GameteGuard is a blend of plant extracts containing antioxidants and membrane stabilizers. Previous studies have shown improvements in post-thaw sperm health with GameteGuard addition to the extender, and therefore it was hypothesized that the addition of GameteGuard to sperm extender would result in increased pregnancy rates per AI in lactating cows. Sperm from 2 ABS Global Inc. production Holstein bulls was extended in egg yolk citrate or egg yolk citrate plus GameteGuard to 30 × 106 sperm/mK (15 × 10⁶ sperm/dose) in a split ejaculate study. Sperm was packaged and frozen according to industry standards. The effectiveness of GameteGuard on pregnancy was assessed by first postpartum insemination of 208 Holstein cows. Over 6 mo, cows were cleared for insemination by the farm veterinarian then 2 farm personnel determined estrus status using visual cues such as disturbed tail chalk. The cows were inseminated within 12 h by one trained farm employee. Cows were inseminated with either Bull 1 or 2, treated or control sperm, where treatment was blind to farm personnel. Bull and treatment combinations were dictated by a calendar day. For example, all cows to be bred on May 15 would use bull 1, red straw (control) while May 16 would use bull 1, blue straw (treated). Cows having postpartum problems and those with reproductive issues, such as ovarian cysts, were excluded from the study. Thirty-five to 45 d post-insemination pregnancy was assessed using transrectal ultrasonography and data were analyzed using Chi-squared. GameteGuard improved pregnancy per AI by 31% (from 38 to 50%; P < 0.05; ~100 cows/treatment) across both bulls. Importantly, of the 124 animals bred within 83 d of calving, GameteGuard improved pregnancy per AI 36.1%.