Francesco Camillo

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.

FERTILITY AT THE FIRST POST PARTUM ESTROUS COMPARED WITH FERTILITY AT THE FOLLOWING ESTROUS CYCLES IN FOALING MARES AND WITH FERTILITY IN NONFOALING MARES

Summary A retrospective study on the reproductive performance of 401 artificially inseminated trotter mares during six breeding seasons is presented. Mares, 279 post partum (PP) and 122 maiden and barren, or nonlactating (NL), were inseminated with fresh semen obtained from four fertile stallions of the same breed. Pregnancy rate (PR) of mares inseminated at the foal heat (182/253, 71.9%) was lower, but not significantly different, than the PR (22/26, 84.6%) of mares inseminated for the first time at the second post partum cycle and similar to the PR at the first and second cycle of NL mares (95/112, 77.8% and 25/33, 75.7%, respectively). PR of mares inseminated at the foal heat was higher, but nonsignificantly different, from PR of the post partum mares not pregnant after artificial insemination (AI) at foal heat and inseminated again at the following estrous cycle. The PR after AI at the foal heat was significantly higher than the PR when the AI was performed at the third or later cycle in NL mares (71.9% vs 22.2%, P

Use of Prostaglandin F2α for Early Pregnancy Termination in the Mismated Bitch

Natural PGF administered at a dose of at least 250 micrograms/kg twice daily subcutaneously for at least 4 days starting no earlier than day 5 of cytologic diestrus induces luteolysis and pregnancy termination in the mated bitch. The resulting shortening of the luteal phase is associated with a shortening of the interestrous interval from 1 to 4 months. Bitches treated with PGF show emesis, diarrhea, and panting within 5 minutes and transient hypothermia which lasts 2 to 3 hours but generally have no further reaction. Bitches with cardiac or respiratory dysfunctions are not considered safe patients for early pregnancy termination with PGF because of the cardiovascular effects of this drug. Bitches treated with this regime early in diestrus resorb their conceptuses; those treated after days 35 to 40 show clinical abortion of viable fetuses.

Presence and distribution of fungi and bacteria in the reproductive tract of healthy stallions

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.

Clinical studies on daily low dose oxytocin in mares at term

The aim of this study was to test whether low dose oxytocin i.v. injection once a day to mares diagnosed as being ready for birth by mammary secretion calcium strip test measurements could be used as a reliable method to induce parturition and/or predict the mare would not foal during the following night if parturition did not occur within 2 h of treatment. Fifty-one near-term Haflinger mares were used and a single injection of 2.5 iu oxytocin was given between 1700 and 1900 h, including 10 mares used as controls which were administered a placebo. Administration of oxytocin resulted in the delivery of a normal foal within 120 min in 95% of mares. Twenty-four out of 38 (63%) treated animals foaled in response to the first oxytocin injection, 9 out of 38 (24%) in response to the second injection and 3 out of 38 (8%) in response to the third treatment. Two out of 38 (5%) treated mares foaled during the night irrespective of treatment whereas 7 out of 10 (70%) control mares foaled during the night. It was concluded that the major advantage of injecting a daily low dose of oxytocin appears to be that such a low dose induces delivery only in mares carrying a mature fetus and which are ready to foal.

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.

Luteolytic effects of prostaglandin F2α on day 8 to 19 corpora lutea in the bitch

Luteolysis was induced in 5 experimental Beagle (8 cycles) and 7 client-owned bitches treated with 150 to 200 microg/kg, sc of prostaglandin F2alpha administered twice daily for 4 d, starting on Days 8 to 19 after the onset of cytological diestrus. Five experimental Beagle bitches had been mated during the estrus preceding treatment, and copulation had been confirmed in 2/7 client-owned bitches presented for termination of unwanted pregnancy. Serum progesterone concentration (mean +/- SD) declined from 26.1 +/- 66 ng/ml before treatment to 0.3 +/- 0.4 ng/ml on the fourth day of treatment One of the 7 client-owned bitches maintained her pregnancy even though serum progesterone concentrations were less than 0.5 ng/ml on the third and fourth day of treatment. Mean (+/- SEM) inter-estrous intervals before and following prostaglandin-induced luteolysis were 207.3 +/- 12.4 (n = 11 cycles in 6 bitches) and 95.5 +/- 20.0 d (n = 6 cycles in the same 6 bitches; P < 0.0001), respectively These results suggest that effective prostaglandin-induced luteolysis can be achieved with administration of 180 microg/kg during the third week of diestrus in pregnant and nonpregnant bitches.