Preliminary results on the use of a novel synthetic extender for artificial insemination in jennies with cooled donkey semen

Abstract

Fertility rates using cooled semen in donkeys are acceptable, however in pursuit of an extender that maintains semen viability beyond 48 hours, we tested a new commercially available synthetic extender developed originally for horses (Beyond^Ⓡ, Minitube). This study aimed to evaluate fertility on artificially inseminated jennies with cooled extended donkey semen stored for 72 hours. Semen samples were collected from four Catalan donkeys of proven fertility (three ejaculates each). For the in vitro trial, semen pools containing one ejaculate from each donkey were divided into three samples and randomly assigned to one of the following treatments: 1) Beyond^Ⓡ (B); 2) Beyond^Ⓡ + 2% of centrifuged egg yolk (BEY); and 3) Equiplus^Ⓡ (Minitube) as control (E). Samples from B and BEY were centrifuged at 600 g for 10 minutes, the pellet resuspended to a final concentration of 70 × 10⁶ sperm/ml. The same dilution was used for E. All samples were stored at 5°C. Total motility (TM) and progressive motility (PM) were evaluated at 0, 24, 48, and 72 h using iSperm^Ⓡ (Aidmics Biotechnology). In the in vivo trial, semen from one donkey were treated with BEY or E and stored at 5°C for 72 h, starting with an initial 1 × 10⁹ PM sperm. Healthy and fertile jennies (n=8) were preovulatory inseminated with either BEY or E. Ovulation was induced (200 µg of buserelin; Busereleq, CIMOL Laboratory) at detection of a preovulatory follicle (size 33 ± 1 mm). The interval between AI and ovulation was ± 12 h. Pregnancy diagnosis was performed 14 days post-ovulation. In the in vitro trial, a general linear model with a compound symmetry structure covariance matrix was used. Treatment, time and their interaction were considered fixed effect while semen pool was considered a random effect. P was set at <0.05. For TM, there were no differences at 0 h. For later times, we observed a treatment-by-time interaction effect (P<0.05). For PM, there were no differences at 0 h. For later times, we observed a treatment effect (P = 0.0001) and time effect (P<0.05), but no interaction. At 72 h, the percentual averages for TM and PM were 22.8% and 1.6% for B; 43,2% and 13% for BEY; and 0,6% and 0% for E, respectively. One pregnancy was obtained from the six inseminated jennies with BEY (16.7%), but no pregnancies from the two ones inseminated with E. These preliminary results show that pregnancies can be achieved with cooled semen treated with BEY and stored for 72 h but the low numbers so far do not allow for drawing conclusions.