Vitrification Using Soy Lecithin and Sucrose: A New Way to Store the Sperm for the Preservation of Canine Reproductive Function

Abstract

Simple Summary: Soy lecithin and sucrose were used at different concentrations to develop and compare different vitrification methods for the cryopreservation of canine semen. All of the sperm quality characteristics were effectively preserved after devitrification when vitrification extenders containing soy lecithin at 1% and 0.25 M sucrose were used. The results suggest that vitrification is effective, fast, and simple for cryopreservation of canine semen. Furthermore, the use of soy lecithin in lieu of animal proteins (e.g., egg yolk) facilitates semen shipping to countries with strict import requirements.Abstract: A challenge in freezing semen for short and long-term availability is avoiding damage to intact spermatozoa caused by the freezing process. Vitrification protocols provide better results through less manipulation of semen and shorter freezing time compared to slow freezing techniques. Our research was aimed at improving vitrification methods for canine semen. Semen quality was determined in 20 ejaculates after collection. Each ejaculate was divided into eight aliquots, each with a different extender. The control extender contained TRIS, citric acid, fructose, and antibiotics. Soy lecithin and sucrose were added to the control extender at different concentrations to make up the test extenders and final concentration of 50 × 10 6 spermatozoa/mL. From each group, a 33 µL (1.65 × 10 6 spermatozoa) suspension of spermatozoa was dropped directly into liquid nitrogen and devitrified at least one week later and evaluated as before. Soy lecithin at 1% and 0.25 M sucrose added to the base vitrification media effectively preserved all sperm qualities. Our results demonstrate the effectiveness of our methods. Vitrification media containing sucrose and soy lecithin cause a minimal decline in quality of canine semen after devitrification. Furthermore, extenders used in our research did not contain egg yolk, which was replaced by soy lecithin, thus allowing for ease of shipping to other countries with strict requirements.

Effect of tris-citric acid-egg yolk-glycerol and vitrified medium with and without ascorbic acid on the quality of post- thaw canine semen

“…According to these two experiments, the quality of the sperm after thawing was comparable to that after freezing in a Tris egg yolk (TEY) extender. Axnér & Lagerson, (2016) and Pipan et al, (2020) concluded that for freezing dog semen, an extender with 1% soybean lecithin extender produced superior post-thaw sperm motility than 2% soybean lecithin.…”

“…The vitrification process was done by adding one portion of the vitrification medium (Tris 2.4 gm + Citric Acid 1.4 g + Fructose 1.0 g + Streptomycin 5 µg + Penicillin 10 kIU is diluted in 100ml distilled Water with Soy lecithin at 1%(Andressa Dalmazzo et al, 2018) and 0.25 M sucrose) to one portion of fresh semen at 37 ˚C immediately after initial analysis (Pipan et al, 2020). After addition of vitrification media, vitrified semen was packed into straw straws (0.5 ml) are maintained at 5 °C for 30 min (equilibration time), then plunged directly into liquid N2 (LN2) for at least one week.…”

Uso da galactose na vitrificação de sêmen ovino em palhetas

“…Para avaliação da integridade física da membrana espermática, foi utilizado o corante supravital eosina/nigrosina (Minitube GmbH, Tiefenbach, Alemanha) (10) . A integridade funcional da membrana espermática foi avaliada através do teste hiposmótico.…”

“…Estas condições provocam a mudança do estado líquido para o estado amorfo da matéria, no qual o fluido que permanece no espaço intracelular adquire uma característica vítrea, impedindo a formação de cristais de gelo, e a ausência de estrutura atômica definida permite adequada dispersão da substância vítrea entre as organelas (6) . Esta técnica encontra-se bem estabelecida em humanos (7) , e experimentalmente apresenta bons resultados em jumentos (8) , equinos (9) , cães (10) e gatos (6) .…”

Antioxidants in assisted reproductive technologies: An overview on dog, cat, and horse

“…This last condition perhaps would be due to that monosaccharides are more easily metabolized by spermatozoa. Recently Zakošek Pipan et al [ 63 ] reported the effects of different sucrose and soy lecithin concentrations utilized to confront different vitrification techniques for canine semen cryopreservation. The results obtained showed that the vitrification media with soy lecithin and sucrose cause minimal wane in the quality of the dog seed after devitrification, as they keep unchanged physiological parameters of the sperm, such as morphology, vitality, total and progressive motility, acrosome, the integrity of the sperm tail membrane, and DNA.…”