Duccio Panzani

Donkey jack (Equus asinus) semen cryopreservation: Studies of seminal parameters, post breeding inflammatory response, and fertility in donkey jennies

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 × 10(6) sperm cells (250 × 10(6) 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.

Clinical use of dopamine antagonist sulpiride to advance first ovulation in transitional mares.

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 S(1); n = 34) or left untreated (Group C(1); n = 15). Group S(1) and C(1) 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 S(1) and C(1) 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 (S(2)), and 47 untreated mares (C(2)) were compared. In Groups S(2) and C(2,) 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.

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.

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.

Clinical use of twice daily injections of buserelin acetate to induce ovulation in the mare.

The difficulties in the prediction of ovulation time in the mare, together with the increasing use of cooled and frozen semen for artificial insemination, have stimulated investigators to search for methods for induction of ovulation. These methods are essentially based on exogenous administration of one of three hormones directly or indirectly involved in the mechanism of ovulation: hCG (Human Chorionic Gonadotrophin), CEG (Crude Equine Gonadotrophin) (Duchamp et al., 1987), or GnRH (Gonadotrophin-releasing hormone). Repeated administration of hCG, which is a heterologous glycoprotein for mares, may stimulate an immune response able to neutralise the effect of the hormone (Roser et al., 1979). Although CEG has not shown this negative side effect, it is not found on the market, and purified extracts are produced and utilised only by few research groups. GnRH, physiologically released in a pulsatile manner, and its analogues have been tested for induction of ovulation in oestrous mares either by repeated injections at given time intervals (Palmer and Quellier, 1988) or as slow-release subcutaneous implants. For this purpose, a slowrelease subcutaneous implant containing the synthetic GnRH analogue deslorelin acetate (OvuplantA) is successfully used in United States and Australia (Meinert et al., 1993; Meyers et al., 1997). The response to OvuplantA administration was similar to that obtained using hCG. However, this formulation is currently not available in the Italian and European market. The efficacy of buserelin, a GnRH analogue also commercialised in Italy, has been recently investigated for induction of ovulation in the mare. The administration of a single dose has not yielded encouraging results (Vidament et al., 1992). On the other hand, Battut et al. (2001) observed that most mares treated twice daily with intravenous (IV) administration of 20 or 40 mg of buserelin ovulated within 48 h. The aim of our study was to evaluate the clinical efficacy of repeated administration of 40 mg buserelin for induction of ovulation in the mare, as compared to a single administration of 2500 IU hCG or a placebo.

Evaluation of Plasma Membrane Integrity of Donkey Spermatozoa

The aim of the study was to develop a hypo-osmotic swelling test (HOS-test) for evaluating plasma membrane integrity in donkey spermatozoa. In the first study, six different hypo-osmotic solutions (fructose or fructose/sodium citrate, 75 or 150 mOsm, or bi-distilled water at 1 : 10 semen : solution ratio, or bi-distilled water at 1 : 3) were compared. The 75 mOsm fructose solution (1 : 10) and bi-distilled water (1 : 3) were chosen for study 2, where two incubation times (5 or 45 min) were tested. Bi-distilled water showed a significantly higher proportion of plasma membrane intact spermatozoa than the fructose solution (p < 0.05), it was thus concluded that the simple incubation for 5 or 45 min at 37 degrees C of one part of semen with three parts of bi-distilled water is an applicable HOS-test in the semen analysis of donkey spermatozoa. Regression analyses showed a significant correspondence of the latest method with Sybr 14- propidium iodide staining.

Effect of Post-Thaw Addition of Seminal Plasma on Motility, Viability and Chromatin Integrity of Cryopreserved Donkey Jack (Equus asinus) Spermatozoa

Pregnancy rates in donkeys after artificial insemination with cryopreserved semen are still low, compared to the horse species. Addition of autologous seminal plasma to frozen-thawed semen appeared to improve pregnancy rates. The aims of this study were to evaluate (1) sperm motility and plasma membrane integrity after thawing (T0) and after one and 2 h (T1 and T2) of post-thaw incubation in either 0% (SP0) or 70% (SP70) autologous seminal plasma and (2) sperm motility, plasma membrane integrity and DNA quality (%COMP-αt) after thawing (T0) and after 2 and 4 h (T2 and T4) of post-thaw incubation in either 0% (SP0), 5% (SP5) or 20% (SP20) homologous seminal plasma. In experiment 1, seminal plasma decreased total and progressive sperm motility and plasma membrane intact spermatozoa immediately after dilution and at all following time points (p < 0.05). In experiment 2, total and progressive motility did not differ between treatments immediately after dilution and between SP0 and SP5 at T2, while they were lower in both SP5 and SP20 than in SP0 at T4. Plasma membrane intact sperm cells did not differ between SP0 and SP5 and were lower in SP20 at all time points. DNA quality was not affected by treatment immediately after dilution and was significantly worse for SP20 after 4 h of incubation (p < 0.05). The post-thaw addition of seminal plasma at the tested concentrations did not improve donkey frozen semen characteristics in vitro over time.

Clinical, ultrasonographic, and endocrinological studies on donkey pregnancy

Although donkey breeding has gained new interest in the past two decades, knowledge about donkey reproduction is still scarce, particularly on jenny pregnancy. The aim of this study was to describe the ultrasonographic and endocrine profiles of the physiological pregnancy in the jenny. The study was performed on 12 pregnancies of 7 Amiata donkeys from Day 10 after ovulation to delivery. Because three pregnancies, respectively at weeks 42, 44, and 45, were considered pathologic and treated pharmacologically, data collected from 2 weeks before diagnosis to the end of pregnancy were removed from the analysis. Average length of the normal pregnancies was 353.4 ± 13.0 days (range, 339-370 days). Timing, dimensions, and development during the first phases of embryonic growth, evaluated using transrectal ultrasound, were similar to that previously described in jennies and mares: first detection of embryonic vesicle was at 11.8 ± 1.3 days of gestation and diameter was 6.5 ± 1.9 mm, loss of spherical shape occurred at 18.5 ± 1.4 days, and embryo and heart beat were first seen at 22.0 ± 1.1 and 25 ± 1.1 days, respectively. The intrauterine growth in the second half of pregnancy, evaluated using the transrectal and transabdominal approach, also showed strong positive correlations, similar to that reported for the mare. The trends of the combined thickness of the utero-placental unit and the echogenicity of the amniotic and allantoic fluids are examples. The diameters (mm) of fetal chest, eye orbit, and aorta increased throughout pregnancy and were 40.6 ± 2.9, 8.7 ± 1.5, and 3.5 ± 0.7, respectively, at week 13, and 190.9 ± 12.0, 21.4 ± 1.5, and 30.6 ± 1.8 at the last evaluation before parturition. In contrast, heart rate decreased as pregnancy progressed. Regression analyses between these parameters and day of gestation were statistically significant (P < 0.001). All fetuses consistently showed some intrauterine activity. Maternal plasma progestagens and estrone sulfate concentrations followed a pattern similar to that seen in mares, although the prepartal progestagen peak was lower in jennies. This study provides a range of ultrasonographic and endocrine values for normal pregnancy in jennies.

Effect of day of transfer and treatment administration on the recipient on pregnancy rates after equine embryo transfer

Effect of day of transfer and treatment administration on the recipient on pregnancy rates after equine embryo transfer D. Panzani & A. Crisci & A. Rota & F. Camillo Published online: 11 July 2009 # Springer Science + Business Media B.V. 2009

Induction of ovulation with buserelin in jennies: In search of the minimum effective dose

The aim of this study was to evaluate the minimum dose of buserelin acetate (buserelin) able to induce ovulation between 24 and 48 h from treatment (positive response) in estrous jennies. Jennies were studied during a total of 172 estrous cycles: ovarian activity was routinely monitored by ultrasound; when the dominant follicle reached a diameter of 33 ± 1 mm, estrous jennies were treated by subcutaneous administration of different doses of buserelin, 3.3mg (N = 11), 1.5mg (N = 21), 0.8 mg (N = 12), 0.4 mg (N = 16), 0.2mg (N = 13), 0.1mg (N = 16), 0.04 mg (N = 14), 0.02 mg (N = 16), or employed as controls (N = 53). Single jennies (P = 0.0001) and GnRH dose (P = 0.003) significantly affected ovulation rates. Ovulation rates between 24 and 48 h of each treated group, except for the 0.02 mg group, was higher than in the control group (P < 0.05). The minimum dose of buserelin effective to induce ovulation in estrous jennies was 0.04 mg.