Optimization of canine sperm cryopreservation by focusing on glycerol concentration and freezing rate

Abstract

The purpose of this study was to improve the quality of frozen-thawed canine spermatozoa through the optimization of glycerol concentration (GC) and freezing rate in the semen freezing protocol. Ejaculates from nine dogs were diluted with an extender containing 0%, 1.5%, 3%, 6%, or 9% glycerol. The suspensions were loaded into 0.25 ml straws, frozen in nitrogen vapor in a closed box, and immersed in liquid nitrogen (LN₂). The freezing rate was controlled by setting the distance from the LN₂ surface to the straws as 1, 4, 7, or 10 cm. Firstly, freezing curves for each GC and freezing rate were analyzed. The analysis showed that the temperature of ice nucleation, freezing point, and immersion were changed with a certain trend depending on the GCs and freezing rates. Secondly, the sperm motility index (MI), viability and mitochondrial (MT) activity were evaluated. At 0 h after thawing, the MI was higher in the 3% and 6% GCs than the 0% GCs (P < 0.05). At 24 h, the 3% GC with 1 cm LN₂ distance (1 cm-3%) and the 7 cm-6% showed higher viability than the other conditions (P < 0.05), and the highest MT activity was obtained in the 1 cm-3%, which was higher than the other conditions (P < 0.05). The present findings indicate that the rapid freezing rate at 1 cm (average - 31 °C/min) with 3% GC provided the optimal condition in this study; use of this condition should reduce the detrimental damage to dog spermatozoa caused by ice crystal formation during freezing.

Keywords: Canine sperm; Cryopreservation; Freezing rate; Glycerol.

Fig. 1

Examples of flow cytometric analyses of dog sperm viability (V) and mitochondrial activity (M). A. Fluorescence images of spermatozoa stained with SYBR14/PI (Viability) and JC-1 (Mitochondrial activity). B. Representative examples of multicolor cytograms in 0 h (a) and 24 h (b) after thawing. Reference data are derived from the same sample frozen with LN₂ distance 1 cm and 3% glycerol concentration. (V-a, b) Cytograms of a SYBR14/PI stain. Three populations are identified: dead sperm (red stained), viable sperm (green stained), and membrane-damaged, ‘moribund’, sperm (red/green fluorescence). (M-a, b) Cytograms for the analysis of mitochondrial activity using JC-1. Two populations are identified. Increasing orange fluorescence indicates higher Δψm (mitochondrial membrane potential); green shows the lower Δψm. Unstained debris (low fluorescence) was distributed in bottom left gates and discarded

Fig. 2

Typical example of freezing curves varying with the distance of LN₂ surface. Diluted semen samples and thermocouple sensors were loaded into straws, which were frozen above a LN₂ surface for 15 min before being directly plunged into LN₂. Temperature changes inside the straws were monitored every 2 s from the start of freezing (cold equilibrium temperature, 4–5 °C) to immersion. The freezing curves of 3% glycerol samples with different LN₂ distances are shown as a representative example

Fig. 3

Effects of freezing conditions on the motility index of frozen-thawed dog spermatozoa. Sperm were evaluated at 0, 12, and 24 h after thawing. Three to six replicate experiments were performed. (A) Box-and-whisker plots of motility index scores. Mean ± SEM. Different letters (a-e) indicate significant differences (P < 0.05). (B) Heatmaps of motility index scores. The color scale from blue to red indicates the motility index scores from the lowest to the highest at each time

Fig. 4

Effects of freezing condition on viability (a) and mitochondrial activity (b) of frozen-thawed dog spermatozoa. Sperm were evaluated immediately (0 h) and 24 h after thawing. Mean ± SEM. Four to eight replicate experiments were performed. Different letters (a–d) indicate significant differences (P < 0.05). MT activity: mitochondrial activity