Effect of different freezing velocities on the quality and fertilising ability of cryopreserved rabbit spermatozoa

Abstract

The freezing step of the cryopreservation protocol negatively influences the quality and fertilising ability of rabbit spermatozoa. This study determines the effect of different rates of freezing on the quality and fertilising ability of rabbit spermatozoa cryopreserved with dimethylsulfoxide (DMSO) (1.75M) and sucrose (0.05M). Ejaculates from meat rabbit line males (n=12) were pooled and each pool (n=7) was split into four aliquots. One group of straws (control, C) was frozen in static liquid nitrogen vapour (5cm above the liquid nitrogen, 10min) and the other groups were frozen at different freezing rates (°Cmin(-1)) from -6°C to -100°C using a programmable freezer: slow (-15°Cmin(-1), S), medium (-40°Cmin(-1), M) or fast (-60°Cmin(-1), F). After thawing (50°C, 12s), the quality was highest (P<0.05) in C and M samples and lowest in S and F samples. F samples presented the lowest litter sizes (P≤0.05) and fertility whilst M samples exhibited the highest values. In conclusion, the freezing rate affects both the quality and the fertilising ability of frozen-thawed rabbit spermatozoa, with both slow (-15°Cmin(-1)) and fast (-60°Cmin(-1)) freezing rates being detrimental for the quality and fertilising ability.

Freezability genetics in rabbit semen

“…Semen characteristics after the freezing-thawing procedures are summarized in Table1, where the dramatic reduction of sperm motility (Rmot=83%) and normal acrosome status (Rnar=74%) can be observed. For Mot-FT, Nar-FT and Live-FT, the means obtained are lower than the values reported for the same line in studies in which the ejaculates are preselected for cryopreservation [8,12]. Important differences of the present study were the assessment of individual, rather than pooled ejaculates, and the lack of ejaculates pre-selection before freezing.…”

“…The percentage of viable (plasma membrane intact) sperm (Live-FT, %) in each frozen-thawed sample was determined using flow cytometry, as described by Mocé et al [12]. Briefly, a sample from each thawed straw was diluted with Tris-BSA to 30 x 10 6 sperm/ mL.…”

Strategies for Highly Efficient Rabbit Sperm Cryopreservation

“…The ice crystal formation during the freezing process damage cells as described above and freezing protocol is also one of the concerns. In rabbit sperms, it was revealed that sperms frozen at slow (−15 °C/min) and fast (−60 °C/min) rates were lower in the quality and fertilizing ability than those frozen at medium (−40 °C/min) rate and in static liquid nitrogen vapor [ 52 ].…”

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

“…Sperm concentration and morphology were evaluated for fresh semen samples, whereas motility (percent total and progressively motile sperm, determined using Computer Assisted Sperm Analyzer system) and plasma membrane integrity (live sperm, determined using flow cytometry for sperm stained with SYBR-14 and propidium iodide) were determined for both fresh and cryopreserved sperm as described by Mocé et al [10].…”

“…Frozen-thawed sperm were inseminated into receptive females using the protocol described by Mocé et al [10]. At parturition, number of young born alive, the number of fetal deaths, and the total number of kits born (number of alive þ number of dead) for each female were determined.…”