All animal procedures were approved by the IACUC at the University of Nebraska-Lincoln. Non-lactating crossbred beef cows between 2 and 6 years in age (Red Angus, Pinsgauer, Red Poll, Herford composites with some Red Angus/Limousin) from the UNL Physiology research and teaching herds were used. To synchronize animal estrous cycles PGF2α (25mg; Lutalyse®, Zoetis Inc., Kalamazoo Michigan, MI) was injected on Day 1 with a subsequent injection of GnRH 36 h later. Ovulation occurs 24 h post-GnRH injection, and on day 13.5 cows will have fully functional mid-luteal phase corpus luteum, equivalent to day 10 in the natural cycle. Cows were fasted for 12–18 h before ovariectomy but retained ad libitum access to water. At day 13.5 cows were treated with an intra-muscular injection of saline (n = 3) or PGF2α (25mg; Lutalyse®) (n = 3). Transrectal ultrasonography was performed immediately before ovariectomy to confirm the presence of corpus luteum. Ovaries were removed via a right-flank approach paralumbar fossa laparotomy, to avoid the rumen and prevent internal hemorrhaging, as previously detailed (Summers et al., 2014 (link); Talbott et al., 2017 (link)). Portions of luteal tissue were snap frozen using liquid nitrogen for subsequent protein analysis.
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