Due to frequent failure of amphibian captive-assurance colonies associated with poor reproduction, it is critical that assisted reproductive technologies be developed as conservation tools for threatened amphibians. We hypothesized that ultrasound could be utilized to monitor follicular development of female salamanders such that hormone protocols could be optimally timed to stimulate oviposition. Using Ambystoma tigrinum (eastern tiger salamander) as a model, female animals were observed via ultrasound throughout their follicular cycle. An ultrasound grading scale was developed for follicular stages based on size (verified with imageJ software)a and echogenicity (0 = little/no development, 1 = minor development, 2 = moderate development, 3 = extensive development).
To test ultrasound management with existing hormone protocols for salamanders in our lab, animals graded 1 (n=9) were treated with a priming dose of 1 IU/g human chorionic gonadotropin (HCG) one week prior to the start of our full ovulatory protocol (1 IU/g HCG; 168 h later 2 IU/g HCG; 24 h later 4 IU/g HCG + 0.1 µg/g luteinizing hormone releasing hormone [LHRH]). Animals graded a level 2 (n=9) were treated with the full ovulatory protocol above, while animals graded a level 3 (n=9) were given a modified ovulatory protocol of the 4 IU/g HCG + 0.1 µg/g LHRH only. Control animals (n=15) were given the full ovulatory protocol; however, they were blindly chosen for treatment and did not receive ultrasonic analysis. In our preliminary trials, 33.3% of animals graded level 1, 83.3% of animals graded 2, and 100% of animals graded 3 oviposited within 18 hours of hormone treatment. In contrast, only 33.3% of control animals oviposited, and time to oviposition was longer (within 72 hours). These preliminary results suggest that ultrasound grading of follicular development can help adaptively manage hormone techniques for salamander species by modifying protocols so they are more efficient based on the physiologic state of the animal.
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