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First published online July 20, 2007
Journal of Experimental Biology 210, 2667-2675 (2007)
Published by The Company of Biologists 2007
doi: 10.1242/jeb.005751

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In ovo temperature manipulation influences embryonic motility and growth of limb tissues in the chick (Gallus gallus)

Christina L. Hammond^*, Biggy H. Simbi and Neil C. Stickland

Department of Veterinary Basic Sciences, The Royal Veterinary College, Royal College Street, London, NW1 0TU, UK

View larger version (8K): [in this window] [in a new window]   Fig. 1. Schematic to show the incubation temperature regime and tissue collection schedule of experiments

View larger version (10K): [in this window] [in a new window]   Fig. 2. Mean number of embryonic movements, not including amnion contractions, per embryo in a 5 min period. **P<0.05, ***P<0.001. Sample sizes: ED5, N=10; ED6, N=22; ED7, N=18; ED11, N=14.

View larger version (9K): [in this window] [in a new window]   Fig. 3. Total embryo wet mass of chicks sampled between ED9 and ED18. **P<0.05, ***P<0.001. Sample sizes: ED9, N=14; ED11, N=14; ED12, N=15; ED15, N=32; ED18, N=24.

View larger version (45K): [in this window] [in a new window]   Fig. 4. (A) Mean combined length of the tarsus and tibia in chicks between ED11 and ED18. **P<0.05, ***P<0.001. Sample sizes: ED11, N=14; ED12, N=16; ED15, N=32; ED18, N=24. (B) Mean femur length between ED12 and ED18. Sample sizes: ED12, N=16; ED15, N=28; ED18, N=22. **P<0.05, ***P<0.001. (C,D) Alcian Blue/Alizarin Red-stained chick hind limbs; red staining shows mineralized bone, while blue staining shows the unmineralized cartilage. (C) Representative hind limbs at ED12; the top limb comes from a chick raised at 38.5°C while the lower limb comes from a control chick. (D) Representative hind limbs at ED15; the limb on the left comes from a chick raised at 38.5°C, with a control limb on the right. Scale bar in C and D represents 5 mm. Sample sizes were ED12, N=12; ED15, N=22. (E,F) Images taken from Micro-CT scans of ED15 femurs. (E) ED15 femur of a chick raised at 37.5°C; left image shows the whole bone while the three right images show, top to bottom, individual scans at 25%, 50% and 75% length of the mineralized portion of the bone (as calculated by the programme); the white portion in the cross-sectional scan represents mineralized bone. (F) As E but for a chick raised on the experimental temperature regime.

View larger version (79K): [in this window] [in a new window]   Fig. 5. (A) Mean number of myofibres and myonuclei per 150 000 µm2 of cross-sectional area of the gastrocnemius muscle at ED18 (N=14). (B) Mean mass of the gastrocnemius at ED18 (N=14). (C) Mean ratio of myonuclei:myofibre in the gastrocnemius at ED18 (N=14). In A–C, **P<0.05, ***P<0.001. (D,E) Representative haematoxylin- and eosin-stained cross sections through the gastrocnemius of ED18 chicks raised at 37.5°C (D) and 38.5°C (E). Scale bars, 50 µm. (F,G) Representative Decorin antibody stained cross sections, through the gastrocnemius at ED18 of chicks raised at 37.5°C (F) and 38.5°C (G).

View larger version (32K): [in this window] [in a new window]   Fig. 6. (A) Mean adipocyte cell cross-sectional area in chicks at ED15 (N=10) and ED18 (N=12). (B) Mean cross-sectional area of the pectoral fat pad at ED18 (N=12). ***P<0.001. (C,D) Haematoxylin- and eosin-stained cross-sections through the pectoral fat pads of ED15 chicks raised at 37.5°C (C) and 38.5°C (D). Scale bars, 50 µm.