| Histology changes and metabolism in limb regenerating of tadpoles of Bufo raddei Strauch in the stage of 27 and 32 are studied in this paper using tissue slice technique and electron extra-thin slice method. At the same time, the distribution of Retinoic Acid (RA) in regenerating limb is determined by Immunochemistry techniques to explore the characteristics and roles of RA in regeneration. The results are as follow.Under laboratory conditions, tadpoles of stage 27 and 32 amputated left limb are all able to regenerate spontaneously after simply amputation. The ability to regenerate lost limbs is affected greatly by different individuals and development stages. For tadpoles amputated at stage 27, the entire left limbs can be restored in almost every individual. The frequency of hind-limb regeneration reaches 100% in this group. But the regenerated limbs are smaller than opposite limb. Length ratio of left tibiofibula to right tibiofibula is 0.892±0.036, and the length ratio of left femur to the right femur of it is 0.914±0.023. For 32TP (indicating tadpoles of the 32nd stages which are amputated left limb at a site between the joint and knee joint), the frequency of hind-limb regeneration reaches 69%. Few individual can regenerate entirely the lost structure with 5 digits. Most have partly ability to regenerate one to three digits, and the average no. of regenerated digits is 2.2±1.3. Length ratio of left tibiofibula to right tibiofibula is 0.848±0.100, and the length ratio of left femur to the right femur of it is 0.916±0.070. From pictures, we can observe that some can reconstruct smaller but entire left limb, some form abnormal regenerated structures such as unobvious regenerated tarsi bones, digits grown in deviated direction, or much smaller regenerated limb in this group. For 32FP, none complete regenerated limb is found in 32FP (indicating the 32nd tadpoles amputated at femur). In this group, the tadpoles remain the slight power to regenerate digits, we hardly see divided digits. Occasional, we can found that apical regenerated limb of tadpole divides and appears to form two digits, but its tarsi bone still looks very slim. The frequency of hind-limb regeneration reaches 78.2%. Few individual can regenerate entirely the lost structure with 5 digits. Most have partly ability to regenerate one to three digits, and the average no. ofregenerated digits is 1.3±0.5. Length ratio of left tibiofibula to right tibiofibula is 0.464±0.127, and the length ratio of left femur to the right femur of it is 0.849±0.079. So, the older of tadpoles of Bufo raddei, the lower ability to regenerate lost limbs.Results from tissue slice technique show that a series of changes including wound healing, blastema formation, redifferentiation and pattern formation are involved in limb regenerating of tadpoles of bufo raddei. And limb regenerating processes include both growth and regeneration, which is differed from the urodeles. The results also show that cells in the regenerated partly are preexisting mesenchymal cells, which are incomplete differentiated.Results from electron extra-thin slice technique show large amount of macrophage and apoptosis cells around the cut surface on the first day after amputation. Mesenchymal cells exist near the wound with high mitosis activities. Cells dedifferention are not obvious. The amount of mesenchymal cells reach the richest on the 8th d and cells differention are distinct. On the 18th d after amputation, active cells mitosis continues, newly differentiated cells have rich rough endoplasmic reticulums with full of the synthesis, indicating a mass of protein synthesis.Results from Immunochemistry techniques show that RA receptor can express in normal developing limb, cells in whole epidermal are strongly positive for RA. On the 2nd d, cells under the wound surface are labeled deep yellowish grown, indicating strongly positive respond. On the 3rd d, blastema begins to form, tissues near the wound are labeled deep yellowish grown, while areas beneath the epidermal are slightly labeled. So RA receptor may play an important role in the formation of blasterma in limb regenerating.
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