Use of Glutamine and Low Density Lipoproteins Isolated from Egg Yolk to Improve Buck Semen Freezing MZ Ali Al Ahmad 1 , G Chatagnon 1 , L Amirat-Briand 1 , M Moussa 1 , D Tainturier 1 , M Anton 2 and F Fieni 1 1 UPSP DGER, Sanitary Risks and Biotechnology of Reproduction, National Veterinary School of Nantes; 2 Groupe Physio-chimie des Emulsions, Laboratoire d’Etude des Interactions des Mole ´cules Alimentaires, INRA, Nantes, France Contents To improve the results obtained with a reference cryopreser- vation extender (control extender: TriladylÒ + 20% (v/v) egg yolk + 6.4% (v/v) glycerol) for freezing caprine semen, glutamine was added to 18 split ejaculates at concentrations of 0, 20, 40, 80 and 120 mM (experiment 1). In experiment 2, glutamine was added to 18 split ejaculates at concentrations of 20, 25, 30, 35 and 40 mM. In the third experiment, the egg yolk was replaced with the low-density lipoprotein (LDL) fraction of egg yolk. The quality of frozen then thawed spermatozoa in each extender was compared using computer- assisted semen analysis. In experiment 1, glutamine at concentrations of 20 mM and 40 mM significantly improved sperm motility compared with the control extender. However, at 120 mM, a significant decrease in motility and velocity was observed. In experiment 2, motility, curvilinear velocity and amplitude of lateral head displacement (ALH) were improved in glutamine at 25 mM compared with the control. In experiment 3, 8% LDL and 25 mM glutamine significantly improved sperm motility, straight line velocity and ALH. In the fourth experiment, the quality of the previously defined freezing extender (TriladylÒ + 8% (v/v) LDL + 25 mM glutamine + 6.4% (v/v) glycerol) was tested by comparing acrosome, tail membrane, plasma membrane and DNA integrity in 18 split ejaculates of frozen then thawed spermatozoa with spermatozoa that had been frozen then thawed in the control extender, and with spermatozoa from fresh, unfrozen sperm. The percentage of spermatozoa with intact acrosomes and tail membranes was significantly higher with the newly defined extender than that observed with the control extender. There was no significant difference in the percentage of spermatozoa with intact DNA between the frozen and fresh semen. Introduction To limit the noxious effects of the freezing–thawing process on spermatozoa due to the formation of intracellular ice crystals and cellular dehydration, which increases the intracellular concentration of solutes (Amann and Pickett 1987), a cryoprotectant such as glycerol is essential (Garner 1991). However, Anchordo- guy et al. (1988) report that some organisms accumulate amino acids in response to cold temperatures. When combined with the traditional cryoprotective agents, these amino acids may play a role in preventing the structural cellular lesions that occur during the freezing and thawing process. Several studies have demonstrated the cryoprotective effect of amino acids, or their precursors, when freezing sperm from rams (Sanchez- Partida et al. 1992), stallions (Koskinen et al. 1989) or humans (Renard et al. 1996). Trimeche et al. (1996) demonstrated the beneficial effect of glutamine when freezing–thawing Poitou jackass sperm. When used in combination with glycerol, egg yolk plays a major role in protecting spermatozoa from the thermal shock caused by freezing (Phillips 1939; Bogart and Mayer 1950). The latter is widely used in combi- nation with ready-to-use commercial media such as Triladyl Ò (Minitu¨b, Tiefenbach, Germany) at a con- centration of 20% (w/v). However, egg yolk presents three disadvantages: First, it presents a health risk, as it is a biological medium and therefore always susceptible to contamin- ation and because it is a culture medium that promotes bacterial growth. Secondly, it presents a technological problem, as it is particularly difficult to exploit in automated industrial processes (breaking eggs, recovering and decomplement- ing the yolk for each semen sample). Lastly, egg yolk contains substances (granules) that prevent the metabolic exchanges of the spermatozoa or reduce their motility (Kampschmidt et al. 1953; Pace and Graham 1974; Watson and Martin 1975). It would therefore be advantageous to replace this complex solution with its component molecules that are respon- sible for its cryoprotective effect (Kampschmidt et al. 1953; Pace and Graham 1974). Many studies have attempted to determine which components of egg yolk are responsible for cellular protection with the objective of preparing a chemically defined freezing extender. Pace and Graham (1974) purified egg yolk using ultracentrifugation and observed that the low-density lipoprotein fraction (LDL) has a cryoprotective effect. This property of LDL was confirmed by several studies that have demonstrated that the best results when freezing bull semen were obtained by replacing the whole egg yolk with 8% (w/v) LDL (Foulkes 1977; Moussa et al. 2002; Amirat et al. 2004; Amirat et al., 2005). The goals of this study were first to assess the addition of glutamine and, secondly the use of LDL as a substitute for egg yolk in the extender used for freezing caprine sperm. This development should eventually make it possible to obtain a chemically defined extender of constant physical quality and with a controlled health risk. Materials and Methods Semen collection Semen collection was undertaken weekly twice by electro-ejaculation of nine bucks (three Saanens and six Alpines), aged between 4 and 7 years. For experi- ments 1 and 2, two ejaculates were obtained from each Reprod Dom Anim 43, 429–436 (2008); doi: 10.1111/j.1439-0531.2007.00930.x ISSN 0936-6768 Ó 2008 The Authors. Journal compilation Ó 2008 Blackwell Verlag