BACKGROUND: The only pre-treatment fertility preservation option for prepubertal girls, who are at risk for infertility due to their diagnosis or treatment, is surgical removal of ovarian tissue for cryopreservation (OTC). We investigated ovarian tissue health following isolation with an ultrasonic advanced energy device (UAED), that has a previously reported thermal spread of ≤2 cm. METHODS: The ovaries of eight Yucatan minipigs were isolated by laparoscopy (1) close dissection with the UAED located up to 2 mm away from the ovarian capsule, (2) far dissection with the UAED located >2 cm away, or by (3) laparotomy for control ovaries using cold scissors. Ovarian cortex tissues were cultured for 4 days to assess tissue health. RESULTS: Ovarian cortex tissue isolated using a UAED produced an altered metabolic ratio in both the far and close dissection compared to control (p < 0.001). There was an increase in folliculogenesis in the control samples over samples isolated with far and close dissection (p < 0.0001), and a reduction in estradiol production in experimental groups (p < 0.0001). CONCLUSIONS: This model defines differences in ovarian tissue health among different isolation techniques. Ongoing work will further define the standard of care surgical technique for OTC.