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Controlled randomized trials comparing liquid stored and frozen semen in TAI protocols are missing. The objective of this study was to compare liquid stored and frozen semen using either an Ovsynch or a Cosynch protocol in a 2 × 2 factorial design. The experiment was performed on 9 commercial dairy farms in Germany from April to October 2016. Lactating dairy cows (n = 1,724; 540 primiparous, 1,184 multiparous) were randomly assigned to 1 of 2 synchronization protocols (i.e., Ovsynch or Cosynch) on a weekly basis to facilitate first timed AI. Cows were inseminated either 12 to 16 h after the second GnRH injection (Ovsynch-56) or concurrent with the second GnRH injection (Cosynch-56). Two different preservation methods for semen were used, i.e., liquid stored semen (10 × 10 Mio. sperm/ straw) and frozen semen (20 × 10 Mio. sperm/ straw). The type of semen used for TAI changed every other week on each farm (i.e., wk 1: frozen semen; wk 2: liquid stored semen; wk 3: frozen semen). The analysis of P/AI at first TAI was performed by logistic regression using the GENLINMIXED procedure of SPSS. There was an overall effect of semen preservation method with liquid stored semen achieving greater P/AI than frozen semen (29.9% vs. 24.0%; P = 0.034). Primiparous cows had greater P/AI than multiparous cows (34.8% vs. 20.2%; P = 0.001). There was also an effect of TAI protocol on P/AI with cows inseminated in the Ovsynch-56 protocol achieving greater P/AI than cows inseminated in the Cosynch-56 protocol (30.4% vs. 23.6%; P = 0.021). Sire had no effect on P/AI (P = 0.899). The effect of semen preservation method differed by TAI protocol. Cows inseminated with liquid stored semen after Cosynch-56 achieved greater P/AI than cows inseminated with frozen semen (20.0% vs. 27.5%; P = 0.032). There
was no effect, however, of semen preservation method after Ovsynch-56 (liquid stored semen: 32.3%; frozen semen: 28.6%; P = 0.330). Liquid stored semen achieved greater P/AI in a TAI protocol with a long time interval between insemination and ovulation (Cosynch-56) compared with frozen semen indicating that liquid stored semen might have a longer viability in the reproductive tract compared with frozen semen.