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 fowl spermatozoa, samples suspended in three cryoprotectants (CPAs), frozen/thawed samples, and frozen/thawed samples maintained in vitro for up to 24 h. The CPAs used were glycerol (Glyc), dimethylacetamide (DMA), and dimethylformamide (DMF). Viability was assayed using two double stains, Eosin + Nigrosin or SYBR-14 + PI (propidium iodide). Semen samples examined with SYBR-14 + PI indicated significant differences in viability between fresh and ready-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 percentages 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 inseminated with semen frozen in DMA reached levels comparable to those obtained from hens inseminated with fresh undiluted semen (88 and 93%, respectively; P > 0.05). In contrast, fertility of eggs from hens inseminated with semen frozen in DMF or glycerol was significantly lower, although still very good, than that observed in eggs from hens inseminated with semen frozen/thawed in 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 viability 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 fertilizing potential of fresh and frozen fowl spermatozoa.