The use of artificial insemination (AI) in buffalo (Bubalus bubalis) is limited by poor ovarian activity during the hot season, seasonal qualitative patterns in semen, low resistance of sperm cells in the female tract, difficulties in estrus detection, and variable estrus duration. Although AI procedures are commonly used in bovine, use of AI has been limited in buffalo. In the zootechnical field, different studies have been conducted to develop techniques for improvement of fertilizing ability of buffalo spermatozoa after AI. In this study, for the first time, the use of alginate encapsulation and cryopreservation of buffalo spermatozoa is described, and the same procedure was performed with Holstein Friesian (Bos taurus) semen. Results obtained from in vitro analyses indicate that the encapsulation process does not have detrimental effects (compared to controls) on quality parameters (membrane integrity, progressive motility, path average velocity) in either species. Similarly, there were no detrimental effects after cryopreservation in either species. The fertilizing potential of encapsulated and cryopreserved semen was evaluated after AI in 25 buffalo and 113 bovine females. Pregnancy rates were not affected in either species. The results of this study show proof of concept for the use of frozen semen controlled-release devices in buffalo.