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Figure 4-6.
(A) Collecting mature unfertilized eggs from an adult female by gently squeezing the abdomen. An anesthetized female is sandwiched between pieces of cotton wool moistened with BSS with its abdomen up. Gently depression of the area of the ovarian lumen toward the urogenital pore using thumb and one finger is applied. (B) Mature unfertilized egg. Oil droplets and cortical alveoli are observed in the cortical cytoplasm. (C) An activated egg by fertilization with thawed sperm. Note that a periviteline space (asterisk) is formed between the egg envelope (chorion) and the vitellus while the cortical alveoli disappears. Animal views are seen in B and C. Scale bars, 100 μm. (D) Collecting a capillary filled with frozen sperm. A cryotube containing capillaries is held with a prechilled Lorna clamp and the cap is opened on a dry ice block covered with aluminum foil in a styrofoam box. Leather gloves are used to avoid frostbite. The capillary is picked up using prechilled fine forceps. If capillaries are frozen together, use the forceps to separate them by poking the junction between them. E. Dilution of thawed sperm suspension. The capillary filled with thawed sperm suspension is attached to the capillary dispenser. The thawed sperm suspension is dispensed into BSS in the lid of a 0.5-mL centrifuge tube.