The success of artificial insemination with frozen semen implies the reduction of the deleterious effects on the cells induced by this technique. These effects can occur as early as during the first dilution in an extender, as well as at any step, during or after the freezing process. In this work, we have compared the modifications induced by Triladyl, low density lipoproteins (LDL) and Biociphos extenders, after dilution and cooling to 4 8C for 1, 4 and 24 h. Alterations in the cell structures were visualized by electron microscopy (EM). More than 80% of spermatozoa were injured after incubation for 4 h in Triladyl, while 3% and 47% were counted in LDL and Biociphos respectively. This latter extender was deleterious to cell membrane integrity after incubation for 4 h or longer. The ultrastructure of frozen spermatozoa was studied by EM of cryofixed-cryosubstituted samples obtained from regular 0.5 ml French straws frozen using our usual protocol. The main differences between samples concerned the size and appearance of the frozen extender veins, while very few cell defects were found to be added by the freezing process at any depth in the straws. After thawing, semen motility was twofold higher (P < 0.05) in Biociphos (64%) and LDL (61%) than in Triladyl (32%) and the cells were less altered in LDL. We concluded that the LDL extender offers a better protection for storage of frozen spermatozoa, and can probably also be used for the preservation of fresh semen for short periods.
IntroductionWhile homogenized whole milk, fresh or reconstituted skimmed milk, or coconut milk have been proposed for the preservation of semen, egg yolk is still commonly used for cryopreservation of bovine spermatozoa in France. Among the extenders, Triladyl or Optidyl are quite popular, despite the fact that, because of the addition of fresh egg yolk, they have variable compositions. Their use has been encouraged, thanks to the excellent protection that they provide for bull spermatozoa (Polge & Rowson 1952).However, this protection is counterbalanced to some extent by known limitations. Egg yolk introduces possible sanitary risks (Ahmad & Foote 1986, Bousseau et al. 1998, Van Wagtendonk-de Leeuw et al. 2000, Vishwanath & Shannon 2000 and several egg yolk components interfere with laboratory biochemical assays and metabolic investigations (Wall & Foote 1999). Furthermore, some studies (Kampshmidt et al. 1953, Pace & Graham 1974, Watson & Martin 1975 have shown that these extenders can reduce the respiration and motility of spermatozoa.For these reasons, many professionals are waiting for substitutes with defined compositions, to at least limit the risks cited above and maintain a high protection of the cells. Among the diverse possibilities, Pace & Graham (1974) purified egg yolk extracts, some of which, such as the low density lipoprotein (LDL) fraction, seemed promising. Their results were confirmed by many investigators (Foulkes 1977, Quinn & Chow 1980, Demianowicz & Strezek 1996, Moussa et al. 2002. However, the nat...
