After freeze-drying, the ultrastructure of boar sperms was observed by optical and electron microscopy. The in vitro development ability of the sperm was also examined with intracytoplasmic sperm injection (ICSI). The rate of male pronuclear formation was (68.52%), for cleavage (59.17%) and for blastocyst formation (19.16%) of the trehalose group (0.2 mol/L), significantly higher than those of the 50 mmol/L EDTA group (64.59%, 56.26% and 15.62%) and the control group (35.36%, 52.33% and 8.60%) (P < 0.05). After storage for 60, 120 and 180 d at 4 degrees C, no significant difference in the in vitro development was observed (P > 0.05). The male pronuclear, cleavage and blastocyst formation after ICSI with freeze-dried spermatozoa incubated for 1 h was superior than those incubated for 2 h (P < 0.05). No significant differences in the structures after stored at 4 degrees C or -20 degrees C (P > 0.05) were observed between the trehalose group and EDTA group. The percent of B grade freeze-dried boar spermatozoa in the trehalose group was higher than that of the EDTA group (P < 0.05). Based on the ultrastructure observation, main cryogenic damage in freeze-dried boar spermatozoa was swelling, damage or rupture in the sperm acrosome, neck and tail.
Animals ; Freeze Drying ; Male ; Semen Preservation ; methods ; veterinary ; Sperm Injections, Intracytoplasmic ; veterinary ; Spermatozoa ; Swine ; Trehalose ; pharmacology

The present study was performed to assess the fertility of frozen-thawed dog semen prepared by freezing with 6% glycerol and thawing at 70degrees C for 8 sec, and to evaluate the least number of post-thaw spermatozoa necessary to achieve pregnancy by intrauterine or intratubal artificial insemination. It was found that the pregnancy rate of intrauterine artificial insemination was 100% using 6% glycerol buffer and thawing at 70degrees C for 8 sec with 5 x 10(7) spermatozoa. Even though the pregnancy rate (80%) and the whelping rate (24.5%) in the 5 x 10(6) spermatozoa inseminated group were lower than those of the 5 x 10(7) spermatozoa group, conception was confirmed with 5 x 10(6) spermatozoa. Although the pregnancy rate of intratubal insemination was low (20%) with 4 x 10(6) spermatozoa, this study is the first report to show the pregnancy rate of intratubal insemination with frozen-thawed ejaculated canine semen. In order to improve the pregnancy rate with intratubal insemination of canine spermatozoa, it is necessary to investigate the optimal insemination site of the uterine tube, the appropriate number of sperm, and the direct effect of buffer on oocytes.
Animals ; Cryopreservation/methods/*veterinary ; Dogs/*physiology ; Female ; Fertility/*physiology ; Glycerol ; Insemination, Artificial/methods/*veterinary ; Male ; Pregnancy ; Pregnancy Outcome ; Semen/*physiology ; Semen Preservation/methods/*veterinary ; Temperature ; Time Factors

The aim of this study was to compare the effects of spermatozoa separation techniques on sperm quality and in-vitro fertilization (IVF) results for cryopreserved bovine semen. Sephadex, glass wool and Percoll gradient separation techniques were used for sperm separation and sperm motility, morphology and membrane integrity were evaluated before and after separation. Also, cleavage and blastocyst developmental rate were investigated after IVF with sperm recovered by each separation technique. The motility of samples obtained by the three separation techniques were greater compared to the control samples (p < 0.05). The percentage of spermatozoa with intact plasma-membrane integrity, identified by 6-carboxyfluoresceindiacetate/propidium iodide fluorescent staining and the hypo-osmotic swelling test, was highest in the glass wool filtration samples (p < 0.05). The cleavage and blastocyst rate of total oocytes produced from glass wool filtration samples were also higher than the control and Sephadex filtration samples (p < 0.05), but were not significantly different from Percoll separation samples. However, a significantly greater number of cleaved embryos produced by glass wool filtration developed to blastocyst stage than those produced by Percoll separation (p < 0.05). These results indicate that spermatozoa with good quality can be achieved by these three separation techniques and can be used for bovine IVF. In particular, it suggests that glass wool filtration would be the most effective method of the three for improving sperm quality and embryo production for cryopreserved bovine spermatozoa.
Animals ; Cattle ; Cryopreservation/methods/*veterinary ; Cytological Techniques/methods/*veterinary ; *DEAE-Dextran ; Female ; Fertilization in Vitro/methods/*veterinary ; *Glass ; Male ; Semen Preservation/methods/*veterinary ; Spermatozoa/*physiology ; Zygote/cytology

Genetically modified (GM) animals are unique mutants with an enormous scientific potential. Cryopreservation of pre-implantation embryos or spermatozoa is a common approach for protecting these lines from being lost or to store them in a repository. A mutant line can be taken out of a breeding nucleus only if sufficient numbers of samples with an appropriate level of quality are cryopreserved. The quality of different donors within the same mouse line might be heterogeneous and the cryopreservation procedure might also be error-prone. However, only limited amounts of material are available for analysis. To improve the monitoring of frozen/thawed spermatozoa, commonly used in vitro fertilization (IVF) followed by embryo transfer were replaced with animal-free techniques. Major factors for assessing spermatozoa quality (i.e., density, viability, motility, and morphology) were evaluated by fluorescence microscopy. For this, a live/dead cell staining protocol requiring only small amounts of material was created. Membrane integrity was then examined as major parameter closely correlated with successful IVF. These complex analyses allow us to monitor frozen/thawed spermatozoa from GM mice using a relatively simple staining procedure. This approach leads to a reduction of animal experiments and contributes to the 3R principles (replacement, reduction and refinement of animal experiments).
Animals ; Benzimidazoles/chemistry ; Cryopreservation/veterinary ; Embryo Transfer/veterinary ; Female ; Fertilization in Vitro/veterinary ; Fluorescent Dyes/chemistry ; Male ; Mice ; Mice, Transgenic ; Microscopy, Fluorescence/*methods/veterinary ; Propidium/chemistry ; Semen Analysis/*methods/veterinary ; Semen Preservation/veterinary ; Spermatozoa/*physiology