Effect of extender, centrifugation and removal of seminal plasma on cooled-preserved Amiata donkey spermatozoa

In the donkey species, the application of cooled semen artificial insemination could aid the survival of endangered breeds. Fifteen ejaculates collected from three Amiata donkeys were used to evaluate the effect of three extenders on spermatozoal motility characteristics after cooling and preservation for up to 72 h. Semen was diluted at a 1:4 semen:extender ratio in INRA96, INRA82 and INRA82 added of 2% centrifuged egg yolk (INRA82-Y) and motility was evaluated by the computerized analyzer CEROS 12.1 at hours 0, 24, 48 and 72. Total motility, and rapid spermatozoa after 24, 48 and 72 h of preservation were higher in INRA82-Y than in INRA96 or INRA82, as was progressive motility after 72 h. INRA82-Y was thus used in a second study, where the effects of centrifugation and of removal of seminal plasma on cooled donkey semen were evaluated on 12 ejaculates from four males. Rapid spermatozoa after 24 and 72 h, and total motility after 72 h were better preserved in the non-centrifuged samples than when seminal plasma was removed, the contrary was true for the proportion of spermatozoa keeping their progressive motility at hour 48. In conclusion, INRA82-Y kept sperm motility characteristics during cooled storage better than INRA82 or INRA96, and removal of seminal plasma during in vitro preservation did not seemed advantageous. Further studies are needed to better understand the changes in motility patterns of donkey spermatozoa caused by seminal plasma and semen extenders, and their relation to fertility.     

Embryo quality and transcervical technique are not the limiting factors in donkey embryo transfer outcome

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 PGF_2α 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.     

Retrospective study of factors affecting multiple ovulations,embryo recovery,quality, and diameter in a commercial equine embryo transfer program

In this study, 198 donor mares of different breeds, ages, and reproductive category were inseminated with fresh, cooled and frozen or frozen and cooled semen at the embryo transfer station or in private artificial insemination centers during 10 breeding seasons. The results of this activity were retrospectively analyzed by Pearson Chi-square test and logistic regression to evaluate factors affecting multiple ovulations, embryo recovery, embryo quality, and embryo diameter. Out of the 661 cycles, 937 ovulations were recorded (mean ovulations/cycle: 1.42 ± 0.58). Ovulation rate and incidence of multiple ovulations were significantly affected by age, breed, and reproductive category. Uterine flushings for embryo recovery were performed between 7 and 10 days after ovulation and resulted in the recovery of 338 embryos (51.1% embryos/cycle and 36.1% embryos/ovulation, respectively). At least one embryo was recovered in 298 flushings (45.1%). The factors affecting embryo recovery were age, breed, reproductive category, type of semen, number of ovulations, and location of artificial insemination. Flushing protocol and day of flushing had no effect on embryo recovery. Age, type of semen, number of ovulations, and day of flushing had a significant influence on embryo diameter (N = 215). None of the factors included in the model had an effect on embryo quality distribution.     

Concentrations of 15-ketodihydro-PGF2α, cortisol, and progesterone in the plasma of healthy and pathologic newborn foals

Information regarding the plasma hormone profiles of prostaglandins (PGs), cortisol (C), and progesterone (P4) during pathologic processes in newborn foals is scarce. The aim of this study was to determine the plasma concentrations of these hormones in diseased foals (n = 40) and healthy at-term foals (n = 24) (Equus caballus) during the first 2 weeks of life. Blood samples were collected daily, before any treatment with nonsteroidal drugs in diseased foals, and plasma was analyzed by radioimmunoassay. 15-Ketodihydro-PGF_2α (PGM) was consistently higher in diseased foals than in healthy foals, probably related to roles of PGs in completing organ maturation and/or the presence of oxidative stress or inflammation. Similar trends were observed for C and P4. In diseased newborns, only PGM was significantly higher in nonsurviving foals, although C showed a similar profile. When specific diseases were considered, the levels of PGM and C were lower in premature foals at 12 h of life, whereas the concentration of P4 was higher than in controls. The results of this study demonstrate the differences in plasma hormone levels between healthy and pathologic newborn foals, particularly during the first 2 d of life, probably reflecting the inability of diseased foals to cope with the transition between fetal and neonatal life.     

15-Ketodihydro-PGF2α and cortisol plasma concentrations in newborn foals after spontaneous or oxytocin-induced parturition

Hormonal changes during early neonatal life play a major role in the physiological processes underlying the maturation of several organs. Since prostaglandins and cortisol are associated with fetal organ system maturation, the aim of this study was to evaluate 15-ketodihydro-PGF_2α (PGM) and cortisol plasma concentrations during the first 21 days after birth in foals born by either spontaneous (24 foals) or low-dose oxytocin (OT)-induced parturition performed after at least 320 gestational days (25 foals) since induction is often considered to be a cause of prematurity. After spontaneous birth, the PGM concentration was significantly (P < 0.05) higher at 20 and 30 min compared to samples taken several hours or days later, while induced foals showed significantly (P < 0.05) higher concentrations at 10, 20, and 30 min. Regarding differences between the two groups, the plasma concentration of PGM was significantly higher 10 (P < 0.01), 20 (P < 0.05), and 30 (P < 0.05) min and 3 h (P < 0.05) after birth in induced foals compared to foals born by spontaneous parturition. It is difficult to determine whether the higher initial PGM concentrations in induced foals is related to higher uterine or fetal PGM release induced by exogenous OT stimulation. Cortisol plasma levels in both groups were higher at birth (P < 0.05) compared to the later sampling times. No differences were observed between the two groups indicating that the induction protocol used does not seem to result in premature foals.