The aim of the current study was to compare the cryoprotective effects of low molecular weight cryoprotectants 6% DMSO, 6% ethylene glycol, 6% 1,2 propanediol and 6% glycerol on the efficiency of ram semen cryopreservation and to test the in vitro fertilizing ability of frozen-thawed ram semen. Ejaculates with a thick consistency, rapid wave motion (3-5), and >75% initial motility were pooled. Sperm were diluted to a final concentration of 1/5 (semen/extender) in 6% glycerol, 6% DMSO, 6% ethylene glycol and 6% 1,2 propanediol using a two-step dilution method. Two hours equilibrated semen was frozen in 0.25-ml straws. As expected, the results of the current study showed that motility, HOST values and the rates of defective acrosomes in sperm were negatively affected by the cryopreservation procedure (P<0.01). In conclusion, 6% DMSO had a deleterious effect on the post thaw ram sperm parameters and embryonic development. Ethylene glycol and 1,2 propanediol successfully protected acrosomal and DNA integrities. Glycerol provided a better cryoprotection to sperm motility and plasma membrane integrity. The results of in vitro fertilization, as assessed by the rate of blastocyst formation, in glycerol group were similar to 1,2 propanediol group. (C) 2016 Elsevier B.V. All rights reserved.