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The study aimed to evaluate the effect of α-chymotrypsin on placental separation as a treatment protocol for retained placenta (RP) in dairy cows and its effect on reproductive performance after placental shedding. The study was conducted on 64 crossbred cows that suffered from retained placenta. Cows were divided into four equal groups: group I (n = 16) treated with prostaglandin F2α (PGF2α); group II (n = 16) treated with PGF2α in combination with α-chemotrypsin; group III (n = 16) treated with α-chemotrypsin only and group IV (n = 16) treated by manual removal of the RP. Cows were under observation after treatment till placental shedding. Placental samples were taken from the non-responsive cows after the course of treatment and examined to observe the histopathological changes in each group. Results revealed that the time of placental dropping showed a significant decrease in group II compared to other groups. Histopathological examination of group II shows that collagen was found as fewer fibres in scattered areas and necrosis appeared as numerous areas widespread in the foetal villi. A few inflammatory cells were infiltrated in the placental tissue and the vascular changes appear as mild vasculitis and mild oedema. Cows in group II have rapid uterine involution, decreased risk of post-partum metritis and improved reproductive performance. It is concluded that PGF2α in combination with α- chemotrypsin is the recommended treatment for RP in dairy cows. This recommendation is warranted, as this treatment was successful in achieving rapid placental shedding, rapid uterine involution, a decreased risk of post-partum metritis and improved reproductive performance., (© 2023 Wiley-VCH GmbH. Published by John Wiley & Sons Ltd.)