentially equal proportions. These results were consistent and provided quantification with the historical records that they were the main contributors to the WPR. The genomic analyses also revealed genome-wide contributions (<10% each) by Brahma, Langshan, and Black Minorca. When viewed on an individual chromosomal basis, contributions varied considerably among stocks.

Asymmetries, heterosis, and phenotypic profiles of red junglefowl, White Plymouth Rocks, and F1 and F2 reciprocal crosses.

During the domestication of farm animals, humans have manipulated genetic variation for growth and reproduction through artificial selection. Here, data are presented for growth, reproductive, and behavior traits for the red junglefowl, a line of White Plymouth Rock chickens, and their F1 and F2 reciprocal crosses. Intra- and intergenerational comparisons for growth related traits reflected considerable additive genetic variation. In contrast, those traits associated with reproduction exhibited heterosis. The role of sexual selection was seen in the evolution of prominent secondary sexual ornaments that lend to female choice and male-male competition. The large differences between parental lines in fearfulness to humans were only mitigated slightly in the intercross generations. Whereas, overall F1 generation heterosis was not transferred to the F2, there was developmental stability in the F2, as measured by relative asymmetry of bilateral traits. Through multigenerational analyses between the red junglefowl and the domestic White Plymouth Rocks, we observed plasticity and considerable residual genetic variation. These factors likely facilitated the adaptability of the chicken to a broad range of husbandry practices throughout the world.

Genomic signatures of 60 years of bidirectional selection for 8-week body weight in chickens.

Sixty years, constituting 60 generations, have passed since the founding of the Virginia body weight lines, an experimental population of White Plymouth Rock chickens. Using a stringent breeding scheme for divergent 8-week body weight, the lines, which originated from a common founder population, have responded to bidirectional selection with an approximate 15-fold difference in the selected trait. They provide a model system to study the genetics of complex traits in general and the influences of artificial selection on quantitative genetic architectures in particular. As we reflect on the 60th anniversary of the initiation of the Virginia body weight lines, there is opportunity to discuss the findings obtained using different analytical and experimental genetic and genomic strategies and integrate them with a recent pooled genome resequencing dataset. Hundreds of regions across the genome show differentiation between the 2 lines, reinforcing previous findings that response to selection relied on standing variation across many genes and giving insights into the haplotype complexity underlying regions associated with body weight.

Correlated responses to long-term divergent selection for 8-week body weight in female White Plymouth Rock chickens: Sexual maturity.

Reported here are correlated responses for reproductive traits to long-term divergent selection (54 generations) for 8-week body weight (BW8). Comparisons involved both selected and relaxed lines. Traits measured were age at first egg (AFE), body weight at first egg (WFE), and ratio of body weight and age at first egg (WAFE). Although sexual maturity was delayed in the selected lines, the effect was more pronounced in the low than high selected and relaxed lines. Selection for low BW resulted in decreases in WFE and WAFE. Correlated responses to selection for high BW were increased WFE and WAFE. Minimum AFE, WFE, and WAFE in relation to sexual maturity were line specific and influenced by selection for BW8. WAFE provided a "yardstick" for target body weights that were optimum for successful attainment of sexual maturity and higher reproductive rates. Such may be line specific. There was opposition between relaxed and artificial selection, resulting in a higher reproductive performance and fitness for the former.

Dual functioning ovaries and atresia in chickens. Is it a coincidence?

During the course of a QTL study involving an intercross between White Plymouth Rock chickens and Red Jungle Fowl, certain reproductive anomalies such as atresia and double ovary-oviduct became evident. Observed in reciprocal pedigreed F1 crosses were 2 full-sib pullets with dual functioning ovaries. One also had complete functioning left and right oviducts. The other had asymmetrical reproductive tracts with a typical left oviduct and a rudimentary right oviduct. In addition, there were 3 females with atresia. Although they had different sires and dams, their grandparents were related, and all 5 traced to a common ancestor. Pedigrees, photos, and biological aspects of these conditions are presented and discussed.

Selection for juvenile body weight in chickens: Standardizing for scaling.

The influence of scaling on phenotypic responses to long-term selection was evaluated in lines of chickens selected for high (HWS) and low (LWS) 8-week body weight. The focus of the experiment was on phenotypic correlations between means and standard deviations, and unadjusted vs. standardized responses. Traits measured were body weight at 4 (BW4) and 8 wk (BW8). Correlations between means and standard deviations for BW8 (the selected trait) were positive and greater for BW4 in LWS than HWS. In both lines, scaling masked the degree more than the pattern of response; however, after standardization, it was greater in LWS than HWS. Selection changed the standard deviation units of juvenile BW with a 2-fold difference in BW8 for both HWS and LWS in which the latter reached a physiological limit. Standardization is a robust method to adjust for scaling, thus enhancing the interpretability of responses in long-term selection experiments. Because of changes in variances associated with those in phenotypic means, this method to correct for part-whole correlations was useful.

Chickens from lines artificially selected for juvenile low and high body weight differ in glucose homeostasis and pancreas physiology.

Artificial selection of White Plymouth Rock chickens for juvenile (day 56) body weight resulted in two divergent genetic lines: hypophagic low weight (LWS) chickens and hyperphagic obese high weight (HWS) chickens, with the latter more than 10-fold heavier than the former at selection age. A study was designed to investigate glucose regulation and pancreas physiology at selection age in LWS chickens and HWS chickens. Oral glucose tolerance and insulin sensitivity tests revealed differences in threshold sensitivity to insulin and glucose clearance rate between the lines. Results from real-time PCR showed greater pancreatic mRNA expression of four glucose regulatory genes (preproinsulin, PPI; preproglucagon, PPG; glucose transporter 2, GLUT2; and pancreatic duodenal homeobox 1, Pdx1) in LWS chickens, than HWS chickens. Histological analysis of the pancreas revealed that HWS chickens have larger pancreatic islets, less pancreatic islet mass, and more pancreatic inflammation than LWS chickens, all of which presumably contribute to impaired glucose metabolism.

Phenotypic responses of chickens to long-term, bidirectional selection for juvenile body weight--historical perspective.

A long-term selection experiment for high (HWS) and low (LWS) BW at 8 wk of age (BW8) was conducted in White Plymouth Rock chickens. Over 54 generations of selection, responses to bidirectional selection were profound. Increase in BW8 in line HWS was linear, and there was a significant quadratic response in line LWS for BW at both 4 and 8 wk of age. Although there is no indication that line HWS has come close to approaching a selection limit in more than 50 generations, selection limits occurred in line LWS chickens at generation 48 for females and generation 50 for males. Evidence also exists that one or more beneficial mutations have occurred in line HWS, aiding in progressive increases in BW8 over generations. Analyses of ratios of BW at 4 wk of age with those at 8 wk of age (ratio 4/8) revealed that LWS females grew proportionately faster through 4 wk of age than LWS males or HWS chickens. Comparisons of the selected lines with contemporary lines in which selection had been relaxed (discontinued) indicated that, in line HWS, the relaxed lines generally regressed toward original (preselection) values, suggesting that the linear response to single-trait selection was at least partially due to continued genetic variance. In LWS chickens, a series of plateaus in selection response occurred, but relaxed contemporary lines still regressed toward preselection values for BW8. In spite of the length of this selection experiment (54 generations), genetic variance and beneficial mutations have allowed continued, linear response to selection for increased BW8. Response to selection for decreased BW8 has been tempered by physiological barriers that have decreased survival of young chicks or the ability of females to reproduce. These findings are discussed in a historical perspective.

Extra-phosphoric effects of superdoses of a novel microbial phytase.

An experiment was conducted to evaluate the influence of a novel microbial phytase on broiler performance from d 0 to 42 and tibia ash at d 21. Male Cobb 500 broilers (n = 2,016) were fed 1 of 7 experimental diets: positive control (PC) formulated to meet or exceed nutrient recommendations; PC plus dicalcium phosphate (PC+DCP) formulated to provide Ca and P at 0.10% above the PC; PC plus 500 U/kg of microbial phytase (PC+500); negative control (NC) with Ca and P reduced from the PC by 0.16 and 0.15%, respectively; and NC plus 500 (NC+500), 1,000 (NC+1,000), or 1,500 (NC+1,500) U/kg of microbial phytase. Diets were fed in 3 phases from d 0 to 21, d 22 to 42, and d 43 to 49 to 32 birds/pen and 9 replicate pens/diet. From d 0 to 21, broilers fed the NC diet had decreased (P ≤ 0.05) BW gain and tibia ash compared with broilers fed all other diets, except tibia ash in birds fed PC+500. Phytase supplementation at 500, 1,000, or 1,500 U/kg to the NC improved (P ≤ 0.05) BW gain and tibia ash comparable with the PC. Feed conversion ratio (FCR) was improved (P ≤ 0.05) in broilers fed NC+1,500 compared with broilers fed all other diets. From d 0 to 49, growth performance was not influenced (P > 0.05) by diet. However, FCR was improved (P ≤ 0.05) in broilers fed 1,500 U/kg of microbial phytase compared with broilers fed the PC, PC+DCP, and NC. There were no differences in performance or tibia ash between broilers fed the PC or PC+DCP, which would indicate the PC diet was sufficient in Ca and P. Therefore, the improvements in FCR in the NC+1500 may be associated with mitigation of the antinutrient effects of phytate rather than improved P utilization.

Phenotypic responses of chickens to long-term selection for high or low antibody titers to sheep red blood cells.

A long-term bidirectional selection experiment was conducted to study antibody response to SRBC. Lines, high antibody selection (HAS) and low antibody selection (LAS), originating from the same White Leghorn base population had undergone 37 generations of selection for either high or low antibody response 5 d after a single intravenous injection of 0.1 mL of a 0.25% suspension of SRBC antigen. Subpopulations, where selection was relaxed, were maintained as contemporaries with the selected lines from generations 16 to 24 [high antibody relaxed (HAR) and low antibody relaxed (LAR)] and 24 to 37. Body weights were obtained at 4, 24, and 38 wk of age and at the onset of lay (BW at first egg). Also measured were age in days to first egg, percentages of hen-day ovulations and normal egg production, and percentages of normal and defective eggs from total ovulation (PNE and PDE). Selection lead to a large divergence in antibody titers between the selected lines, with a plateau reached in line LAS. Line HAS and HAR females displayed higher antibody titers, lower BW4, and matured at older ages than those from LAS and LAR (P < 0.05). Correlations between BW at 4 wk and antibody titers were different between the selected lines, being positive in line LAS and negative in line HAS. Quadratic regression models fit well with antibody titers, BW4, and PNE, with limiting values for these traits calculated based on regression curves. For line HAS, plots showed that an increased tendency of antibody titers was followed by decreased BW4 and increased PNE. For line LAS, however, antibody titers and BW4 decreased in parallel while PNE increased. It appears that at the phenotypic level there was a resource balance between immune response, growth, and reproductive traits, which during long-term selection, individuals altered their dynamic of resource allocations to satisfy certain needs.

Effect of incubation temperature on nutrient transporters and small intestine morphology of broiler chickens.

This study evaluated the effects of elevated incubation temperature on posthatch nutrient transporter gene expression, integrity of the intestinal epithelium, organ development, and performance in Ross 308 broiler chickens. Ross × Ross 308 fertile eggs (n = 900) were incubated at different eggshell temperatures during development. From embryonic day (ED) 1 to ED12, all eggs were incubated at 37.1°C, whereas from ED13 to ED21, the eggs were divided into 2 groups for incubation at 37.4°C (S) or 39.6°C (H). Performance characteristics were measured at day of hatch (DOH) and d 7, 14, 21, 30, and 42. Small intestine and residual yolk sacs were collected at DOH and d 2, 4, 6, and 10 and weighed individually. Intestinal samples from the duodenum, jejunum, and ileum were evaluated for mucosal morphology and relative nutrient transporter gene expression. No significant differences were found in performance or organ weights. The intestinal morphology results showed a temperature × age interaction in duodenum villus height (P = 0.02) and crypt depth (P = 0.05) and in ileum villus height-to-crypt depth ratios (P = 0.02). There was a main effect of temperature, resulting in deeper crypts (P = 0.02) in the jejunum of chicks incubated at H compared with S. In the nutrient gene expression evaluation, peptide transporter (PepT1) showed a temperature × age interaction. On DOH and d 2, 4, and 10, PepT1 expression was similar between chicks incubated at S and H. However, on d 6, chicks incubated at S had significantly higher expression of PepT1 than those incubated at H. This study presents the effects of elevated incubation temperature on small intestine morphology and relative expression of nutrient transporter mRNA in high-yield broiler chicks, which can be important for the availability of nutrients and distribution of energy.

Antibody response of chickens to sheep red blood cells: crosses among divergently selected lines and relaxed sublines.

Crosses were made among lines of chickens that had undergone 30 generations of selection for high or low antibody response 5 d after an intravenous injection with SRBC, and between sublines in which selection was relaxed in generation 24. Antibody responses at 5, 10, and 14 d after injection were measured in the 4 lines and in reciprocal crosses among them. Divergence between the high and low lines selected for SRBC antibody was immediate and increased during selection. Although significant in both cases, separation of the relaxed subline from its respective selected line was greater in the high than the low line. Five-day SRBC titers of the relaxed lines and the crosses were intermediate to the high and low selected lines, with the direction and magnitude of heterosis being line dependent. A high proportion of chickens from low line mating combinations did not have detectable antibody titers at 10 and 14 d postinoculation with SRBC, precluding statistical analysis of these data. Results are discussed in the context of intra- and interlocus effects on the selected trait.

The effect of claw and beak reduction on growth parameters and fearfulness of two Leghorn strains.

Commercial equipment used by the turkey industry at hatch sterilizes the germinal tissue of the claw with microwave energy and the beak tissue with infrared energy. This effectively trims the claws and beaks of the birds. Two strains of Leghorn chicks (1,200 each strain) were utilized to test this technique on chickens. Half were subjected to the claw reduction (RC) technique at hatch, and half retained intact claws (IC). The beaks of one-third of the birds in the claw treatments were reduced (BT) at hatch using the infrared technique, one-third were precision trimmed at 7 d, and one-third were not trimmed (IB). Body weight, weight gain, feed intake, feed conversion, mortality, and fearfulness were measured. Rearing followed standard commercial feeding and husbandry procedures. The RC birds had significantly lower BW, except from 3 to 6 wk, and had significantly lower feed consumption from 8 to 18 wk. The 1 d BT birds experienced significantly lower BW from 3 to 14 wk and ate less total feed by 4 wk. Mortality was less than 2.1% for all treatments. From 6 to 8 wk, fearfulness score (a subjective scale of 1 to 10) peaked at 8 to 10 for IC birds and 3 to 4 for RC birds. By 16 to 18 wk, fearfulness score subsided to 2 to 3 for RC and 6 to 8 for IC. Beak trimming and reduction of claw growth at hatch allowed pullets to be grown to sexual maturity on less feed and with a lower level of observed fearfulness using standard husbandry practices.

Lung fibroblasts undergo apoptosis following alveolarization.

In the rat lung, primary saccules are transformed into alveoli from postnatal Days 4 to 13, after which time there is a 20% reduction in the number of lung fibroblasts as the interstitial volume of the alveolar walls decreases. Our objective was to determine whether apoptosis is a factor in the observed decrease in the number of interstitial lung fibroblasts beyond Day 13. We used both histologic and flow cytometric assays to detect in lung fibroblasts the DNA fragmentation and condensation that are characteristic of apoptosis. In addition, we evaluated levels of bcl-2 and BAX messenger RNAs (mRNAs) using a reverse transcriptase-polymerase chain reaction (RT-PCR) assay. Apoptotic cells were quantitated in glycol methacrylate-embedded sections of neonatal rat lungs using the terminal transferase dUTP-digoxygenin nick end-labeling (TUNEL) method. Although TUNEL-positive interstitial cells were observed in the lungs of rats ranging in age from 10 to 16 d, a dramatic increase in apoptotic cells was seen on Day 17. Although diminished in number, TUNEL-positive cells were still present on Day 28. Hoechst-stained apoptotic bodies were observed in isolated lung cells that were vimentin-positive and factor VIII-negative, which identified the apoptotic cells as fibroblasts as opposed to endothelial cells. Flow cytometric analysis of freshly isolated lung fibroblasts stained with Hoechst 33342 indicated a 24% increase in chromatin condensation in cells from 17-d versus 16-d rats. DNA fragmentation was also quantitated by flow cytometry in freshly isolated fibroblasts labeled with BODIPY-conjugated dUTP in the presence of terminal deoxynucleotidyl transferase. The percentage of lung fibroblasts containing fragmented DNA was 51.4 +/- 13.4 in 17-d, 36.9 +/- 8.6 in 18-d, and 13.8 +/- 5.4 in 19-d rat pups. Finally, evaluation by RT-PCR indicated that on postnatal Day 17, mRNA for bcl-2, which inhibits apoptosis, was decreased to 73.5 +/- 11.4% (P < 0.001) of Day 5 controls; whereas mRNA for BAX, which enhances apoptosis, was increased to 243.0 +/- 102.0% (P < 0.001) of Day 5 values. These results demonstrate that rat lung fibroblasts undergo apoptosis after the completion of alveolarization, and suggest that this decrease in fibroblast number plays an important role in the thinning and remodeling of the alveolar walls of the lung.

Transcriptional regulation of tropoelastin expression in rat lung fibroblasts: changes with age and hyperoxia.

Elastic fibers are thought to provide structural support for secondary septa as the lung undergoes the transition from the saccular to the alveolar stage. The synthesis of the soluble precursor of elastin, tropoelastin, occurs during a finite developmental period. We have investigated the developmental regulation of tropoelastin gene transcription and mRNA expression in fetal and postnatal rat lung fibroblasts and have assessed the changes in tropoelastin gene expression caused by hyperoxic exposure during secondary septal development. With the use of an RT-PCR assay and intron-specific primers to detect heterogeneous nuclear RNA (hnRNA) and intron-spanning primers to detect mRNA in freshly isolated rat lung fibroblasts, tropoelastin gene expression was found to be upregulated late in gestation. From days 18 to 21 of gestation, there was a 4.5-fold increase in tropoelastin hnRNA (P < 0.0001) and a 6-fold increase in mRNA (P = 0.002). After birth, tropoelastin expression was downregulated. Signals decreased from fetal day 21 to postnatal day 2 for both tropoelastin hnRNA (P = 0. 021) and mRNA (P = 0.043). Tropoelastin hnRNA decreased further from days 2 to 6 (P = 0.04). Both tropoelastin hnRNA and mRNA were again upregulated during alveolarization from days 9 to 11, indicating that, once upregulated, transcription of the tropoelastin gene is not constant but varies with fetal and postnatal age. Exposure to >95% oxygen, when initiated on postnatal day 2 or 3 and continued until day 11, significantly diminished the developmental increase in tropoelastin hnRNA (P < 0.005) and mRNA (P < 0.05) normally seen on days 9-11, indicating that the postnatal upregulation of tropoelastin gene expression is inhibited by hyperoxic exposure in the early postnatal period.

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Postnatal age at onset of hyperoxic exposure influences developmentally regulated tropoelastin gene expression in the neonatal rat lung.

Upregulation of tropoelastin (TE) gene expression in rat lung interstitial fibroblasts normally occurs during alveolar septation. TE message increases at the end of the first week of life, peaks on days 9-11, and returns to barely detectable levels over the next 7-10 days. Our previous in situ hybridization studies indicated that exposure of pups to > 95% oxygen from 3 to 13 days of age interfered with the increased in TE gene expression in interstitial fibroblasts normally seen during septation. However, when the pups were returned to room air, lung fibroblast TE message levels increased, exceeding levels seen in control lungs during the exposure. In addition, TE message levels remained elevated for a week after levels in control lungs had returned to background. A possible interpretation of these results was that the developmentally regulated increase in TE messenger RNA (mRNA) was downregulated by the hyperoxic exposure but resumed when the pups were returned to a normoxic environment. We report herein the results of a subsequent study conducted to determine whether continued hyperoxic exposure beyond day 13 would further delay the peak in TE mRNA. Rat pups were exposed to 95% O2 from 5 to 17 days of age. TE and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) message levels in lung interstitial fibroblasts were assessed by in situ hybridization. As observed in pups exposed from 3 to 13 days, hyperoxic exposure from days 5 to 17 also extended the period during which TE mRNA levels were elevated. After exposure, TE message levels were 99%, 262%, and 223% of controls on days 19, 21, and 23 respectively. In addition, delaying the exposure 2 days until the pups were 5 days old resulted in an upregulation of TE message, relative to control values, during the hyperoxic exposure. In hyperoxic pups, values for TE message expression were 105%, 152%, 168%, and 144% of control pups on days 9, 11, 13, and 16 respectively. The influence on peak TE message expression of postnatal age at the time of exposure was further explored to verify the results of the 3-13 and 5-17 day exposures. When pups were exposed continuously from 2, 3, 4, 5, or 6 days until 11 days of age, the results of both in situ hybridization and Northern blot analysis confirmed our previous observations, demonstrating that the postnatal age at which hyperoxic exposure is initiated influences TE message expression in the developing lung.