Background: Semen cryopreservation involves preserving the sperms so they can be used later. This technique can prove to be a pathway for restoring the number of endangered species as their sperm can be preserved and used for fertilization later on.
Objective: The propose of study is to find out the optimum thawing time, temperature and post thaw incubation period of cryopreserved buffalo sperm.
Methods: Three buffalo bulls of the 1-2 year age were selected at Semen Production Unit, Qadirabad, Pakistan. Semen was collected by artificial vagina at 42 °C and 18 qualified ejaculates (two ejaculates/bull/week) for 3 weeks (replicate) were cryopreserved following a standard protocol. Frozen semen was divided in different groups, with each group being thawed at different temperature and time. Group I was subjected to slow thawing at 4-5°C, 2nd  group  to moderate thawing at 35ºC-37 ºC , 3rd group to  rapid thawing  while the 4th one was subjected to air thawing. Moreover, optimum incubation period of cryopreserved sperm was evaluated by thawing and incubating 42 straws in a water bath at 37ºC in with 2 straws being assessed every hour up till 10 hours. 
Results: Thawed semen samples were assessed based on different semen quality parameters. Acrosome integrity, plasma membrane integrity and progressive motility of cryopreserved buffalo sperm were significantly higher (P<0.05) in Group II which was subjected to moderate thawing. However, sperm quality parameters remained same (P > 0.05) with different incubation time (from 1 hr to 11 hr at 37°C).  
Conclusion: In conclusion, moderate thawing is effective in protecting the sperm cell from freeze thaw damages during cryopreservation. Sperm functional quality parameters were very acceptable after even 5 h of incubation indicated the usefulness of these incubated semen straws for field insemination in areas where availability of liquid nitrogen containers is problematic. Digital thermos maintained at 37°C could be alternative for short distance travelling for taking semen doses at insemination site. Further research efforts are needed to verify the fertilizing ability of this straw incubated (37°C) semen.