A comparison of liquid and lyophilized egg yolk plasma to low density lipoproteins for freezing of canine spermatozoa.

The current study aimed to explore the potential usefulness of liquid or lyophilized egg yolk plasma (EYP) as a substitute for low-density lipoproteins (LDL) for cryopreservation of canine spermatozoa. In the first experiment, a total of 20 ejaculates harvested from six Beagles were frozen in extenders containing 6% LDL (control) or liquid or lyophilized EYP at one of three concentrations (20%, 40% or 60%). Motility parameters were assessed 10 min after thawing using computer-assisted sperm analysis. For both liquid and lyophilized EYP, the 40% concentration yielded motility similar (p > 0.05) to that observed with the control extender. In the second experiment, 12 ejaculates collected from the same six dogs were frozen in 6% LDL (Control), 40% liquid EYP or 40% lyophilized EYP extenders. Spermatozoal membrane integrity (hypo-osmotic swelling test [HOSt] and SYBR14/propidium iodide [PI] staining), acrosome integrity (FITC-Pisum sativum agglutinin staining) and DNA integrity (acridine orange staining) characteristics were evaluated 10 min after thawing. Both liquid and lyophilized 40% EYP-based extenders successfully preserved all assessed integrity parameters as efficiently as the control. Results of this study suggest that lyophilized EYP is a viable alternative to LDL in freezing extenders for dog semen.

Liposomes as an alternative to egg yolk in stallion freezing extender.

Egg yolk is normally used as a protective agent to freeze semen of equine and other species. However, addition of egg yolk in extenders is not without disadvantages and the demand to find cryoprotective alternatives is strong. The objective of this study was to test the cryoprotective capacities of liposomes composed of egg yolk phospholipids. Two experiments were conducted: 1) the first to determine the optimal composition and concentration of liposomes to preserve post-thaw motility and membrane integrity of spermatozoa; 2) the second to assess in vivo the cryoprotective capacities of these liposomes. In Experiment 2, post-thaw motility and membrane integrity of spermatozoa were also analyzed. Experiment 1 demonstrated that liposomes composed of phospholipids E80 (commercial lecithins from egg yolk composed mainly of phosphatidylcholine and phosphatidylethanolamine) and of Hank's salts-glucose-lactose solution (E80-liposomes) were the most efficient in preserving post-thaw motility. The optimal concentration was 4 % (v/v). In Experiment 2, fertility rate after artificial insemination of semen frozen with E80-liposomes was 55 % (22/40) compared with 68 % (27/40) with the control extender containing egg yolk (EY) (p = 0.23). Post-thaw motility parameters were higher with EY than with E80-liposomes (p < 0.0001). For post-thaw membrane integrity no difference was observed between the two extenders (p = 0.08). Liposomes composed of egg yolk phospholipids appeared to be a promising alternative to replace egg yolk in semen freezing extenders in equine species.

The advantages of using a combination of LDL and glutamine in comparison with TRIS egg yolk and Equex® STAMP extenders in the cryopreservation of canine semen.

Twenty semen samples taken from 5 dogs were frozen in liquid nitrogen at -196 °C in four different extenders: one control extender based on 20% egg yolk, 6% LDL alone (low density lipoproteins: the active cryoprotective principle in chicken egg yolk), 6% LDL combined with 20 mmol glutamine, and Equex® (a reference extender that we wish to compare with the LDL-glutamine combination). After thawing, spermatozoal motility was evaluated using a HAMILTON THORNE CERROS 12 image analyzer; the percentage of motile spermatozoa was 27.7% in the egg yolk extender (p<0.05), 49.9% with 6% LDL alone (p>0.05), 54.7% in the 6% LDL+20 mmol glutamine extender, and 47.9% with Equex® (p>0.05). The motility parameters (VAP, VCL, VSL and ALH) were also superior in the 6% LDL+20 mmol glutamine extender in comparison with the other extenders. Finally, the spermatozoa were generally better protected during freezing with the 6% LDL+20 mmol glutamine association than with the egg yolk, 6% LDL, or Equex extenders in terms of the flagellar plasma membrane (HOS test), DNA (Acridine orange test), and acrosome integrity (Spermac® test: no significant difference). The Equex® extender obtained the best results for the acrosome, followed by 6% LDL+20 mmol glutamine (FITC-PSA test: p<0.05 between each extender).

Effect of low-density lipoproteins, spermatozoa concentration and glycerol on functional and motility parameters of bull spermatozoa during storage at 4 °C.

An extender has been developed with low-density lipoproteins (LDLs) that eliminates the microbial risks associated with the use of whole egg yolk. The objective of this study was to assess the effects of substituting egg yolk with LDLs for use as an extender in sperm preservation at 4 °C, as well as on spermatozoa motility, plasma membrane and acrosome integrity, at two different concentrations (80×10(6) and 240×10(6) sperm per ml) for 8 days and to evaluate glycerol toxicity in both extenders. A total of 12 ejaculates were collected from three bulls. Spermatozoa motility was examined using computer-assisted semen analysis. Plasma membrane integrity was determined using the hypo-osmotic swelling test and acrosome integrity with the fluorescein isothiocyanate-Pisum sativum agglutinin test. The semen was subsequently divided into four aliquots and diluted with Tris-egg yolk-glycerol (TEG), Tris-egg yolk without glycerol (TE), LDL with glycerol (LDL(+)) and LDL without glycerol (LDL(-)), at 80×10(6) and 240×10(6) sperm per ml. This study showed that the LDL(+) and LDL(-) extenders were more effective at preserving spermatozoa motility, plasma membrane integrity and acrosome integrity than TEG and TE (P<0.05) during 8 days of incubation. After 3 days of incubation, a toxicity of glycerol was observed in TEG, whereas no significant difference was observed between LDL(+) and LDL(-). We can therefore conclude that the LDL extender can be used to refrigerate semen at 4 °C instead of TEG and TE at 80×10(6) and 240×10(6) sperm per ml for elite bulls. This finding can be used to define a policy for the storage of high-quality bull semen.

Freezing canine sperm: comparison of semen extenders containing Equex and LDL (Low Density Lipoproteins).

Chicken egg yolk is held as an excellent cryoprotective agent for freezing canine semen. Recent advances have enabled the extraction of low density lipoproteins from egg yolk, which are responsible for the cryoprotective abilities of the latter. The objective of this article was to compare 3 semen extenders for freezing canine semen: 2 containing egg yolk (Tris egg yolk and Equex STAMP) and one containing 6% LDL. After freezing and thawing 20 ejaculates from 5 different dogs, the 6% LDL extender produced 50% mobile spermatozoa, compared with 48% with the Equex extender and 27.7% with the extender containing egg yolk alone (EY). In vitro functional tests demonstrated that the integrity of the plasma membrane (hypoosmotic test) was respected in 65-66% of spermatozoa as a function of the extender; DNA integrity was respected in more than 97% of the spermatozoa. The Equex extender provided superior acrosome integrity (FITC/PSA test): 68.4% compared with 55.1% with LDL and 53.3% with egg yolk. However, the 6% LDL extender resulted in fewer spermatozoal anomalies (Spermac test), with 54.6% normal spermatozoa compared to 53.6% for Equex and 53.3% with the egg yolk. All six of the bitches inseminated artificially via the intra-uterine route (Scandinavian technique) using semen frozen in the 6% LDL extender became pregnant. The LDL extender resulted in percentages of mobile spermatozoa and movement characteristics that were as good if not better than those obtained with the reference extenders following thawing. The 6% LDL extender appears to have the same cryoprotective qualities as the reference diluent, Equex STAMP.