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Embryo cryopreservation ensures that genetic biodiversity is preserved over time. This study evaluates the survival of donkey embryos subjected to slow freezing and vitrification after thawing and in vitro culture. Seven-day-old in vivo produced donkey embryos were subjected to slow freezing (SF, N = 14) or vitrification (VIT, N = 22). After one year of cryopreservation, embryos were warmed, washed and 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 and after 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 the area and growth. The survival rate was not influenced by the procedure but by the 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%) (P < 0.05). Embryo diameter class at recovery did not significantly influence the survival rate. 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 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. • Slow freezing and vitrification procedures resulted in similar embryo survival rates. • Area and growth percentage were statistically higher in the slow freezing than the vitrified embryos after 68h IVC. • Embryo quality was more negatively affected by vitrification. [ABSTRACT FROM AUTHOR]

Many of the equid species can interbreed to produce viable but usually infertile offspring, with the mule (♂donkey [2n=62] × ♀mare [2n=64]) being the commonest of these hybrids. Despite its reduced ability to breed successfully, the mule has been shown capable of carrying and rearing horse and donkey foals using between-species embryo transfer (ET). These pregnancies have provided insight into the early development of the conceptus by highlighting the role of the uterine environment on endometrial cup development and lifespan (Allen et al., Reproduction, 1993); 98(1), 55-60). The aim of this preliminary study was to see if a mule (2n=63) was capable of carrying a mule pregnancy to term, which to the authors' knowledge has not previously been reported. Mule embryos were produced in vivo, by inseminating donor horse mares with fresh donkey semen. The uteri of donor mares were flushed on either Day 7.5 or 8 post-ovulation and three grade 1 expanded blastocysts and one grade 1 early blastocyst were recovered. In Argentina, two embryos were transferred using the Wilsher ET technique into each of two anestrous recipient muleson day 8 after initiating treatment with IM estradiol 17β for 3 days followed by long-acting progesterone every 7 days until day 100 of gestation. In Italy, two embryos were simultaneously transferred by a conventional ET technique into a cyclic recipient mule on day 5 after ovulation. The transfers resulted in two singleton pregnancies, in Argentina, and a twin pregnancy, which was manually reduced to a singleton, in Italy. Repeated ultrasound examinations showed normal conceptus and, subsequently, fetal development to term. Spontaneous delivery of three female mule foals occurred on Days 355, 342, and 335 of gestation, respectively and no placental abnormalities were recorded. No complications occurred during either parturition or peripartum, and the resulting mule fillies developed normally to weaning. To the best of the authors' knowledge, this is the first report of mules foaling mules after embryo transfer. More studies are warranted to describe the hormonal profile of mule-in-mule pregnancy, especially equine Chorionic Gonadotrophin (eCG) which is known to be influenced by both maternal and fetal factors. [ABSTRACT FROM AUTHOR]

Horses and Donkeys are known to differ phenotypically and karyotypically, although they can interbreed freely (Allen et al., Journal of Heredity, 1997;88(5), 384-392). The present study aimed to describe the outcome in terms of pregnancy rates of mares and jennets, after AI with either pooled horse or donkey semen of proven fertility. Semen was collected from four fertile horse (N=2) or donkey (N=2) stallions. Each insemination dose consisted of 1 × 109 total spermatozoa, at a 1:1 ratio for the two stallions of the same species, diluted with INRA96 to a final volume of 10 ml and with subjective total motility > 85%. AIs were performed every 48 hours until the detection of ovulation. A first pregnancy diagnosis was performed on day 10 after ovulation and repeated every day until embryo detection or day 16. At the first detection, embryos were collected for other studies. Differences between groups were examined using Fisher's Exact test, and differences were considered statistically significant when P<0.05. Results of the pregnancy rates are presented in Table 1. No statistical differences were found (P>0.05) when comparing interspecific artificial inseminations (mare x donkey versus jennet x stallion), or the male effect (horse versus jack). In the literature, the interspecies mating of a mare to a jack donkey is reported to be as fertileor even more than the intraspecies mating (Allen et al., Journal of Heredity, 1997;88(5), 384-392); by contrast, the cross between the jennet and the horse stallion is reported to be considerably less fertile. In our study, jennets gave much poorer results than mares, and hinny hybrids (jennet x horse) resulted in the lowest number of pregnancies. Further studies are required to better investigate the mechanism involved in this apparently poorly compatible gamete interaction between jennets and stallions. [ABSTRACT FROM AUTHOR]

Kisspeptin (KPs) are the most potent stimulating neurotransmitter of GnRH release from GnRH neurons. The administration of KP stimulates LH and/or FSH release. In small ruminants KPs analogues were able to induce an LH surge followed by ovulation in both cyclic and acyclic animals while in mares were able to lead to an increase of the LH/FSH plasma levels but not ovulation. Aim of this study was to compare the endocrinological effect, ovulatory and pregnancy rates effect of C6 KPs analogue in jennies. Ovarian activity of 9 Amiata jennies was monitored daily by transrectal ultrasound for 3 complete estrus cycles in which jennies in estrus were assigned, alternatively, to one of the 3 treatment groups: 50 nmol of the Kps analog C6 twice, 24 hours apart (C6 group); 0.4 mg buserelin acetate once (Bu group); 2 mL of saline once (CTRL group). Blood samples for [LH] were collected g-1 (-24h) g0 (h0, before treatment), h2, h4, h6, h8, h10, h24 (before second treatment with C6), h26, h28, h30, h32, h34, h48and every 24 hours until ovulation. Jennies were inseminated once at h24 with fresh extended semen of a donkey stallion. Pregnancy diagnoses were performed 14 days after ovulation. Significant higher plasma [LH] were found after induction between Bu and CTRL group at h6 and h8 (P<0.05), while tendentially higher differences were found between Bu/C6 groups and CTRL at h10. Five/9, 4/9 and 2/9 jennies of the Bu, C6 and CTRL groups, ovulated between 24 and 48 hours after induction, respectively (P>0.05). Pregnancy rates after artificial insemination were not different among groups (CTRL: 6/9, 66.7%; C6: 7/9, 77.8%; Bu: 6/9, 66.7%; P>0.05). In this study C6 showed to be able to tendentially and limited in time increase plasma [LH], compared to CTRL. Ovulation rates of C6 after induction were comparable to Bu even if not different to CTRL. Pregnancy rates were comparable to what reported in literature for donkey fresh extended semen for each group. Further studies using higher doses and/or more animals are needed. [ABSTRACT FROM AUTHOR]

Abstract: The aims of this study were (1) to evaluate motility parameters of donkey jack (jack; Equus asinus) semen cryopreserved in INRA-96 (INRA; IMV Technologies, France, 2% egg-yolk enriched) using either glycerol (GLY) or ethylene glycol (EG) as a cryoprotector; (2) to compare in vitro the postthaw re-extension with homologous seminal plasma (SPL) or INRA; (3) to compare fertility in donkey jennies (jennies; Equus asinus) timed artificially inseminated with jack semen cryopreserved using GLY or EG, re-extended with INRA; (4) to compare fertility in jennies timed artificially inseminated with jack semen cryopreserved using GLY re-extended with SPL, INRA, or not re-extended (NN); and (5) to describe some preliminary results of the inflammatory uterine response postbreeding. Semen from two jacks was collected and frozen in an INRA-2% egg yolk extender added of either 2.2% GLY or 1.4% EG. Postthaw motility was evaluated by a computer-assisted motility analyzer. Uterine inflammatory response and fertility were evaluated after artificial insemination (AI) of 13 jennies with frozen-thawed semen, either further extended with INRA (Group GLY-INRA, 13 cycles, and EG-INRA, 8 cycles), or with SPL (Group GLY-SPL, 13 cycles), or not re-extended (GLY-NN, 5 cycles). In each cycle, jennies were bred twice with 500 × 106 sperm cells (250 × 106 from each jack), at fixed times after induction of ovulation, and uterus was flushed at 6 and 10 h after first and second breeding, respectively. Cells in the recovered fluid were counted and distinguished as polymorphonuclear neutrophils (PMN) or other cell types. Total and progressive motility did not differ between cryoprotectants, but were higher when semen samples were re-extended in INRA, compared with SPL (P < 0.05). Pregnancy was diagnosed by transrectal palpation and ultrasonography examinations at 14 and 16 days postovulation. In 7/13 (53.8%) jennies and 12/39 (30.4%) cycles postbreeding intrauterine fluid accumulation was observed, with no differences between treatments (P < 0.05). Polymorphonuclear neutrophil numbers and concentrations were higher in the first flushing compared with the second, and PMN concentration was higher in GLY-SPL than in GLY-INRA (P < 0.05). Pregnancy rates in GLY-SPL, GLY-INRA, EG-INRA, and GLY-NN were 8/13, 3/13, 2/8, and 1/5, respectively. There was no significant difference either between the two cryoprotectants re-extended in INRA, or between re-extension groups. There was however a trend for GLY-SPL to improve pregnancy rates compared with GLY-INRA (P = 0.055). These results indicate that it is possible to obtain similar postthaw sperm motility and pregnancy rates using GLY or EG as a cryoprotectant for donkey semen, and that in the conditions of this study the re-extension in SPL of thawed semen before AI showed a trend toward the improvement of fertility and increased PMN concentration in uterine flushings. [Copyright &y& Elsevier]

Abstract: Embryo transfer (ET) in the donkey resulted in a very low recipient pregnancy rates. The aim of these studies was to investigate if nonsurgical transfer techniques or donkey embryo quality affect donkey recipient pregnancy failure. In Study 1, the impact of transfer technique was investigated by evaluating if cervical catheterization is associated with prostaglandin release and suppression of luteal function and if donkey recipients would become pregnant after nonsurgical transfer of horse embryos. Four jennies, from 5 to 8 d after ovulation, were submitted to a sham transcervical ET and to evaluation of PGFM and progesterone plasma concentrations. Five 8 d horse embryos were nonsurgically transferred into synchronized donkey recipients (HD). Cervical stimulation caused a transient PGF2α release in two of four jennies in the absence of a significant decrease in progesterone plasma concentration. All transferred horse embryos resulted in pregnancies in the jenny recipients. In Study 2, donkey embryo viability was investigated by 1.2 meters, 6-diamidino-2-phenylindole (DAPI) staining of 10 embryos and by the transfer of 6 and 12 donkey embryos in synchronized mare (DH) and donkey (DD) recipients, respectively, of known fertility. The estimated proportion of dead cells in DAPI stained embryos was 0.9% (range 0–3.9%) and below what is considered normal (20%) for horse embryos. Three of six and six of 12 of the DH and DD ETs, respectively resulted in pregnancies at 14 and 25 d (50%), a higher pregnancy rate than previously reported after DD ET. The overall results of this study suggest that the transcervical technique for ET and donkey embryo viability are not the reasons for the low pregnancy rates that have previously been described in donkey recipients, and that nonsurgical ET in donkeys can result in acceptable results. [Copyright &y& Elsevier]

Abstract: Artificial photoperiod treatment is currently the best method to hasten the first ovulation of the breeding season in winter anoestrus mares. However, this is not easy to apply in large herds of mares and, to be effective, has to be planned in the northern hemisphere in December at the latest. Pharmacological treatments have been proposed as alternatives: GnRH agonists, progesterone or its synthetic agonist Altrenogest, and dopamino-antagonists, as pherphenazine, domperidone or sulpiride. Dopamino-antagonists protocols, beginning at a given day of the year, gave controversial results in terms of hastening ovulation. The aim of this study was to evaluate the efficacy of an up-to-21-d long dopamine antagonist (sulpiride) treatment on mares at the beginning of the spring transition for its ability to hasten estrous cyclicity. In Study 1, 49 seasonally-acyclic standardbred mares, maintained in paddocks under natural photoperiod, were treated with 1 mg/kg/d sulpiride at the evidence of the first follicle with of 25 mm in diameter until ovulation for a maximum of 21 d (Group S1; n = 34) or left untreated (Group C1; n = 15). Group S1 and C1 mares showed a follicle of 35 mm in diameter after 8 and 22 d (median; P < 0.05) and ovulated after 18 and 43 d, respectively (median; P < 0.05). Twenty-two/26 and 6/15 mares of the Group S1 and C1 ovulated within 30 d from the beginning of the treatment, respectively (P < 0.05). All the mares of the study cycled until Autumn, unless they became pregnant. In Study 2, pregnancy rates after the first ovulation of the year of 22 acyclic standardbred mares maintained in paddocks under natural photo-period, treated following the same protocol as Study 1 (S2), and 47 untreated mares (C2) were compared. In Groups S2 and C2, 63.6% and 61.7% of the mares became pregnant after the first cycle (P > 0.05) and 50.0% and 61.1% of the remaining became pregnant in the following cycles (P > 0.05), respectively. Beginning with sulpiride treatment when follicles were 25 mm in diameter resulted in a significant advancement of cyclicity in non-photo-stimulated mares. Pregnancy rates after artificial insemination of treated mares were similar to those of untreated animals. [Copyright &y& Elsevier]

Abstract: Sixty-three embryos were recovered out of 83 estrous cycles (75.9%) and 98 ovulations (64.3%) of five Pantesca jennies, 2 to 5 yr old, naturally mated or artificially inseminated with fresh semen. Embryo recovery rate was influenced by number of ovulations per cycle (133% and 63% for double and single ovulations, respectively), by the day of embryo recovery attempt (12%, 83%, and 75% at Days 7, 8, and 9 after ovulation, respectively), and by the repetition of the embryo recovery attempt on successive cycles (60%, 79%, and 100% for cycles 1 to 7, 8 to 14, and 15 to 24, respectively). All recovered embryos but three were classified as good or excellent. Of 58 nonsurgical embryo transfers to Ragusana jenny recipients, 13 (22.4%), 10 (17.2%), and 9 (15.5%) resulted in a pregnancy at Days 14, 25, and 50, respectively. Recipients’ pregnancy rate was not influenced by the evaluated parameters: embryo quality and age, media employed to wash embryos, days after ovulation of the recipient, experience of the operator. Between 14 and 50 d of pregnancy, 4 of 13 (30.7%) embryos were lost with an influence of the days from ovulation of the recipient: recipients at Days 5 or 6 kept all pregnancies (N=7), whereas recipients at Days 7 or 8 lost 3 of 4 pregnancies, as one of the two recipients at Day 3. More studies are needed before embryo transfer could be considered a reliable tool to preserve endangered donkey breeds. [Copyright &y& Elsevier]

Sperm vitrification has been recently developed, but fertility trials have not been performed yet in equine species. In this study, a new warming technique for vitrified donkey semen was developed and the uterine inflammatory response and fertility were compared to conventional freezing. In Experiment 1, sperm was vitrified in straws and warmed in 3 ml of extender or in a water bath at: 37 °C/30 s; 43 °C/10 s; and 60 °C/5 s. Sperm motility, plasma and acrosome membranes and DNA integrity were compared between treatments. In Experiment 2, jennies were inseminated twice (500 × 106 sperm) in the uterine body either with vitrified or frozen semen (2 cycles/jenny). Pregnancy rates and the uterine inflammatory response (polymorphonuclear neutrophil concentration; PMN) were evaluated after artificial insemination (AI). No differences between warming in extender/water bath were found and 43 °C/10 s was better than lower temperatures in terms of total (53.8 ± 13.2%) and progressive sperm motility (41.4 ± 11.4%). No differences in PMN concentration (× 103 PMN/ml) were found between vitrified (276.8 ± 171.6) or frozen (309.7 ± 250.7) semen after AI. However, PMN decreased faster (P < 0.05) using vitrified semen. Pregnancy rates were greater for vitrified (22%) than frozen semen (10%) but not statistically different. In conclusion, donkey sperm vitrified in straws could be directly warmed in a water bath at 43 °C/10 s, reducing the uterine inflammatory response obtained after AI and promoting positive pregnancy outcomes. These findings confirm the possibility to use vitrified semen as an alternative for AI in jennies. [ABSTRACT FROM AUTHOR]

Abstract: Oocyte transfer is a potential method to produce offspring from valuable mares that cannot carry a pregnancy or produce embryos. From 2000 through 2004, 86 mares, 19.2±0.4 yr of age (mean±S.E.M.), were used as oocyte donors in a clinical program at Colorado State University. Oocytes were collected from 77% (548/710) of preovulatory follicles and during 96% (548/570) of cycles. Oocytes were collected 21.0±0.1h after administration of hCG to estrous donors and cultured 16.4±0.2h prior to transfer into recipients’ oviducts. At 16 and 50 d after transfer, pregnancies were detected in 201 of 504 (40%) and 159 of 504 (32%) of recipients, respectively, with an embryo-loss rate of 21% (42/201). Pregnancy rates were similar (P >0.05) for cyclic and noncyclic recipients and for recipients inseminated with cooled, fresh or frozen semen. One or more recipients were detected pregnant at 16 and 50 d, respectively, for 80% (69/86) and 71% (61/86) of donors. More donors &#60;20 than ≥20 yr (mean ages±S.E.M. of 15.5±0.4 and 23.0±0.3 yr, respectively) tended (P =0.1) to have one or more pregnant recipients at 50 d (36/45, 80%; 28/45, 62%, respectively). Results of the program confirm that pregnancies can consistently be obtained from older, subfertile mares using oocyte transfer. [Copyright &y& Elsevier]

This study compared the outcome of two new timed artificial insemination (TAI) protocols in a milk-producing donkey farm. Ninety Amiata jennies were inseminated at the moment of ovulation induction with hCG, with fresh-transported semen that had been stored at room temperature from 3 up to 6 h, with an approximate average storage time of 4 h and a half. In both protocols, on Day 0 jennies were treated with alfaprostol (PGF2α), and on Day 7 they were checked by ultrasound (US) and, if in estrus, they were treated in order to induce ovulation and were then artificially inseminated. In the slow-short TAI protocol, jennies not already inseminated were treated again with PGF2α at Day 14. On day 21 US was repeated and estrus jennies were induced to ovulate and inseminated. In the fast-long TAI protocol, US was performed once a week in jennies not already inseminated and if found in estrus, they were induced to ovulate and inseminated, while those not in estrus were treated again with PGF2α. This protocol was repeated for up to nine weeks. The rates of inseminated/treated, pregnant/inseminated and pregnant/treated jennies were 76%, 56% and 43% for the slow-short TAI protocol and 94%, 47% and 44% for the fast-long TAI protocol. The age class and the lactation status of the jennies had no significant effect on synchronization success or final pregnancy rate. This study demonstrates that it is possible to achieve reasonable pregnancy rates through simplified TAI protocols in jennies, reducing animal handling to a minimum. • Ninety milk producing jennies were submitted to TAI using fresh semen according to a slow-short or a fast-long protocol. • The pregnancy rates observed were similar for both TAI protocols although the long one was more time-consuming. • Reasonable pregnancy rates were achieved using TAI in jennies with a short-time stored semen and reducing animal handling. • This is the first report of AI outcome in donkey using fresh semen stored at room temperature up to 6 h after collection. [ABSTRACT FROM AUTHOR]

Abstract: A saprophytic bacterial flora is present on the penis and the distal part of the urethra of stallions. Little is known about the fungal flora of their reproductive tract. As micro organisms play an important role in mares fertility, the aim of the study was to describe the distribution of fungi and bacteria in the normal genital apparatus of stallions. The microbic flora of the reproductive tract of 11 healthy, fertile stallions was evaluated, collecting samples from 5 different locations: urethral fossa, penis/internal lamina of the prepuce, urethra pre- and post-ejaculation, and semen. For fungal examination samples were taken on 3 different occasions (N = 165), while for bacteriologic examination samples were taken on one occasion only (N = 55). There was a statistical difference in the presence of filamentous fungi between urethral fossa or penis/prepuce (45.4%) and urethra pre- or postejaculation or semen (15.1%, 6.0%, and 0.0%, respectively). Yeasts were isolated in 9.1% of the samples, never in semen. The most represented mycelial fungi were Penicillium spp., Aspergillus spp., Scopulariopsis spp., Trichosporon spp. and Mucoracee. The proportion of samples showing a total bacterial count ≥10 000 colony forming units (CFU) was higher for urethral fossa than for urethra pre- or postejaculation or for semen. Some bacterial growth was always observed in all locations, including the ejaculate. Differences between sampling locations were observed also for Staphylococci, both coagulase positive and negative. Salmonella enterica Abortus equi and sulphite reducing clostridia and other pathogens (including Klebsiella spp. and Pseudomonas spp.) were never isolated. Escherichia coli and coliforms always showed a low or absent flora. These data add information to the literature. [Copyright &y& Elsevier]