Chiara Sabatini

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.

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.

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.

Post-thaw Addition of Caffeine and/or Pentoxifylline Affect Differently Motility Characteristics of Horse and Donkey Cryopreserved Spermatozoa

Post-thaw sperm motility and viability can be poor and thus partially responsible for the low fertility results after AI. In order to improve sperm survival during freezing, equine and donkey semen extenders have been supplemented with a variety of substances: different cryoprotectants, antioxidants, and cholesterol loaded cyclodextrins (e.g. 1-5). Moreover, with respect of equine semen, caffeine and pentoxifylline, two methylxantine derivative phosphodiesterase inhibitors, have been also tested. Through the inhibition of the enzyme cyclic-adenosine-monophosphate (cAMP) phosphodiesterase these substances increase its intracellular concentration, which in turn generates adenosine triphosphate, the source of energy required for sperm motility (6-7). When pentoxifylline was added to equine semen post-thaw, sperm motility parameters were improved (8). The same effect was not observed when 2mM caffeine was employed (9). The addition of phosphodiesterase inhibitors to donkey frozen-thawed semen has not been evaluated yet. The aim of the study was to evaluate if the post-thaw addition of different concentrations of either caffeine or pentoxifylline, or of the two molecules combined, improves motility parameters of cryopreserved stallion and donkey spermatozoa.

Postmating Endometritis and Pregnancy Rate Were Not Affected by the Addition to Frozen-Thawed Semen of Filtered Seminal Plasma When Mares Without Evidence of Endometritis Were Artificially Inseminated Once 40 Hours Post-Gonadotropin-Releasing Hormone Treatment

ABSTRACT The main aim of this study was to evaluate the effect of the addition of seminal plasma (SP) to frozen‐thawed semen on postmating endometritis (PME) and embryo recovery rate in mares when only one artificial insemination (AI) is performed. Forty hours following induction of ovulation, 15 fertile Standardbred mares were submitted to a single AI per cycle with frozen semen obtained from one of two different stallions, on two cycles, according to two different protocols: routine AI (200–280 × 106 frozen‐thawed spermatozoa in 2 mL) and SP AI (200–280 × 106 of frozen‐thawed spermatozoa in 2 mL to which 7.8 mL of frozen‐thawed SP was added). Six and 20 hours after AI, mares were evaluated by ultrasound for the presence of uterine fluid. Six hours after AI, mares were also subjected to uterine lavage for the evaluation of the presence and number of inflammatory cells. Eight days after ovulation, pregnancy was diagnosed by embryo recovery. There was a significant effect of treatment on subjective motility, which was lower when SP was added (20%; interquartile range [IQR] 10) compared with undiluted semen (45%; IQR 10) (P < .05). There was no significant effect of stallion or treatment on PME or embryo recovery rate. In the mares and conditions of this study, the addition of SP to frozen‐thawed semen had no effect on post‐AI uterine inflammation and pregnancy rate. HighlightsSeminal plasma was added to frozen‐thawed semen before artificial insemination of mares.Treatment reduced sperm subjective motility.There was no significant effect of treatment on postmating endometritis.There was no significant effect of stallion or treatment on embryo recovery.

Reproductive parameters of donkey jacks undergoing puberty

In male donkeys, puberty and the related events have been poorly characterized. The aim of this study was to evaluate the age at which male donkeys reach puberty, and characterize age associated changes in testicular size, testicular blood flow, serum testosterone concentration and semen quality. Every two months, starting at 6 months and finishing at 24 months of age, five male donkeys born in May to July were subjected to B-mode ultrasound examination to assess testicular size and scrotum content and blood serum sampling for testosterone concentration. From the age of 8 months, pulsed Doppler was employed to evaluate blood flow in the testicular artery. Testosterone serum concentration was evaluated via RIA. From the age of 12 months, monthly semen collections were attempted and semen was evaluated for sperm number, motility and morphology. Onset of puberty was defined as the first ejaculate containing ≥50 × 106 spermatozoa with ≥10% total motility. One of the donkeys was excluded from the statistical analyses due to a hydrocele presented during the study. Testes width was affected by age (P < 0.0001) and after an initial plateau increased linearly from 10 months of age. Pulsatility and resistivity indexes were also affected by age (P < 0.01), being significantly higher at 14 months than at 24 months. Testosterone serum concentration was affected by age (P < 0.0001) and was significantly lower at 6 months (0.1 ng/ml) compared to 22-24 months (≥0.8 ng/ml). Spermatozoa appeared in the ejaculate at a mean age of 18.7 months and puberty was attained between 19 and 20 months of age (mean: 19.5 months), between January and February. In conclusion, late spring born Amiata donkey colts reached puberty at 19-20 months of age. Puberty was accompanied by changes in testicular size, testicular blood flow and serum testosterone concentration.

Cooled semen artificial insemination in donkeys: field results

Success of first breeding is a major concern for goat breeders, since failure to fertilize does increases unproductive time and breeding costs. Fertility rates after artificial insemination of young goats are highly variable and rather low. Breeders generally breed does that are older than 5 months and weight more than 32 kg. However, sexual precocity is highly variable between does. Up to now, there is no known biomarker for sexual precocity. A better characterization of the pubertal stage of maturity could help optimizing time for first breeding. Our objective was to analyze the serum metabolome of doe kids, just before the first breeding, in order to characterize the pubertal stage of maturity and identify biomarkers of sexual precocity. Weekly blood sampling was performed on twenty 6-to 7-month-old does born in February for 5 weeks before their first contact with bucks in September. Progesterone assays and metabolome analysis using 1H Nuclear Magnetic Resonance Spectroscopy were performed on the serum samples. No spontaneous ovulatory cycle was observed before breeding based on progesterone assays. All does had reached the pubertal stage of maturity at breeding since all got pregnant. Metabolome analysis allowed the identification of 109 spectral bins in sera. Between week 1 and 5 preceding buck introduction, 32 buckets showed significant variations (t-test, p < 0.05): i.e. inosine, formate, lactate and creatinine decreased, while threonine, tryptophan, isoleucine and trimethylamine oxide significantly increased. Metabolites with significant variations between the 5 considered weeks could be biomarkers of sexual precocity; studies are in progress to identify them.