Summary
Five experiments were conducted to evaluate damage incurred in each processing step for cryopreservation of stallion spermatozoa. In Experiment 1, semen was centrifuged for 9 centrifugation times and the percentage of spermatozoa recovered after each treatment was calculated and spermatozoal motion characteristics analysed. Recovery of spermatozoa was ≥80% when spermatozoa were centrifuged for ≥10 min. Experiment 2 evaluated spermatozoa cryopreserved at 5 different concentrations in each of 2 extenders (skim milk‐egg yolk‐glycerol, SM‐EYG; and lactose‐EDTA, LAC). In SM‐EYG, TMOT and PMOT were higher at spermatozoal concentrations of 20, 200 and 400 times 106/ml (51%/41%, 52%/44%, 50%/43%, respectively) than for samples frozen at ≥800 times 106 spermatozoa/ml (41%/35%, 32%/27%; P<0.05). Spermatozoa frozen in LAC at a concentration of 20 times 106/ml resulted in the highest TMOT and PMOT (43% and 30%, respectively, P<0.05). The effect of freezing rate on motion characteristics of spermatozoa was evaluated in Experiment 3. The VCL of spermatozoa frozen in SM‐EYG was the only parameter affected by freezing rate (P<0.05). Experiment 4 evaluated motion characteristics after cryopreservation of spermatozoa in different sized straws (0.5 or 2.5 ml) in each of 2 extenders (SM‐EYG and LAC). In SM‐EYG, PMOT (38%) and VCL (109 μm/s) were highest when spermatozoa were frozen in 0.5 ml straws (P<0.05). In Experiment 5, spermatozoa thawed immediately after cryopreservation or thawed after storage in liquid nitrogen for 24–48 h were evaluated. There was no effect of length of storage in liquid nitrogen on spermatozoal motion characteristics (P<0.05). Experiment 6 evaluated the effects of cooling time to 5°C (0, 2.5 and 5 h) on motion characteristics of spermatozoa cryopreserved in 2 extenders (SM‐EYG and LAC). TMOT and PMOT were effected by cooling time, and there was a cooling‐time‐by‐extender interaction (P<0.05). In SM‐EYG, TMOT and PMOT were higher if spermatozoa were cooled to 5°C prior to initiation of freezing than if freezing was initiated at 20°C (P<0.05).
A suggested protocol for cryopreservation of stallion spermatozoa would include: 1) centrifugation at 400 g for 14 to 16 min; 2) extension at 23°C with SM‐EYG to 400 times 106 spermatozoa/ml; 3) cool to 5°C for 2.5 h; 4) package in 0.5 ml straws at 5°C; 5) freeze in liquid nitrogen vapour at −160°C; and 6) thaw for 30 s in 37°C water.
The use of fluorescein-conjugated Pisum sativum agglutinin (FITC-PSA) was evaluated for its ability to distinguish acrosome-intact from acrosome-damaged stallion spermatozoa. Incubation of fresh (acrosome-intact) and frozen-thawed (acrosome-damaged) spermatozoa with FITC-PSA resulted in acrosome-intact spermatozoa that exhibited no fluorescence, while acrosome-damaged spermatozoa exhibited fluorescent staining over the rostral portion of the head and equatorial segment. Experiments using mixtures of various ratios of acrosome-intact and acrosome-damaged spermatozoa determined the precision (intrasample coefficient of variation), and linearity (increased percentage of spermatozoa with PSA binding, with increased percentage of frozen-thawed spermatozoa in a sample) of FITC-PSA binding. The binding of FITC-PSA increased in samples as the portion of frozen-thawed (acrosome-damaged) to fresh (acrosome-intact) spermatozoa increased. A positive correlation existed (r = 0.98, P less than 0.05) between the percentage of FITC-PSA bound sperm and the proportion of damaged spermatozoa added to a sample. Location of PSA lectin binding on acrosome-damaged spermatozoa, determined by electron microscopy using gold-conjugated PSA, was to components of the outer acrosomal membrane and acrosomal matrix. These results demonstrate that FITC-PSA binding may be useful in determining acrosomal integrity of fresh and frozen-thawed stallion spermatozoa.
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