Effect of Cryodiluent and Time of Glycerol Addition on Cryopreservation and In Vitro Fertilization of Domestic Cat Epididymal Spermatozoa

Abstract

Sperm cryopreservation and assisted reproduction are powerful tools for the conservation of endangered species. The domestic cat has been a useful model for studying wild felid reproductive biology due to the limited availability of endangered individuals for experimental research. Here, we investigate the effect of cryodiluents (TEST vs. Biladyl) and the timing of glycerol addition (before vs. after refrigeration, in one vs. three steps, respectively) on post-thaw sperm quality (motility, acrosome integrity) and their subsequent in vitro fertilization (IVF) ability with homologous oocytes. The results showed no statistically significant differences in sperm traits when samples were cryopreserved in TEST or Biladyl, or when glycerol was added in one or three steps. Motile sperm and intact acrosomes were significantly correlated before and after cryopreservation, indicating consistent relationships in fresh and thawed samples. The use of Biladyl significantly reduced IVF rates after cryopreservation compared to fresh sperm. Cryopreservation in TEST led to IVF rates that were not significantly different from those of fresh sperm. Using swim-up after thawing, or adding 1 mM pentoxifylline, did not enhance IVF results. Overall, a TEST cryodiluent with 4% glycerol added in one step is a reliable option for preserving epididymal cat spermatozoa.

Keywords: cryodiluent; cryopreservation; glycerol; in vitro fertilization; pentoxifylline; sperm; swim-up.

Figure 1

Comparison of cryodiluents (TEST vs. Biladyl) used for domestic cat epididymal sperm. Sperm were suspended in cryodiluent with 4% glycerol, refrigerated at −0.125 °C/min and stored in straws loaded at 5 °C. (A) Sperm motility index (SMI), (B) percentage of spermatozoa with intact acrosome. The X axis indicates the cryopreservation steps: (1) after recovery (fresh), (2) after refrigeration, (3) after thawing, and (4, 5, 6 and 7) at 90, 150, 210 and 270 min of incubation post-thaw in air at 37 °C. There are no significant differences (p > 0.05) between TEST (n = 33) and Biladyl (n = 32). (a–c) Different letters within diluent indicate significant differences (p < 0.05) over time. Analysis was carried out using split-plot ANOVA.

Figure 2

Comparison of the effect of glycerol addition system (one step vs. three steps) on domestic cat epididymal sperm cryopreservation using TEST cryodiluent. Glycerol final concentration was 4%. (A) Sperm motility index (SMI) and (B) percentage of spermatozoa with intact acrosomes. The X-axis represents the cryopreservation steps: (1) after recovery (fresh), (2) after refrigeration, (3) after thawing, and (4, 5, 6, and 7) at 90, 150, 210, and 270 min of post-thaw incubation in air at 37 °C. An asterisk (*) indicates significant differences (p < 0.05) between the two systems (one-step, n = 15; three-step, n = 10) at each cryopreservation step. (a–d) Different letters within diluent indicate significant differences (p < 0.05) over time. Analysis was carried out using split-plot ANOVA.

Figure 3

Correlations between sperm parameters (% motile sperm, % spermatozoa with intact acrosomes, and % normal sperm) before (fresh) vs. after (thawed) cryopreservation. (A) % motile sperm in fresh and frozen-thawed samples, (B) % spermatozoa with intact acrosomes in fresh and frozen-thawed samples, (C) % motile sperm in fresh samples and % spermatozoa with intact acrosomes after thawing, (D) % spermatozoa with intact acrosomes in fresh samples and % motile sperm after thawing, (E) % normal sperm in fresh samples and % spermatozoa with intact acrosomes after thawing, (F) % motile sperm and % spermatozoa with intact acrosomes after thawing. R is the Pearson correlation coefficient, and p is the associated critical level.

Figure 4

Effect of centrifugation and swim-up or addition of pentoxifylline after thawing on domestic cat epididymal sperm motility. (A) Effect of centrifugation. (B) Effect of addition of 1 mM pentoxifylline. Steps after thawing were: 1, immediately after thawing; 2, 1 h after thawing; 3, 2 h after thawing. At each step after thawing, an asterisk (*) indicates significant differences (p < 0.05) between control and treatment. Analysis was carried out using split-plot ANOVA. SMI: Sperm motility index.

Figure 5

IVF of domestic cat in vitro matured oocytes with cat epididymal spermatozoa cryopreserved in two diluents (TEST vs. Biladyl) using two glycerol addition methods (one-step [4%] vs. three-step [0 + 8%]) or freshly collected in Hepes-Tyrode’s medium (control group). (a,b) Different letters indicate significant differences (p < 0.05) between treatments (n = 3 each). Analysis was carried out using split-plot ANOVA.