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

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 purpose was to optimize the vitrification for porcine embryos cryopreservation. Blastocyst/Morula (5-6th day-embryos) were collected from superovulated Bama mini-pigs (sows/gilts). We compared different cryopreservation methods, cryopreservation tools, thining of zona pellucida (ZP) and recipient breeds on the efficiency of porcine embryo cryopreservation. The results showed that: in embryo survival rate and blastocyst cell number, there were no significant differences between cryopreservation method I [embryos were vitrified by two step method with open pulled straw (OPS) and glass micropipette (GMP) in solution 1 (TCM199 + 20% FBS + 10% EG + 10% DMSO) for 3 min, and solution 2 (TCM199 + 20% FBS + 20% EG + 20% DMSO + 0.4 mol/L SUC) for 1 min, stored in liquid nitrogen] and method II[Blastocysts were cultured for 25 min in NCSU23 + 7.5 microg/mL cytochalasin B, centrifuged at approximately 13 000 xg for 12-13 min, and recovered back into pNCSU23. They were then equilibrated for 5 min in 2 mol/L ethylene glycol in pNCSU23, washed quickly in the vitrification medium, 8 mol/L ethylene glycol, 7% polyvinylpyrrolidone (PVP) in pNCSU23, loaded into OPS/GMP, and plunged into liquid nitrogen]. GMP vitrification method was more suitable and efficient than OPS method (P < 0.05) in embryo survival rate (83.8% vs 77.6%) and blastocyst cell number (53.1 vs 47.5) after thawing. Thining of ZP did not increase the survival rate, but significantly improved blastocyst cell number in the survival blastcysts (60.1 and 46, P < 0.01). Local pig breeds (Fengjing sows) were more suitable as recipients for embryo transfer of vitrified/warmed blastcysts, which can improve pregnant rate and embryo efficiency.
Animals ; Blastomeres ; cytology ; Cryopreservation ; methods ; veterinary ; Embryo Transfer ; veterinary ; Embryo, Mammalian ; Swine ; Swine, Miniature ; Vitrification ; Zona Pellucida ; physiology

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

291 embryos (Blastocyst/Morula) from 20 donor sows were vitrified by two step method with OPS (open pulled straw) in solution I (TCM199 + 20% FBS + 10% EG + 10% DMSO) for 3min, and solution II (TCM199 + 20% FBS + 20% EG + 20% DMSO + 0.4mol/L SUC) for 1min, stored in liquid nitrogen for 3 months, and transferred into 8 recipient sows after warming, one recipient sow was pregnant and 8 alive piglets were born. This is the first paper to report getting alive piglets by vitrification in China.
Animals ; Blastocyst ; physiology ; Cryopreservation ; methods ; veterinary ; Embryo Transfer ; Female ; Pregnancy ; Swine ; embryology

Nano-cryopreservation may become a new way in the next generation of cryopreservation technology. However, research using nanoparticles in oocytes vitrification has not been reported in the literature. In this study, HA nanoparticles with different diameters were added into cryoprotectant and M II-stage porcine oocytes were vitrified by Cryotop. The results showed that nanoparticles improved the survival rate of cryopreserved M II-stage porcine oocytes, but the difference between nanoparticles with different diameters of was not significant. In order to study the mechanism of nano-cryopreservation, the cooling rate of cryoprotectant was measured by ultra-fast temperature measurement system and the melting enthalpy of cryoprotectant was measured by differential scanning calorimeter (DSC). The results showed that the adding of nanoparitcles could not increase the cooling rate of cryoprotectant, but could decreases the amount of ice crystals during freezing and warming. Therefore, the mechanical injury within and outside cells might be effectively reduced.
Animals ; Cell Survival ; physiology ; Cryopreservation ; methods ; veterinary ; Cryoprotective Agents ; pharmacology ; Durapatite ; pharmacology ; Female ; Fertilization in Vitro ; methods ; veterinary ; Metaphase ; Nanoparticles ; Oocytes ; cytology ; Swine ; Vitrification

Thanks to the increasing use of flow cytometry in research in veterinary spermatology, many new membrane integrity assays have been developed over the past decade. These assays are important because of their superior ability to forecast fertility when compared with other tests, such as sperm motility. This major component of the sperm quality assessment has generated new investigations with the aim of developing tests that can detect membrane damage in a very early state. Using phospholipid transposition tests, early changes in membrane permeability and fluidity can be assessed in a large number of spermatozoa using fluorescent probes in combination with flow cytometry.
Andrology ; methods ; Animals ; Cell Membrane ; physiology ; Cell Membrane Permeability ; physiology ; Cryopreservation ; methods ; Male ; Phospholipids ; metabolism ; Spermatozoa ; cytology ; metabolism ; ultrastructure ; Veterinary Medicine ; methods