Effect of cooling and freezing, the two first steps of a freezing protocol, on the fertilizing ability of the rabbit sperm

Abstract

-The effect of different phases of a freezing protocol on the fertilising ability of rabbit spermatozoa was evaluated. An extender containing 1.75 M DMSO and 0.05 M sucrose (final concentration) was used to freeze rabbit sperm. In the first experiment, the results obtained with fresh and cooled (5 ºC for 45 min) sperm were compared; no differences were observed between fresh and cooled semen for any of the parameters studied: fertility rate (78% vs. 91% for fresh and cooled sperm, respectively), and normal embryos two days after insemination (6.8 vs. 8.5 normal embryos for fresh and cooled sperm). In the second experiment, the results obtained with fresh semen and sperm which had passed the first two steps of a freezing protocol (5 ºC for 45 min and -30°C for 30 min, and thawed at 50 °C for 15 s) were compared; the differences between them were obtained for fertility rate (94% vs. 61% for fresh and frozen sperm, respectively) and normal embryos two days after insemination (7.8 vs. 3.8 fresh and frozen sperm). These observations indicated that the differences in the results obtained with fresh and cryopreserved sperm were produced during the second step of the freezing protocol, and that apparently no toxic effect of DMSO was produced.embryo / fertility / freezing / rabbit / sperm Reprod. Nutr. Dev. 42 (2002) [189][190][191][192][193][194][195][196] 189

Reducing the time rabbit sperm are held at 5 °C negatively affects their fertilizing ability after cryopreservation

“…Indeed, rabbit sperm tolerate cooling rates of at least À7.3 C/min during the first minute of cooling and retain their fertilizing ability [4]. Our results show that rabbit sperm need to be first cooled to 5 C before freezing, as freezing the sperm directly from room temperature resulted in considerable lower (1.5-2-fold lower) in vitro assessed sperm quality measurements than samples that were first cooled to 5 C and then cryopreserved.…”

“…Rabbit sperm contain twice the amount of cholesterol in their membranes than do bull or ram sperm [2], which makes rabbit sperm membranes less susceptible to cold shock damage [3] and, therefore, allows them to be cooled more rapidly to 5 C without affecting sperm motility, membrane integrity, or fertilizing ability [4], than coldshock sensitive sperm from bulls or rams. Current protocol for cryopreserving rabbit sperm is packaging the sperm into straws and then placing the straws into a 5 C chamber for 45 minutes before freezing [5].…”

Effect of the primary cooling rate on the motility and fertility of frozen-thawed rabbit spermatozoa

“…In general, rapid cooling and freezing of semen (cold shock) influences acrosome morphology and the percentage of post-thaw live and motile sperm (Blackshow, 1954;Gilmore et al, 1998;Watson, 2000). To avoid this severe irreversible damage, freezing semen in domestic animals requires a 2-step protocol (primary cooling and freezing) in which samples are suspended in an appropriate extender and gradually cooled from room temperature to 5°C, followed by freezing (Mocé and Vicente, 2002;Barbas and Mascarenhas, 2009). The appropriate conditions for cooling and freezing must be investigated for each species.…”

Comparison of Glycerol, Lactamide, Acetamide and Dimethylsulfoxide as Cryoprotectants of Japanese White Rabbit Spermatozoa

“…In early research on freezing of rabbit spermatozoa, spermatozoa frozen with glycerol resulted in extremely low fertility [8][9][10][11]. After 1980, studies employing acetamide [12][13][14][15] or DMSO [12,[16][17][18][19][20][21][22] as a cryoprotectant have been encouraging. H o w e v e r , t h e r e i s a l i t t l e i n f o r m a t i o n o n cryoprotectants when freezing Japanese white rabbit spermatozoa.…”