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.

ABSTRACT. This study was performed to evaluate the use of dimethylacetamide (DMA) and dimethyl sulfoxide (DMSO) in boar sperm cryopreservation. Semen from eight boars was cryopreserved following treatment with 3, 5, and 7% DMA and DMSO, and 3% glycerol (control). After thawing, sperm conventional parameters and membrane integrities were evaluated. There were no significant differences among different DMA concentrations in all evaluations. Membrane intactness were higher in 5% and 7% DMSO than 3% DMSO (P<0.05). Sperm motility of 5% DMSO was lower than that of 3% glycerol (P<0.005), and membrane intactness were lower in 5% DMA and DMSO than 3% glycerol (P<0.05). DMA and DMSO didn't improve sperm quality and glycerol remains the most useful for boar sperm cryopreservation. Glycerol (at a concentration of 3%) is the most commonly used cryoprotectant (CPA) for boar sperm [3], but it has toxic effects as well as contraceptive effects on sperm [8]. Its detrimental effects have stimulated the study of alternative CPAs such as N,N-dimethylacetamide (DMA) or dimethyl sulfoxide (DMSO) [2,10]. Due to its highly hydrophilic nature and low molecular weight, DMA reduces the formation of intracellular ice crystals and increases membrane permeability, thus decreasing osmotic damage [2]. The penetration of DMSO is also rapid, due to its lower molecular weight relative to glycerol [10] and DMSO has been used to successfully cryopreserve sperm [9]. However, there are few studies on the uses of DMA and DMSO for freezing boar semen. Therefore, the objective of this study was to evaluate the effectiveness of DMA and DMSO as possible replacement for glycerol in boar semen cryopreservation.Three CPAs were used in this study: glycerol, DMA, and DMSO (all from Sigma-Aldrich, St. Louis, MO, U.S.A.). Glycerol was used at a final concentration of 3% as a control, and the others at final concentrations of 3, 5, and 7%. We first compared the three concentrations of DMA and DMSO (n=5), and the concentrations showing the best result for each CPA were then compared with 3% glycerol (n=8).Ejaculate from 8 Duroc boars was collected and processed according to the straw freezing procedure [1] with some modification. Sperm-rich fractions were extended in Beltsville Thawing Solution (BTS) and were slowly cooled to 15°C for 3 hr. After centrifuging, the semen pellet was resuspended in lactose-egg yolk (LEY) extender. After further cooling to 5°C for 90 min, LEY-extended semen was aliquot for treatment with each CPAs and then two parts LEY-extended semen were mixed with one part freezing extenders (LEY extender containing 1.5% Equex STM [Nova Chemical Sales, Scituate Inc., MA, U.S.A.] and the CPAs described above, v/v). The semen was loaded into 0.5-ml straws (IMV, L'Aigle, France), and held in liquid nitrogen vapor 3 cm above the liquid for 20 min. The straws were then plunged into the liquid nitrogen. After thawing the straws at 37°C for 20 sec [5], thawed semen was evaluated for sperm motility [13], morphological defect, viability by eosin-nigrosin st...

The present study evaluated whether pentoxifylline added to the freezing extender could improve the sperm characteristics and function in canine frozen semen. Also the conception rate following AI with frozen-thawed semen was investigated. The beneficial effects of pentoxifylline supplementation were visible in motility, viability, acrosome reaction, and tail swelling patterns. Especially, highest sperm viability and function were obtained in the forzen semen supplemented with 1mM pentoxifylline. The follicle size measured by ultrasonography was 6.48 mm, 11.52 mm and 8.9 mm on 11, 13 and 15 days after the onset of natural estrus, respectively and ovulation occurred on 13 and 15 days. The pregnancy rates in bitches inseminated with frozen semen on natural and induced estrus were 71.4% and 75.0%, respectively. There was no significant difference between the pregnancy rates in bitches inseminated with frozen semen following natural and induced estrus, but the litter size was slightly increased in natural cycle.