Factors Affecting Cryopreservation of Domestic Cat (Felis catus) Epididymal Spermatozoa

Abstract

Sperm cryopreservation is a powerful tool for the conservation of endangered species, but its application requires adapting protocols to particular species, due to differences in sperm structure, function, and cryosensitivity. Research on the biology of endangered felids primarily relies on the domestic cat as an experimental model. Semen from live animals can be collected using several methods. However, in animals that die due to roadkill or in the field, spermatozoa must be retrieved from the epididymis. Differences may exist in the cryosensitivity of epididymal and ejaculated sperm due to the influence of secretions from accessory genital glands. We analyzed the effects of several factors on the motility and acrosomal integrity of cryopreserved cat epididymal spermatozoa, including cooling rate, storage system, time and temperature of straw loading, and the freezing method in nitrogen vapors. There were no significant differences in motility or acrosomal integrity at thawing between fast (-0.5 °C/min) or slow (-0.125 °C/min) cooling rates or between loading straws at room temperature versus 5 °C. Post-thaw motility was significantly higher when using straws compared to pellets and when freezing in nitrogen vapors at two levels rather than at a single level. Additionally, interactions between the loading temperature of straws and both motility and acrosomal integrity were not significant. These results are important for standardizing protocols to cryopreserve feline epididymal sperm, facilitating the rescue of genetic material from endangered species in the field.

Keywords: cryopreservation; domestic cat; epididymis; freezing; refrigeration; sperm.

Figure 1

Cooling curves used with domestic cat spermatozoa: fast, cooling samples over 30 min (−0.5 °C/min) and slow, cooling samples over 120 min (−0.125 °C/min).

Figure 2

Diagrams of containers used for freezing straws over nitrogen vapors: (A) Freezing at one level, 5 cm from liquid nitrogen surface; (B) freezing using two levels, 7.5 and 2.5 cm from liquid nitrogen surface.

Figure 3

Acrosome integrity and morphological abnormalities in domestic cat spermatozoa evaluated using Coomassie blue-stained slides. Acrosome: (1) intact normal, (2) intact abnormal, (3) damaged and (4) missing. Morphology: (A) normal, (B) macrocephalic, (C) bent midpiece, (D) aplasic midpiece, (E) bent principal piece, (F) coiled tail.

Figure 4

Effect of cooling rate (−0.5 and −0.125 °C/min) on motility and percentage of sperm with intact acrosomes of cat epididymal spermatozoa suspended in TEST 4% glycerol, stored in straws loaded at 5 °C, and frozen using two levels on nitrogen vapors. (A) Sperm Motility Index (SMI) and (B) percentage of sperm with intact acrosome. Timing of cryopreservation: (1) fresh, (2) after refrigeration, (3) after thawing, (4, 5, 6, 7) after incubation post-thaw for 90, 150, 210, and 270 min at 37 °C. For each cooling rate, different letters between bars indicate statistically significant differences (p < 0.05) in time. No significant differences were found between cooling rates (−0.5 vs. −0.125 °C/min) at any time during cryopreservation (n = 21).

Figure 5

Comparison of storage vessels (straws or pellets) used for cat epididymal sperm cryopreserved in TEST with 4% glycerol and refrigerated for 120 min. (A) Sperm Motility Index (SMI) and (B) percentage of sperm with intact acrosome. Timing of cryopreservation: (1) fresh, (2) after refrigeration, (3) after thawing, (4, 5, 6, 7) at 90, 150, 210, and 270 min incubation post-thaw at 37 °C. Different letters between the bars indicate statistically significant differences (p < 0.05) over time (1–7), for each storage system (straws or pellets). * indicates statistically significant differences (p < 0.05) between pellets and straws (n = 19).

Figure 6

Comparison of straws loaded at different temperatures. Cat epididymal spermatozoa were cryopreserved in TEST 4% glycerol, after refrigeration for 120 min, with straws loaded at room temperature or at 5 °C. (A) Sperm Motility Index (SMI) and (B) percentage of spermatozoa with intact acrosome. Timing of cryopreservation: (1) fresh, (2) after refrigeration, (3) after thawing, (4, 5, 6, 7) at 90, 150, 210, and 270 min post-thaw at 37 °C. For each straw loading system (room temperature or 5 °C), different letters between bars indicate statistically significant differences (p < 0.05) over time. * indicates statistically significant differences (p < 0.05) between the two straw loading systems. Straws loaded at room temperature (n = 10), straws loaded at 5 °C (N = 10).

Figure 7

Comparison of one- or two-level freezing in nitrogen vapors of domestic cat sperm cryopreserved in TEST 4% glycerol, refrigerated for 120 min, and stored in straws loaded at 5 °C. (A) Sperm Motility Index (SMI) and (B) percentage of sperm with intact acrosome. Timing of cryopreservation: (1) fresh, (2) after refrigeration, (3) after thawing, (4, 5, 6, 7) at 90, 150, 210, and 270 min incubation post-thaw at 37 °C. For each freezing method (one level or two levels), different letters between bars indicate statistically significant differences (p < 0.05) in time. * indicates statistically significant differences (p < 0.05) between the two freezing methods (one or two levels) (n = 15).