Transvaginal collection and ultrastructure of llama (Lama glama) oocytes

Ultrasound-guided transvaginal follicle aspiration has been described as a noninvasive and repeatable procedure for oocyte collection in several species, but its use has not been described for any of the members of the family, Camelidae. A study was designed to determine the feasibility of an ultrasound-guided transvaginal approach for oocyte collection in llamas. Fifteen non-pregnant, adult female llamas (10 non-stimulated and 5 superstimulated) were examined by transrectal ultrasonography with a 7.5-MHz linear-array transducer to determine the number and diameter of follicles available for aspiration. After caudal epidural anesthesia was induced, the 7.5-MHz linear-array transducer was fastened to a long rigid handle and inserted intravaginally. The free hand was placed into the rectum to manipulate the ovaries, one at a time, in position against the vaginal wall over the face of the transducer. A 20-gauge, 55-cm-long, single-lumen needle was advanced through the vaginal fornix and into follicles > or = 3 mm in diameter. Follicular contents were aspirated using a regulated vacuum pump (flow rate = 33 mL/min; approximately 150 mm Hg) into a tube containing 3 mL of phosphate buffered saline and 0.2% BSA. Fluid was filtered (75 microm mesh), and oocytes were located and morphologically evaluated using a stereomicroscope. Overall, 134 follicles were aspirated, and 76 oocytes were collected (collection rate = 57%). Thirty-two oocytes (42%) were surrounded by multiple layers of compacted granulosa cells and had homogenous dark ooplasm; 13 oocytes (17%) were surrounded by the corona radiata layer only and had heavily granulated ooplasm; 9 oocytes (12%) were denuded and had homogenous dark ooplasm; and 22 oocytes (29%) were denuded and displayed signs of ooplasm degeneration. The ultrastructure of llama oocytes was similar to that of cattle except for conspicuous accumulation of large lipid droplets in the cytoplasm. Twenty-four hours after follicle aspiration, the ovaries were examined by transrectal ultrasonography and intrafollicular hematomas were detected in 3 llamas (9 of 48 follicles aspirated). Results demonstrate the potential utility of a transvaginal ultrasound-guided technique for oocyte collection and in vitro embryo production in llamas. Oocytes of llamas bear an ultrustructural resemblance to those of cattle, but are distinguished by a predominance of cytoplasmic lipid.