First pregnancies in jennies with vitrified donkey semen using a new warming method.

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 ±â€¯13.2%) and progressive sperm motility (41.4 ±â€¯11.4%). No differences in PMN concentration (×103 PMN/ml) were found between vitrified (276.8 ±â€¯171.6) or frozen (309.7 ±â€¯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.

Cryopreservation of donkey embryos: Comparison of embryo survival rate after in vitro culture between conventional freezing and vitrification.

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.

New simplified protocols for timed artificial insemination (TAI) in milk-producing donkeys.

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.

Radical hysterectomy for stage IB1 cervical carcinoma: lymph node metastasis as a prognostic factor.

PURPOSE: Lymph node metastasis is one of the most important factors influencing prognosis and further therapy in patients with cervical carcinoma. The aim of this study was to confirm the impact of nodal metastasis od disease-free interval in women with stage IB1 cancer of the uterine cervix. PATIENTS AND METHODS: From June 1986 to December 1999 269 patients with stage IB1 cervical carcinoma were operated on. Two hundred thirty-six (87.84%) patients had class III and 33 (12.16%) class II radical hysterectomy, according to Piver's classification. RESULTS: The median number of the removed lymph nodes was 21. Positive lymph nodes were found in 71 (25.28%) patients. All patients with positive lymph nodes received postoperative adjuvant external beam radiotherapy. Patients with bulky nodal disease received also chemotherapy.Overall 5-year disease-free interval in 212 patients was 80%. Five-year disease-free interval for patients without lymph node metastasis was 91%, while it was 40% in those with lymph node metastasis (p < 0.0001). CONCLUSION: Surgical staging of cervical cancer, which includes pelvic and para-aortic lymphadenectomy,together with pathological data, can provide potentially useful information for the radiation oncologist and precise analysis of survival and prognostic risk factors.

Therapy of atherosclerotic arteriopathy of lower limbs. Aspects and results.

As far as therapy is concerned, atherosclerotic arteriopathy may be divided into acute and chronic forms. In acute embolic forms, therapy should be surgical. Only in cases of peripheral embolism or polyembolism, and in rare cases for which vascular surgery cannot be adopted, can thrombolysis be carried out with UK. In acute thrombotic forms, therapy should be medical, because a thrombus of recent formation is rich in fibrin and may be lyzed by UK. Total recanalization takes place in 61% of cases treated, partial recanalization in 23%. Subsequently perviousness is maintained by adequate antithrombotic therapy. In chronic arteriopathy, the thrombus is lacking or almost lacking in fibrin and thrombolytic therapy is not indicated. Special therapeutic combinations are used containing platelet inhibitors (ticlopidine), antifibrin drugs (subcutaneous heparin), minor fibrinolytic agents (mesoglycan) and hemorheological drugs (pentoxyphylline). This therapy seems to give good results, as showed by the low percentage in amputation calculated on 2,565 patients treated and kept under observation for 5 years. Finally let us consider chronic progressive arteriopathy. This term indicates a very advanced stage, characterized by a gradual irreversible change for the worse leading towards gangrene. As a last resort, before amputating, a thrombolytic therapy with UK was tried to see if with strong fibrinolysis continued for 3 days amputation might be avoided. In a pilot study carried out on 12 patients, the angiographic data showed only partial lysis in small arteries or arterial branches. Clinical data showed reduction or disappearance of pain at rest in 80% of cases. In 70% of cases gangrene disappeared if it was initial and superficial, it was delimited if already in progress.