Successful Cryopreservation of Domestic Cat (Felis catus) Epididymal Sperm after Slow Equilibration to 15 or 10°C

Abstract

To support conservation strategies in wild species, simple but highly reproducible procedures of sperm cryopreservation are required for an application under field conditions. We used epididymal sperm of the domestic cat to optimize a sperm freezing procedure for felid species, particularly questioning the demand for sperm cooling to 4°C. We equilibrated sperm during slow cooling to only 15 or 10°C in a Tes-Tris-fructose extender with final concentrations of 4.7% (v/v) glycerol and 10% (v/v) of the water-soluble fraction of hen's egg yolk (low-density lipoproteins). Subsequently, sperm were frozen over liquid nitrogen. Total and progressive motility (mean ± SD) after thawing was 60.7 ± 8.6% and 53.9 ± 9.6% in samples cooled to 15°C or 61.6 ± 9.5% and 55.3 ± 9.9% in samples cooled to 10°C. Therefore, a one-step addition of glycerol to sperm at room temperature together with the freezing extender, the use of cryovials (loaded with diluted sperm aliquots of 300 μl), an equilibration period of 40 min comprising slow cooling to 15°C at a rate of approximately -0.14 K/min before rapid freezing over liquid nitrogen, yielded satisfying results. Cooling, freezing and thawing rates were exactly characterized as a prerequisite for further optimization and to provide a repeatable protocol to other practitioners.

Semen cryopreservation and radical reduction capacity of seminal fluid in captive African lion (Panthera leo)

“…However, neither the percentage of total nor progressive motile lion sperm after thawing is satisfactory when compared with the outcome of cryopreservation in the domestic cat [16,30] or even more in farm animal species. Swanson [29] proposed a minimum of 10 × 10 6 motile sperm for one artificial insemination procedure in felids.…”

“…A study from Crosier et al [31] on cheetah semen revealed that sperm exposure to glycerol for 1 hour at room temperature (∼22 °C) before a cooling phase of 3.5 hours to 5 °C had only a minimal effect on sperm motility after cryopreservation. In the light of this finding, we tested a procedure with a one-step addition of glycerol at ambient temperature, followed by a short equilibration on slow cooling for 40 minutes to only 15 °C before freezing, which had recently been proven to be successful with epididymal semen from the domestic cat [16]. After thawing, we determined 31.5 ± 14.1% (mean ± SD) motile lion sperm (median 31.0, range 2.2%-56.5%) for samples frozen in cryovials.…”

“…Cryopreservation of semen was performed according to the recently published protocol for domestic cat epididymal sperm [16]. A Tes-Tris-fructose-buffer (Test) was prepared according to Schmehl et al [22] with addition of 15% (v:v) of the water-soluble fraction of hen's egg yolk (containing low-density lipoproteins) according to Lengwinat and Blottner [23].…”

“…Vials and straws were thermally insulated in a water bath or polystyrene box, respectively. After 40 minutes, a temperature of ∼15 °C was reached and vials were frozen in the vapor of liquid nitrogen in a polystyrene box [16]. Straws were frozen rapidly either by lowering them to the bottom of a container (dry shipper) or in liquid nitrogen vapor.…”

“…Based on these results, we first aimed to test a field-friendly cryopreservation method for African lion (Panthera leo) semen collected by urethral catheterization (UC) or electroejaculation (EE). The method was successfully applied on epididymal sperm of domestic cats [16] and comprised a one-step addition of glycerol together with the freezing extender to sperm at ambient temperature. The diluted semen was also packaged at ambient temperature either in vials or straws to compare both options.…”