Production and Transfer of IVF Embryos from Individual Inhibin-Immunized Cows by Ultrasound-Guided Transvaginal Follicular Aspiration

Abstract

Ultrasound-guided transvaginal follicular aspiration (UTA) was performed on 8 multiparous Japanese Black cows which had not been successful in a conventional superovulation program. These cows were actively immunized with a synthetic peptide replica of porcine inhibin αN1-26 (pINH) conjugated with rabbit serum albumin (RSA) using Freund's complete adjuvant. Booster injections of the peptide were given at 6, 10, 14 and 20 wk after the primary injection. Twelve follicular aspirations were performed between 10 and 33 wk after the primary injection. Cumulus-oocyte complexes (COCs) were aspirated from follicles ≥ 2 mm in diameter. The recovered COCs were classified into 5 categories according to the surrounding cumulus cells: Grade 1 (> 4 layers), Grade 2 (3 to 4 layers), Grade 3 (1 to 2 layers), Grade 4 (denuded) and Grade 5 (expanded and fluffy cumulus cell mass or degenerated ooplasm). The recovered COCs available for in vitro fertilization (IVF; Grades 1, 2, 3 and 4) were matured with granulosa cells (1×10⁶ cells/ml) for 20 to 23 hr, fertilized with frozen-thawed spermatozoa, and cultured in vitro. At day 7 after insemination, a portion of the embryos were transferred to Holstein heifers. There were great variations among donors in the number of recovered COCs and in the numbers of COCs in each grade, and also in the developmental ability of fertilized COCs. IVF procedures were carried out on 685 oocytes and produced 120 embryos (17.5%). The transfer of 36 fresh IVF embryos resulted in 20 pregnancies (55.6%) at 60 days after the transfer. This study has demonstrated first, that the number of COCs collected from individual inhibin-immunized cows by UTA greatly varies among donors. Second, recovered COCs vary greatly in the classification of the morphological categories of surrounding cumulus cell mass among donors and had different developmental abilities when they were cultured after fertilization in vitro. Third, the production of pregnancy in previously non-productive problem donors is possible using the UTA technique.