Modifications of bull spermatozoa induced by three extenders: Biociphos, low density lipoprotein and Triladyl, before, during and after freezing and thawing

Abstract

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...

Comparison of various semen extenders and addition of prostaglandin F2 on pregnancy rate in cows

“…The first to find the AndroMed ® extender preferable were Aires et al (2003). In accord with the present findings, others confirmed this assessment for various ruminant species (Herold et al, 2003;Amirat et al, 2005;Janett et al, 2005;Akhter et al, 2010;Mohan and Atreja, 2014;Chelucci et al, 2015), although for sheep semen there are also controversial assessments (Fukui et al, 2008;Emamverdi et al, 2013). Many insemination organizations are hesitant to switch from the customary egg yolk to a phytoextender, because they are concerned that the latter may not be compatible with semen from all bulls and that, with the recently recommended substantial extension of the pre-freezing equilibration time, egg yolk seems to be indispensable.…”

Effect of sire and extender on sperm motility and share of live or dead sperm in bulls’ fresh ejaculate and in AI doses after thawing

“…Nevertheless, the use of egg yolk as a cryoprotectant has recently been restricted in some countries for reasons of hygiene risk (Thun et al 2002). Moreover, some researchers (e.g., Amirat et al 2005) have demonstrated that extenders based on egg yolk can have negative effects on sperm respiration and motility due to other specific substances they contain. Pace & Graham (1974) purified egg yolk by ultracentrifugation and found that a fraction of egg yolk known as »low-density lipoprotein« (LDL) has a cryoprotective effect on the integrity of the plasma membrane, as well as on the percentage of normal spermatozoa and sperm motility (Bencharif et al 2010) and preserves bull semen and maintains its fertility during freezing, storage, and thawing (Amirat et al 2004).…”

Efficacy of egg yolk based and egg yolk free soya bean milk based extenders for cryopreservation of Zebu cattle and buffalo semen

“…The efficacy of lecithin based extenders is still a matter of debate. Many previous studies have reported either comparable or higher sperm motility and plasma membrane and acrosomal integrity (Thun et al, 2002;Amirat et al, 2005;Rastegarnia et al, 2013;Singh et al, 2013;Chaudhari et al, 2015;Kumar et al, 2015;Ansari et al, 2016, Layek et al, 2016 with similar or even better fertility rates (Bousseau et al, 1998;Gil et al, 2000;Akhter et al, 2010;Akhter et al 2012;Beura et al, 2014) for bovine semen cryopreserved using lecithin or plant derived soya bean based commercial extenders, while some studies showed better efficiency of egg yolk based extenders over soya lecithin based extenders (Celeghini et al, 2008;Veerabramhaiah et al, 2011;Crespilho et al, 2012).…”