The present study aimed to determine the effect of administrating prostaglandin F2α (PGF2α) and GnRH at the time of artificial insemination (AI) on the pregnancy per artificial insemination (P/AI) and the pregnancy survival rate of dairy cows. A number of 830 lactating Holstein cows were randomly divided into four groups. Cows in group 1 (n=200) treated with 150 µg d-cloprostenol. In group 2 (n=212), cows received 10 µg buserelin acetate, and group 3 (n=205) was treated with both 150 µg d-cloprostenol and 10 µg buserelin acetate. In addition, 213 cows were assigned as control group which received normal saline as placebo (group 4). To measure progesterone, milk samples were collected at the insemination day and five days later. Pregnancy diagnosis was performed 28 and 60 days after the insemination, and the size and number of corpus luteum (CL) and twin pregnancies were recorded. Hormone therapies had no effect on the P/AI, pregnancy survival rate, and the size and number of CL. The P/AI ratio in groups 1, 2, 3 and 4 were 38.50%, 42.92%, 41.46% and 40.84%, and the pregnancy survival rates in groups 1, 2, 3 and 4 were 84.42%, 86.81%, 88.23% and 83.91%, respectively. The probability of a twin pregnancy was significantly higher in group 1 (15.58%) than other groups. There was no significant difference between groups in terms of the offspring gender. In conclusion, the administration of d-cloprostenol or buserelin acetate at the time of AI had no effect on P/AI and pregnancy survival rate in dairy cattle under no heat stress condition, while the administration of d-cloprostenol increased the probability of twin pregnancies.