Critical developmental windows for morphology and hematology revealed by intermittent and continuous hypoxic incubation in embryos of quail (Coturnix coturnix)

Abstract

Hypoxia during embryonic growth in embryos is frequently a powerful determinant of development, but at least in avian embryos the effects appear to show considerable intra- and inter-specific variation. We hypothesized that some of this variation may arise from different protocols that may or may not result in exposure during the embryo's critical window for hypoxic effects. To test this hypothesis, quail embryos (Coturnix coturnix) in the intact egg were exposed to hypoxia (~15% O2) during "early" (Day 0 through Day 5, abbreviated as D0-D5), "middle" (D6-D10) or "late" (D11-D15) incubation or for their entire 16-18 day incubation ("continuous hypoxia") to determine critical windows for viability and growth. Viability, body mass, beak and toe length, heart mass, and hematology (hematocrit and hemoglobin concentration) were measured on D5, D10, D15 and at hatching typically between D16 and D18 Viability rate was ~50-70% immediately following the exposure period in the early, middle and late hypoxic groups, but viability improved in the early and late groups once normoxia was restored. Middle hypoxia groups showed continuing low viability, suggesting a critical period from D6-D10 for embryo viability. The continuous hypoxia group experienced viability reaching <10% after D15. Hypoxia, especially during late and continuous hypoxia, also inhibited growth of body, beak and toe when measured at D15. Full recovery to normal body mass upon hatching occurred in all other groups except for continuous hypoxia. Contrary to previous avian studies, heart mass, hematocrit and hemoglobin concentration were not altered by any hypoxic incubation pattern. Although hypoxia can inhibit embryo viability and organ growth during most incubation periods, the greatest effects result from continuous or middle incubation hypoxic exposure. Hypoxic inhibition of growth can subsequently be "repaired" by catch-up growth if a final period of normoxic development is available. Collectively, these data indicate a critical developmental window for hypoxia susceptibility during the mid-embryonic period of development.

Fig 1. Experimental protocol for acute and chronic hypoxic treatments during the incubation of quail embryos.

The shaded regions indicate hypoxic exposure.

Fig 2. Cumulative viability rates of normoxic (control) and treatment groups of quail embryos measured at Days 10, 15, and Hatch.

Table 1 provides n-values of normoxic and each treatment group. Insert contains specific comparisons, test statistics and significance values. Comparisons not shown in the inset were not significant (P>0.05)

Fig 3

Wet body mass (A) and dry body mass (B) changes during incubation in quail embryos measured on D10, D15, and hatch for control and early, middle, late and continuous hypoxia groups. Mean ± 1 standard error is plotted. n values for each population are provided in Table 1. Statistical differences between each incubation group mean within a developmental time are indicated by letters (separate lower case, upper case or upper case italic for each developmental day).

Fig 4

Changes in A) Beak length and (B) Third toe length during incubation in quail embryos measured on D10, D15, and hatch for control and early, middle, late and continuous hypoxia groups. Statistical differences between each incubation group mean within a developmental time are indicated by letters (separate lower case, upper case or upper case italic for each developmental day). Mean ± 1 standard error is plotted. n values provided in Table 2.

Fig 5

Heart mass (A, B) and heart mass to body mass ratio (C, D) changes during incubation in quail embryos for control and early, middle, late and continuous hypoxia. (Mean ± 1 standard error is plotted. n values provided in Table 3. Statistical differences between each incubation group mean within a developmental time are indicated by letters (separate lower case, upper case or upper case italic for each developmental day).

Fig 6

Developmental changes in (A) hematocrit and (B) hemoglobin concentration during normoxic and hypoxic incubation in quail embryos. Mean ± 1 standard error is plotted. Mean ± 1 SEM is plotted. n values for each stage are the same as provided for body mass in Table 1. Statistical differences between each incubation group mean within a developmental time are indicated by letters (separate lower case, upper case or upper case italic for each developmental day).