In vitro comparison of fowl sperm viability in ejaculates frozen by three different techniques and relationship with subsequent fertility in vivo

A series of experiments was conducted to compare the viability of fresh fow l spermatozoa, samples suspended in three cryoprotectants (CPAs), frozen/th awed samples., and frozen/thawed samples maintained in vitro for up to 24 h . The CPAs used were glycerol (Glyc), dimethylacetamide (DMA), and dimethyl formamide (DMF). Viability was assayed using two double stains, Eosin + Nig rosin or SYBR-14 + PI (propidium iodide). Semen samples examined with SYBR- 14 + PI indicated significant differences in viability between fresh and re ady-to-freeze preparations (fresh, 83%; Glyc, 73%; DMA, 74%; DMF, 72%; P < 0.05). In contrast, Eosin + Nigrosin did not detect any difference at this stage (fresh, 88%; Glyc, 86%; DMA, 87%; DMF, 88%; P > 0.05). The percentage s of viable spermatozoa in frozen/thawed ejaculates stored in vitro for 0, 4, and 24 h were generally higher in samples treated with glycerol than in those treated with DMA or DMF, irrespective of the technique used to assess sperm viability (P < 0.05). Fertility in eggs obtained from hens inseminat ed with semen frozen in DMA reached levels comparable to those obtained fro m hens inseminated with fresh undiluted semen (88 and 93%, respectively; P > 0.05). In contrast, fertility of eggs from hens inseminated with semen fr ozen in DMF or glycerol was significantly lower, although still very good, than that observed in eggs from hens inseminated with semen frozen/thawed i n DMA (79 and 76%, respectively; P < 0.05). Finally, the double stain SYBR- 14 + PI was proven more effective than Eosin + Nigrosin to assess sperm via bility in fresh, stored, and frozen fowl semen. However, additional tests ( e.g., morphology, acrosomal status, motility) remain necessary to develop a working model of in vitro sperm analysis capable of revealing the fertiliz ing potential of fresh and frozen fowl spermatozoa. (C) 1999 Academic Press .