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.

Fatty acid composition, glutathione peroxidase and superoxide dismutase activity and total antioxidant activity of avian semen.

This work demonstrates that spermatozoa from five avian species (chicken, turkey, guinea fowl, duck and goose) are all characterised by high proportions of polyunsaturated fatty acids, from 46 (turkey) to 55% (duck) of total. For each of the species, the most abundant fatty acids were arachidonic (20:4n-6) and docosatetraenoic (22:4n-6) acids, representing between 22 (turkey) and 40% (chicken) of total. Significant activities of the major isozymes of superoxide dismutase and glutathione peroxidase, which protect against the peroxidation associated with high degree of fatty acid unsaturation, were found in spermatozoa from all species. The seminal plasma also had these activities and showed additional mechanisms for protecting spermatozoa from peroxidation. In general terms, these lipid and enzyme proteins were similar between the five avian species and different from those reported for mammalian sperm.

Comparison of assessment of fowl sperm viability by eosin-nigrosin and dual fluorescence (SYBR-14/PI).

The kinetics of fowl sperm viability/mortality following short-term and long-term in vitro storage were studied using 2 different staining methods: eosin/nigrosin (observed under light microscopy) and SYBR-14/PI (dual fluorescence). Based on data obtained at 0, 30 min and at 2, 4 and 24 h (T0, T30, T2, T4, and T24) after in vitro storage (4 degrees C, agitated) of fresh or frozen-thawed semen, the dual association SYBR-14/PI was more effective than eosin/nigrosin (P < 0.05) staining for the detection of sperm viability/mortality at early stages (30 min) in nonfrozen ejaculates stored above 0 degree C. In cryopreserved preparations, the 2 techniques were comparable for assessing viable spermatozoa immediately after thawing, but higher percentages (P < 0.05) of nonviable spermatozoa were detected by the SYBR-14/PI procedure for up to 4 h of in vitro storage post thawing (4 degrees C, agitated). Finally, comparable results were observed between the 2 techniques 24 h after beginning in vitro storage post thawing. It is concluded that the dual association SYBR-14/PI procedure is more effective (or, at least, more rapid) than eosin/nigrosin staining for the assessment of sperm viability/mortality in both fresh and cryopreserved samples of fowl semen. However, in the latter case, the thawing stage needs to be followed by a period of in vitro storage lasting at least 4 h to allow for easier discrimination between viable and nonviable populations of spermatozoa.