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In vitro fertilization is a reliable laboratory technique for generating large numbers of synchronously developing embryos. This synchronicity enhances Xenopus laevis as a powerful model organism for studying vertebrate embryonic development, molecular, cellular, and disease mechanisms. In conventional in vitro fertilization protocols for X. laevis, male frogs are euthanized, and both testes are harvested. In order to reduce the number of required males, a procedure was developed for unilateral gonadectomy in male X. laevis, which allows sequential use of male frogs' gonads for in vitro fertilizations. This approach has been successfully used to generate embryos, yielding equally good fertilization outcomes with sperm from either the surgically removed or the contralateral testis. The surgical process involves the administration of MS-222 anesthesia, use of Meloxicam and Bupivacaine analgesia, and post-operative care and monitoring, thus allowing reduction in animal use and supporting best practices in animal care across multiple experimental conditions and laboratory setting.
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42475430 ???displayArticle.link???J Vis Exp
Figure 1: Schematic diagram for surgical removal of one testis.
Figure 2: Male X. laevis survival surgery for single testis collection. (A) Frog body partially submerged in an anesthetic at the beginning or in water at the end of the procedure, ensuring that the head remains above the water level. (B) Small incision, approximately 1 cm in length. (C) Intestines and abdominal fat body are gently pulled out until the testis can be seen. The kidney-shaped testis is dissected out. (D) For suturing, square knots are recommended. Muscle and skin layers are sutured separately. (E) Three sutures are appropriate for the incision length. (F) Frog is returned into a separate housing tank with low water for 24 h to enable easy breathing. (G) The frog is returned to a standard housing tank on the recirculating system.
Figure 4: Equally effective development resulting from use of surgically removed and contralateral gonads and reduction in frog usage. (A) Independent clutches of embryos derived from surgically removed gonads. (a,b) Fifteen representative 4-cell stage embryos from two clutches. (a’,b’) The same fifteen embryos at tailbud stage. (B) Embryos derived from using contralateral gonads. (c,d) Fifteen representative four-cell embryos. (c’,d’) The same fifteen embryos at tailbud stage. (C) Total number of male frogs used in 2 month periods from January 2023 to April 2026. Gonadectomies were begun in November 2024 and resulted in a significant reduction in male frog usage.
