A suitable method for the cryopreservation of donkey semen would be very valuable for the ex situ management of genetic diversity in this species. This report uses a variety of observation and trials to evaluate the effect of cryoprotectants in per-cycle pregnancy rates (PC) in equids females (jennies (donkey) and mares (horse)). This was explored by (1) comparing the results of insemination of jennies and mares with cooled or frozen donkey semen, (2) examining the possible toxic effect of the cryoprotectant (CPA) glycerol in these two species and (3) studying alternative solutions. Donkey and horse semen was either used immediately, or cooled according to some steps of the pre-freezing procedure or frozen and thawed. The pre-freezing procedure included semen dilution, centrifugation, resuspension in milk or in INRA82+2% egg yolk+various % CPA (expressed as final concentrations in extended semen (v/v)) and then cooling to 4 degrees C. PC was similar in mares and jennies inseminated with donkey semen cooled to 4 degrees C in milk. However, the PC was significantly higher in mares than in jennies when donkey semen was frozen with 2.2% glycerol (36%, n=50 cycles vs. 11%, n=38 cycles; P<0.01). Increasing the concentrations of glycerol (0, 2.2, 3.5, 4.8%) before cooling stallion semen resulted in a progressive decrease in mare PC (87, 53, 53, 13% (n=15 cycles for each concentration); P<0.0001). The addition of 2.2% glycerol before cooling donkey semen decreased the PC measured in jennies to 0. The replacement of glycerol by 2% dimethylformamide increased the fertility obtained in jennies with cooled donkey semen (PC: 67%, n=12 cycles) but did not increase the fertility obtained with frozen-thawed donkey semen (PC: 11%, n=28 cycles with dimethylformamide vs. 0%, n=16 cycles with glycerol). In conclusion, this study clearly shows that the ability of jennies to conceive after AI with donkey frozen semen is lower than that of mares. Glycerol affects the fertility of donkey and stallion spermatozoa as early as during the pre-freezing procedure. In consequence, the glycerol level must be low in frozen equine semen to provide good fertility. The toxic dose of glycerol for donkey spermatozoa seems to be almost half that for stallion spermatozoa. Whether this greater sensitivity of donkey spermatozoa to glycerol is responsible for the low success of semen cryopreservation in jennies is not so obvious because replacement of glycerol by dimethylformamide was not much more effective in terms of fertility.

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Results on procedures for freezing stallion semen and the subsequent fertility during 20 years are presented. The present system applied in French National Stud includes: (1) a freezing protocol (dilution in milk, centrifugation and addition of freezing extender (INRA82+egg yolk (2%, v/v)+glycerol (2.5%, v/v) at 22 degrees C, a moderate cooling rate to 4 degrees C and freezing at -60 degrees C/min in 0.5-ml straws); (2) selection of ejaculates showing post-thaw rapid motility >35%; and (3) an insemination protocol (mares examined once daily, two AI of 400 x 10(6) spermatozoa 24 h apart before ovulation, sufficient number of straws to have the possibility to perform six AI of 400 x 10(6) total spermatozoa, i.e. 2.4 x 10(9) total spermatozoa available per mare per season). This system was applied to >110 stallions per year, the average post-thaw motility of ejaculates was 50% (>1800 ejaculates) before selection. The semen freezability was defined as the number of selected ejaculates divided by the total number of ejaculates frozen. Of the stallions, 5, 4, 5, 21 and 64% had semen freezability of 0-10, 10-33, 33-60, 60-90 and over 90%, respectively. Per-cycle pregnancy rate was 45-48% (>1500 mares per year, 1.8 cycles per mare) and foaling rate 64%. In comparison, per-cycle pregnancy rate and foaling rate of mares hand-mated to stallions were 57-59% and 64%, respectively. The average number of straws used was 32-35 (1.75 x 10(9) total spermatozoa) per mare per season. According to our results and the literature, the most important factors for improving fertility of frozen equine semen include: (1) a low concentration of glycerol (2-3.5% final concentration); (2) a suitable base extender for freezing like Lactose-Glucose EDTA or INRA82; (3) a post-thaw motility >30-35%; and (4) a sufficient number of spermatozoa per mare per season (1.5-2 x 10(9) total spermatozoa for two to three cycles) divided into small units. Numbers of spermatozoa, lower than 750.10(6) total spermatozoa per cycle, could result in lower per-cycle pregnancy rate with higher additional costs for management of mares. Because there are no particular regulations on quality and quantity of equine semen in the European Community, there is a need for the uniformity of information about frozen semen. A codification is suggested, based on the number of spermatozoa available per mare per season, the post-thaw motility and the final glycerol concentration.

The aims of this study were to evaluate the effects of cooling rate to 4 degrees C and temperature at the time of centrifugation/glycerol-addition (freezing extender: INRA82 + 2% egg yolk + 2.5% glycerol) on postcentrifugation recovery rate, post-thaw motility and per-cycle fertility. When centrifugation/glycerol-addition was performed at 4 degrees C (14 ejaculates), a moderate cooling rate (37 degrees C to 4 degrees C in I h) resulted in higher post-thaw motility (45%) than when using a slow cooling rate (37 degrees C to 4 degrees C in 4 h) (39%; P<0.05). When centrifugation/glycerol-addition was performed at 22 degrees C (37 degrees C to 22 degrees C in 10 min) (10 ejaculates), post-thaw motility was lower when spermatozoa were frozen directly from 22 degrees C (23%) than when spermatozoa were cooled to 4 degrees C (22 degrees C to 4 degrees C in 1 h) before freezing (47%; P<0.0001). When centrifugation/glycerol-addition was performed at 22 degrees C (before cooling at a moderate rate), as opposed to 4 degrees C (after cooling at a moderate rate), a significant improvement of 1) recovery of spermatozoa after centrifugation (P<0,0001), 2) post-thaw motility of spermatozoa at thawing (40% vs 36% (n < or = 291 ejaculates/group), P<0.0001) and 3) per-cycle fertility (56% vs 42% (n > or = 190 cycles/group), P<0.01) was observed. In conclusion, centrifugation/glycerol-addition at 22 degrees C followed by cooling to 4 degrees C at a moderate rate results in an improvement of post-thaw motility, spermatozoa recovery rate and per cycle fertility.

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