Quercetin and genistein, plentifully present in fruits and vegetables, are flavonoid family members that have antioxidative function and plant-derived phytoestrogen activity. The antioxidative effects of quercetin and genistein on boar sperm characteristics and in vitro development of IVF embryo were investigated. The sperm motility was increased by addition of genistein 50 μM for 6 hr incubation compared to control (p<0.05). The sperm viability was increased by addition of quercetin 1 and 50 μM and genestein 1 and 50 μM for 3 hr incubation. In addition, the sperm viability seemed to be increased dose-dependantly by addition of quercetin or genistein 1 and 50 μM, respectively (p<0.05). The membrane integrities were not increased by quercetin or genistein treatments for 3 hr or 6 hr incubation period except for quercetin 1 μM for 3 hr incubation. In mitochondrial activities, addition of quercetin 50 μM for 6 hr incubation increased mitochondrial activity but decreased at 100 μM concentration compared with control (p<0.05). When porcine IVF embryos were cultured in PZM-3 medium supplemented with low concentrations of quercetin (1∼10 μM), the developmental rates to morula and blastocyst increased but significantly decreased at high concentrations of quercetin (25∼50 μM). The highest developmental rate to blastocysts among all concentrations of quercetin was shown at quercetin 10 μM (p<0.05). The developmental rates to morula or blastocysts at low (0.01∼1 μM) and high (5∼10 μM) concentrations of genistein were not significantly different among all treatment group and genistein did not affect on IVF embryo development. These results suggest that quercetin and genistein seem to have positive effects at certain concentrations on sperm characteristics such as motility, viability and mitochondrial activity. In addition, low concentrations of quercetin (1, 5 and 10 μM) in this experiment, seem to have beneficial effect on porcine IVF embryo development but genistein did not affect on it at all given concentrations (0.01∼10 μM).

To investigate the biochemical nature of changes in vaginal physiology during estrus and pregnancy, we examined the cytology and viscosity, and monitored the protein expression profile in vaginal mucus during estrus and pregnancy. The viscosity progressively decreased from estrus to pregnancy. Cell type analysis revealed that white blood cells progressively increased from estrus to pregnancy, while red blood cells progressively decreased during pregnancy. The cornification index (CI) was higher in estrus than in pregnancy. Protein mass spectrumetry identified the presence of ribosome-binding protein 1, GRIP 1 (Glutamate receptor-interacting protein 1)-associated protein 1, DUF729 (Domain of unknown function729) domain-containing protein 1, prolactin precursor, dihydrofolatereductase, and MMP (Matrix metalloprotease)-9 in vaginal mucus. MMP-2 and MMP-9 proteins in the vaginal mucus were active throughout estrus and gestation, as measured by a gelatinase assay, but most abundant in the vaginal mucus on day 0 of estrus. Results from ELISA of MMP-2 and MMP-9 were in accordance with the gelatinase assay. In light of the crucial role of metalloproteinases in extracellular matrix remodeling, the level of MMP-9 in vaginal mucus might be useful as an indicator of estrus and pregnancy to increase the efficiency of reproduction.

The MTT assay is one of superior evaluation methods widely used to analyze the viability of metabolically active cell. It can be used to determine the percentage of viable sperm through measurement of the reduction of MTT granules at mitochondria in sperm tail. The purpose of this study is to determine the optimal condition of a simple and easy MTT assay to validate boar sperm viability and compare the accuracy of this test with microscopic examination. The MTT reduction rate for sperm viability were analyzed in microtiter plates (96 well) from 1 hr to 5 hr incubation periods at using spectrophotometer (microplate reader) at 550 nm wavelength. The remainder of semen sample was simultaneously examined to compare the correlation of accuracy between MTT assay and other sperm parameters. Those sperm parameters were included the motility, survival rates, membrane integrity, mitochondria activity and acrosome integrity. The OD values of MTT assay (MTT reduction rates) did not greatly change at 1 hr to 5 hr incubation periods in different proportion of live and freeze-killed sperms (dead sperm). The MTT reduction rates or survival rates were decreased according to the different concentration of live and dead sperm. The linear regression at 1 hr and 4 hr incubation periods in sperm MTT assay was y=291.55x-72.176 and y= 180.64x-44.569, respectively. There are high correlation between 1 hr and 4 hr incubation periods (p<0.001). The results of MTT assay and other sperm parameters has a positive correlation (p<0.01 or 0.05). The correlation coefficients for MTT assay was 0.88115 for motility, 0.89868 for survival rates, 0.91722 for membrane integrity and 0.77372 for acrosome integrity, respectively. In conclusion, the MTT assay can be used as a reliable and efficient evaluation method for boar sperm viability. It can be use practical means to evaluate the quality of boar sperm by a fast, inexpensive and easy method.

Serial ultrasonography was performed to measure the normal appearance of uterine during estrous cycle and to determine whether the unterine appearance was related to the sex hormone, progesterone and estrogen. The uterine appearances, shape, diameter and echogenicity were daily monitored with ultrasonography in 9 Miniature Schnauzer dogs undergoing II estrous cycles. During proestrus and estrous, the uterus became hypoechoic but developed hyperechoic luminal echo. In the longitudinal view, the shape of the uterus occasionally changed from rectangular to coiled or serpentine, compared to other stages of the cycle. The diameter of the uterus during proestrus and estrous was larger (range: 0.600.86 mm) than other stages (range: 0.480.62 mm) of the cycle. The rising estrogen concentrations (range: 14.5116.86 pg/ml) in plasma during proestrus correlated with changes in the uterus (p<0.05). Progesterone concentrations were 0.080.15 ng/ml at the onset of proestrus, but rose 1.061.26 ng/ml at the end of proestrus. There was no relation to progesterone concentration from onset of estrus (p>0.05). There was dramatical changes in normal uterus and sex hormone during estrous cycle. Especially, the appearance, shape and diameter of uterus were related to plasma estrogen concentration during proestrus, correlated with other stages of the cycle.