• donkey; artificial insemination; cryopreservation

Donkey breeding activities in Europe are increasing, requiring the use of newer technologies for the management of reproduction in this species. Furthermore, being many donkey breeds at risk of extinction, it is important to study procedures for preserving donkey genetics indefinitely. Aims of this Ph-D thesis were to improve the knowledge on the management of reproduction and on the application of Assisted Reproductive Technologies (ARTs) in donkey species.
In Chapter 2, three different combinations of prostaglandin F2α (PGF2α) and gonadotropin-releasing hormone (GnRH) analogs were studied in order to apply a Timed Artificial Insemination (TAI) protocol in jennies. Estrus or diestrus status diagnosed by ultrasonography, without the use of a teaser stallion, was compared with serum progesterone concentration in order to assess the accuracy of the ultrasound-based diagnosis. The comparison of the three TAI protocols showed a trend for difference in pregnancy rates per synchronized jennies comparing the lower result (11% for the PPG protocol; PGF2α, PGF2α, and GnRH) with the best one (56% for the PGPG protocol; PGF2α, GnRH, PGF2α, and GnRH). Ultrasound itself was confirmed to be highly accurate for the determination of the estrus or diestrus status in the studied jennies.
In Chapter 3, ninety milk producing jennies underwent 2 different TAI protocols based on the use of a weekly alternance of the administration of PGF2α and hCG and lasting 3 or 9 weeks. Fresh diluted seme, stored up to 6 hours, was employed for AI that was done at the time of hCG administration. The rates of inseminated/treated, pregnant/inseminated and pregnant/treated jennies were 76%, 56% and 43% for the 3 weeks TAI protocol and 94%, 47% and 44% for the 9 weeks TAI protocol, showing not advantages in prolonging the protocol for more than 3 weeks. It is well known that jennies’ pregnancy rates after frozen semen AI are very low.
In Chapter 4, three different AI protocols were used to compare pregnancy rates in jennies bred with frozen-thawed semen: release of the semen in the uterine body after re-extension in INRA96 or intrauterine horn deep insemination with or without re-extension in homologous seminal plasma.
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Although the re-extension in seminal plasma was found to be detrimental for all the semen kinematic parameters observed overtime, there was a tendence toward a higher proportion of pregnancy rates when using deep horn AI with re-extension in seminal plasma, compared to in the body of the uterus AI after re-extension in INRA96 (60% vs 10%, respectively; P<0.06). Comparing AIs with or without re-extension in seminal plasma, pregnancy rates were statistically significant (P<0.05). These results suggest the opportunity to deeply investigate the role of seminal plasma in AI with frozen semen in equids.
In Chapter 5, cryopreservation of donkey embryo was investigated in order to develop protocols for genetic biodiversity preservation overtime. Aim of this study was to evaluate the survival of donkey embryos subjected to slow freezing or vitrification after thawing and in vitro culture. Thirty-six seven-day-old in vivo produced donkey embryos were subjected to slow freezing (SF) or vitrification (VIT). After one year of cryopreservation, embryos were placed in incubation for in vitro culture (IVC). In order to assess the embryo viability, the quality grade and developmental stage were recorded after thawing, 24 and 48 h of IVC. Eleven embryos (SF = 4 and VIT = 7) were incubated under a time-lapse camera, for up to 68 h, in order to determine their area and growth. The survival rate was not influenced by the cryopreservation procedure but by the embryo developmental stage: after 48 h of IVC, blastocyst survival rate (1/8, 12.5%) was significantly lower compared to both morulas (8/12, 66.7%) and early blastocysts (11/16, 68.7%) survival rates (P < 0.05). In terms of the embryos that were judged to be alive after 48 h of IVC, quality grade 1 was observed in 7/8 (88%) and 4/12 (33%) of the SF and VIT embryos, respectively (P < 0.05). After time-lapse analysis, the IVC embryo area as well as growth percentage were statistically higher in the SF than in the VIT embryos (P < 0.05). In conclusion, no difference in survival rates was found between the two cryopreservation procedures, although embryo quality was more negatively affected by vitrification. The development of Assisted Reproductive Techniques for donkeys remains a challenge and more studies are needed to better understand the possible applications of ARTs in this species.