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E. Blesbois and D. Hermier

Summary. Qualitative and quantitative characterization of lipoproteins found in seminal plasma from domestic cocks was performed after isolation by density gradient ultracentrifugation. Trigyceride-rich lipoproteins (very low, intermediate- and low density lipoproteins) were not detectable in seminal plasma. High-density lipoproteins (HDL), identified on the basis of size, chemical composition and protein moiety, were present at a concentration of 66 μg/ml. A fraction possibly corresponding to VHDL (very high density lipoproteins, 77% protein, 23% lipid) was also detected but appeared contaminated by a protein-rich opalescent material. Since HDL contains mostly phospholipid and cholesterol, the physiological role of these lipoproteins on the storage of fowl spermatozoa was studied. Replacing seminal plasma with a solution containing chicken HDL at physiological concentration (66 μg/ml) had no effect on fertilizing ability of spermatozoa stored at 4°C for 24 h. However, higher concentrations of HDL (560 μg/ml) had deleterious effects on spermatozoa stored in vitro.

Keywords: lipoproteins; chicken; seminal plasma; sperm storage; in vitro

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E. Blesbois and M. de Reviers

Summary. This paper describes the effects of whole seminal plasma and of dialysed seminal plasma on the fertilizing ability of fowl spermatozoa stored for 24 h at 4°C. The fertilizing ability of fowl semen diluted 1:1 with Beltsville Poultry Semen Extender and stored for 24 h at 4°C was enhanced after replacement of the homologous seminal plasma by the diluent (89 versus 77% fertilization rate). Better results were obtained with seminal plasma dialysed against water before sperm storage to discard the < 1 kDa or the < 50 kDa fractions. It was concluded that low molecular weight seminal plasma fractions could damage the fertilizing ability of spermatozoa during storage at 4°C, whereas high molecular weight fractions appeared to enhance fertilizing ability.

Keywords: seminal plasma; fertilizing ability; fowl

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E Blesbois, I Grasseau and F Seigneurin

The ability to survive cryopreservation varies in spermatozoa from different bird species. Among the biological factors potentially responsible for such differences, species variations in membrane fluidity have a role in the restoration of the physiological state after freezing. Membrane fluidity may be assessed by measuring fluorescence polarization anisotropy with a fluorescent dye. Anistropy values are proportional to membrane rigidity and consequently inversely proportional to membrane fluidity. In the present study, polarization anisotropy of spermatozoa originating from species differing in the freezability of their semen (chicken, turkey and guinea fowl) was measured in addition to lipid composition (cholesterol/phospholipid ratio), sperm viability (membrane permeability to eosine) and morphological integrity before and after cryopreservation.

The percentages of viable and normal spermatozoa in fresh sperm were highest in the chicken (87%), lowest in guinea fowl (64%), and intermediate in turkeys (69%). Anisotropy values were highest in guinea fowl (0.205), lowest in chickens (0.155), and intermediate in turkeys (0.180). As a consequence, membrane fluidity was highest in chickens and lowest in guinea fowl. Cryopreservation significantly decreased sperm viability and morphological integrity and increased anisotropy in all species but did not change the inter species hierarchy. Initial cholesterol/phospholipid ratios were lower in chickens than in guinea fowl, and intermediate in turkeys (0.25, 0.26 and 0.29, respectively). Cryopreservation induced a severe decrease in cholesterol/phospholipid ratios in turkeys and guinea fowl.

Sperm membrane fluidity in chickens, turkeys and guinea fowl behaves as an indicator of sperm freezability in these species. Inter species differences for this parameter may be partly explained by differences in initial cholesterol/phospholipids content of spermatozoa. On the other hand, the rigidifying process induced by cryopreservation is not related to lipid damage by the same mechanisms.

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M Lemoine, I Grasseau, J P Brillard and E Blesbois

Chicken spermatozoa may remain in the female oviduct for a prolonged period before induction of the acrosome reaction on contact with the inner perivitelline layer (IPVL). By contrast, the acrosome reaction may be induced very rapidly in vitro in the presence of IPVL and Ca2 +. In the present study, we examined the extent to which the chicken acrosome reaction can be induced in media of various compositions in the presence or absence of IPVL and/or Ca2 + and other factors known to be efficient in mammals. We also compared the efficacy of perivitelline layer (PL) taken at various states of oocyte maturation in initiating the reaction. The acrosome reaction was induced in less than 5 min in the presence of Ca2 + and IPVL. Incubation of spermatozoa in different saline media (Beltsville poultry semen extender (BPSE); Dulbecco's modified eagle medium; NaCl-TES buffer) without IPVL showed a significant induction of acrosome reaction in BPSE supplemented with 5 mM Ca2 + and in the three media after supplementation with Ca2 + and Ca2 + ionophore A23187. By contrast, the acrosome reaction was never induced without Ca2 +. BSA, NaHCO3, and progesterone did not stimulate the acrosome reaction. Ca2 + plus PL taken at various physiological states (follicle IPVL, ovulated IPVL, oviposited IPVL, and/or outer perivitelline layer) strongly stimulated the acrosome reaction, the latest states being the most efficient. Although PL induced the acrosome reaction in the presence of extracellular Ca2 +, it was not possible to induce hyperactivation in chicken spermatozoa. Taken together, these results emphasize the central role of Ca2 + in the in vitro initiation of the acrosome reaction in chickens and show specific features of this induction in birds.