Bull semen in vitro fertility after cryopreservation using egg yolk LDL: a comparison with Optidyl®, a commercial egg yolk extender
Introduction
The goal of any sperm freezing is the production of a bank of sperm cells to be used for artificial insemination (AI), an important tool for distribution of the genetic potential of males. However, various biochemical and anatomical compartments in the sperm cells (acrosome, nucleus, mithochondria, axoneme, plasma membrane) may be altered during freezing and thawing. Consequently, the first aim of sperm-freezing protocols, including the use of extenders, is to prevent lethal intracellular ice crystal formation and to reduce membrane damage during and after cryopreservation.
The use of hen egg yolk in semen dilution was first reported for its beneficial effect as diluent for low-temperature storage of bull spermatozoa. Egg yolk helps the sperm cells in resisting against cold shock, in association with other components [1], [2], [3]. Egg yolk extender provides an excellent protection for bull semen against cold shock and is widely used commercially as Triladyl® (mini tüb) or Optidyl® (Biovet, France).
Egg yolk was usually used at the concentration of 20% (w/v). Laboratory studies revealed that this concentration make results difficult to standardize and interfered with biochemical assays and metabolic investigations, which could be overcome by removing some components in the egg yolk by centrifugation [4]. Furthermore, the presence of substances in yolk that inhibit respiration of spermatozoa or reduce their motility, increases demands to replace whole egg yolk by the cryoprotective fraction [5], [6], [7].
There have been many attempts to find out which component in egg yolk provides cell protection with the aim to prepare chemically defined extender. Pace and Graham have purified egg yolk using ultracentrifugation and observed that the low-density fraction (LDL) had a cryoprotective action [5]. Many investigations [8], [9], [10], [11] confirmed that LDL have a cryoprotective action in the egg yolk. Moussa et al. [11] obtained better results in terms of motility and movement characteristics when replacing whole egg yolk by 8% (w/v) LDL.
Most studies have reported results of LDL effect on bull semen motility but they never evaluated the fertility of semen after freezing in LDL. The ability of spermatozoa to fertilize after cryopreservation is an important factor of high-pregnancy rates in cattle after insemination [12]. Furthermore, no correlation has been established between motility and fertility of mammalian semen [13], [14], [15], [16]. Consequently, the measure of spermatozoa motility is an insufficient predictor of in vitro and in vivo fertility in many species [17], [18], [19]. Parameters like in vitro fertilization, hypoosmotic swelling (HOS) test, and the integrity of the acrosome could be associated to the motility results.
The aim of the present study was to evaluate and to compare bull semen fertility after the freeze–thaw process with whole egg yolk Optidyl® (Biovet, France, usually used by many French artificial insemination centers) or with LDL. Fertility parameters were evaluated as the cleavage rate and embryo development. Also, integrity of the acrosome and spermazoa membrane integrity were checked. Acrosome function is essential for the fertilizing ability of spermatozoa because acrosomal enzymes allow them to reach the oocyte plasma membrane [20]. Damage of the acrosomes has been reported to be associated with a lower fertilizing capacity. In addition, semen motility was evaluated after cryopreservation in Optidyl® and compared to semen cryopreserved in LDL.
Section snippets
LDL extraction
Fresh hen eggs were manually broken. Yolks were separated from the albumen and were carefully rolled on a filter paper to remove chalazes and traces of albumen adhering to the vitellin membrane. The latter was then disrupted with a scalpel blade and yolk was collected in a beaker, which is placed, on ice. LDL were extracted from yolk according to Moussa et al. [11]. Egg yolk was diluted with an isotonic saline solution (0.17 M NaCl) (w/w) and stirred for 1 h before centrifugation at 10,000×g for
Semen motility
The motility and VCL, ALH and VAP (movement characteristics) were significantly higher (P<0.05) in semen frozen in extender containing LDL as compared to semen frozen in the Optidyl® (Table 1). No significant difference was observed for VSL and LIN between the two extenders. These results indicated that in our experimental conditions, LDL extender provided a better protection than Optidyl® during the freeze–thaw process.
Acrosome integrity
After exposure of permeabilized spermatozoa to FITC-labeled PSA, two types
Semen motility
The percentages of post-thaw motilities were twofold higher in LDL extender than in Optidyl®. Extenders differ in composition the following way: Optidyl® contains 20% (w/v) egg yolk, and egg yolk contains 50% dry matter, 66% of which are LDL. This means that Optidyl® naturally contains 6–7% (w/v) LDL. This concentration is equivalent to the one used in our LDL extender. These results were observed for any of the eleven bulls tested and they confort those obtained by Moussa et al. [11] who
Conclusion
After cryopreservation in LDL extender, a higher percentage of motile spermatozoa (>50%) and a higher cleavage rate were obtained than when using Optidyl®. The blastocyst rate was similar in both semen extenders. The acrosome integrity and that of plasma membranes were also maintained. Freezing bull semen in LDL extender offers a high number of functional spermatozoa available for artificial insemination and allows to reduce the number of spermatozoa in the straws. Moreover, this LDL extender
Acknowledgements
The authors express thanks for the technical skills of Gérard Chatagnon, Nina Couvrand, Myriam Larrat, Frédérique Nguyen and Djemil Bencharif.
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