Glycerol addition and conservation of fresh and cryopreserved ram spermatozoa

Abstract

. 2002. Glycerol addition and conservation of fresh and cryopreserved ram spermatozoa. Can. J. Anim. Sci. 82: 347-356. Fresh extended ram semen has a short fertile lifespan whereas acceptable fertility with cryopreserved semen is achieved only by laparoscopy, which limits widespread artificial insemination in sheep. Although glycerol is considered essential for freezing spermatozoa, it is often included in extenders for short-term storage at above-freezing temperatures. To test the hypothesis that glycerol reduces the function of fresh sperm, ram semen was divided into two aliquots and diluted with commercial extenders that were identical, except that one contained 7% glycerol (n = 6). In a second experiment, ram semen was prepared for cryopreservation by a one-step dilution with a 7% glycerol extender or gradually, with a two-step protocol, to test the hypothesis that the method and time of glycerol addition affects sperm quality after freezing and thawing (n = 7). For both experiments, semen was diluted in a synthetic oviductal fluid (SOF-m) and sperm quality was assessed by computerassisted motility, viability and chlortetracycline fluorescence (CTC) patterns (an indicator of capacitation status). The presence of glycerol did not affect the quality of fresh sperm (P > 0.27). For cryopreserved sperm, the method of glycerol addition also did not affect thawed sperm. However, a decrease in sperm motility and viability, and different distribution of CTC patterns occurred due to the duration of time in extender and in SOF-m (P ≤ 0.0002). Cryo-capacitation was also observed. In conclusion, the presence of glycerol in the extender did not reduce ram sperm quality during conservation of the semen at 5°C or when it was used to completely and rapidly dilute the semen before cooling for cryopreservation. La semence de bélier fraîche (conservée à 5°C) a une durée de vie limitée tandis que la semence cryoconservée ne donne des taux de fertilité acceptables que lorsque la laparoscopie est utilisée, ce qui limite l'expansion de l'insémination artificielle chez l'ovin. Même si le glycérol est essentiel à la congélation des spermatozoïdes, il est souvent inclus dans les diluants destinés à la conservation de courte durée à des températures au-dessus du point de congélation. Afin de tester l'hypothèse selon laquelle le glycérol réduit la fonction des spermatozoïdes frais, la semence de bélier a été divisée en deux aliquotes et diluée avec deux diluants commerciaux identiques, à l'exception du fait qu'un contenait 7% de glycérol (n = 6). Dans une seconde expérience, la semence de bélier était diluée en une seule étape avec un diluant contenant 7% de glycérol ou graduellement, en deux étapes afin de tester l'hypothèse selon laquelle la méthode et le temps d'addition du glycérol affectent la qualité spermatique lors de la congélation-décongélation (n = 7). Dans les deux expériences, la semence était diluée dans un fluide synthétique mimant l'oviducte de la brebis (SOF-m) et la qualité de la semence était évaluée selon la motilité...

Semen cryopreservation: Successes and persistent problems in farm species

“…Regardless, these data provided evidence that partial or full cryopreservation induces precocious capacitation of bull sperm. Similar findings have also been reported for different species, including mice (Fuller and Whittingham 1996), sheep (Pérez et al 1996;Morrier et al 2002), and pigs (Gillan et al 1997). Capacitation, though a necessary step toward fertilisation, is known to destabilise the sperm membranes (Harrison 1996).…”

Current status and advances in ram semen cryopreservation

“…Pelufo et al (2015) demonstrated that the best procedure for ram semen cryopreservation in hypertonic disaccharide-containing diluents is the addition of glycerol after the cooling process, at 5 o C, than before it at 30 o C. Differences between 30 o C and 5 o C for spermatozoa quality were noticed just for HOST (hypo-osmotic swelling test), as plasma membrane showed higher sensitivity to glycerol addition at 30 o C. Moreover, Anel et al (2003) suggested that freezing of ram semen in a test-fructose-egg yolk medium containing 4% glycerol in a two-step dilution (to 2% at 35 o C and to 4% at 5 o C) along with a programmable biofreezer (-20 o C/min), results in good field fertility rate. Furthermore, according to Gil et al (2003) the addition of glycerol in a traditional milk extender supplemented with 5% egg yolk at 15 o C does not present any improvement in thawed semen quality compared to 5 o C. In contrast with the two steps dilution protocol studies, Morrier et al (2002) reported that the addition of glycerol either immediately after collection (one-step) or at 5° C (two-steps) does not affect motility and viability of frozen-thawed ram sperm.…”

Conservation of fresh ram spermatozoa at 5°C in the presence of seminal plasma

“…At 8 and 24 h post-collection, one sealed straw of semen was cut open (with clean scissors) and diluted (about 32-fold; to 50 × 10 6 sperm mL -1 ) in synthetic oviductal fluid (SOF-m; Morrier et al 2002). The SOF-m was composed of 108 mM NaCl, 7.2 mM KCl, 1.2 mM KH 2 PO 4 , 25 mM NaHCO 3 , 5 mM CaCl 2 , 0.5 mM MgCl 2 , 1.5 mM glucose, 3.3 mM sodium lactate, 0.33 mM sodium pyruvate, 1 mM glutamine, 20 µM penicillamine, 10 µM hypotaurine, and 50 mg L -1 gentamycin.…”

“…For the assessment of sperm motility, three 2-µL samples of sperm in SOF-m (~50 × 10 6 cells mL -1 ) were deposited into a 10-µm Makler counting chamber (Sefi-Medical Instruments, Haifa, Israel) placed on the motility analyser (Hamilton Thorn Research, Beverly, MA) as described by Morrier et al (2002). At least 200 cells from five random fields were recorded.…”

“…The CTC fluorescence assay used was conducted as described previously (Morrier et al 2002). In brief, the CTC solution was 5 mM cysteine and 750 µM CTC diluted in a buffer of 20 mM Tris base and 130 mM NaCl (pH 7.8).…”