Collection, Handling, Fixation, and Processing of Tammar Wallaby (Macropus eugenii) Embryos

INTRODUCTION

This protocol describes methods for collecting and handling tammar wallaby (Macropus eugenii) conceptuses. Zygotes, cleavage stages, diapausing blastocysts, and unexpanded blastocysts are ~250 μm in diameter and are collected by flushing either the oviduct or uterus. Later stages are collected by opening the uterus. Late preimplantation stages must be handled carefully because their large size (>2 cm in diameter) results in high surface tension within the yolk sac, especially on the delicate avascular area. When handled too roughly, the embryonic vesicle tends to split and then rapidly collapse; such embryos do not reinflate. If embryos are to be cultured, all steps during collection must be carried out aseptically. In all cases, solutions should be prewarmed to 37°C and embryos should be kept warm by use of a heating stage. Standard fixatives, fixation times, and washing protocols used for mouse embryos are also used for tammar embryos. Methods for embedding tammar conceptuses in paraffin wax depend on the size and stage of the conceptus.