Melatonin modulates the functions of porcine granulosa cells via its membrane receptor MT2 in vitro.

Melatonin (N-acetyl-5-methoxytryptamine) is documented as a hormone involved in the circadian regulation of physiological and neuroendocrine function in mammals. Herein, the effects of melatonin on the functions of porcine granulosa cells in vitro were investigated. Porcine granulosa cells were cultivated with variable concentrations of melatonin (0, 0.001, 0.01, 0.1, 1.0, and 10ng/mL) for 48h. Melatonin receptor agonist (IIK7) and antagonist (Luzindole, 4P-PDOT) were used to further examine the action of melatonin. The results showed optimum cell viability and colony-forming efficiency of porcine granulosa cells at 0.01ng/mL melatonin for 48-h incubation period. The percentage of apoptotic granulosa cells was significantly reduced by 0.01 and 0.1ng/mL melatonin within the 48-h incubation period as compared with the rest of the treatments. Estradiol biosynthesis was significantly stimulated by melatonin supplementation and suppressed for the progesterone secretion; the minimum ratio of progesterone to estradiol was 1.82 in 0.01ng/mL melatonin treatment after 48h of cultivation. Moreover, the expression of BCL-2, CYP17A1, CYP19A1, SOD1, and GPX4 were up-regulated by 0.01ng/mL melatonin or combined with IIK7, but decreased for the mRNA levels of BAX, P53, and CASPASE-3, as compared with control or groups treated with Luzindole or 4P-PDOT in the presence of melatonin. In conclusion, the study demonstrated that melatonin mediated proliferation, apoptosis, and steroidogenesis in porcine granulosa cells predominantly through the activation of melatonin receptor MT2 in vitro, which provided evidence of the beneficial role of melatonin as well as its functional mechanism in porcine granulosa cells in vitro.

Effect of Salvia miltiorrhiza polysaccharides on boar spermatozoa during freezing-thawing.

Salvia miltiorrhiza polysaccharides (SMPs) were extracted from S. miltiorrhiza in this study. The aim of the present study was to evaluate the effect of SMP on the motility of boar sperm, including the antioxidant effect of SMP on boar sperm and the effect of SMP on the in vivo fertilizing ability of frozen-thawed boar sperm. Fifty ejaculates from 5 Swagger boars were collected and diluted with an extender, which contained 3% glycerol (v/v) with five concentrations of SMP (0.2, 0.4, 0.6, 0.8, and 1.0mg/mL). The semen was frozen in 0.25mL straws at 1.0×10(9) cells/mL. Sixty gilts were inseminated using fresh semen, frozen semen with 0.4mg/mL of SMP and frozen semen without SMP. The results indicate that the addition of SMP to the extender results in a higher percentage of motile sperm post-thaw (P<0.05). The activities of superoxide dismutase, lactate dehydrogenase, glutamic-oxalacetic transaminease and catalase were all determined to be significantly higher than the control group after adding SMP to the extender (P<0.05). The artificial insemination (AI) results demonstrated that the litter size was significantly higher in the 0.4mg/mL of SMP group than in the control group (P<0.05). In conclusion, during the process of freezing, SMP can protect boar sperm from peroxidative damage and increase sperm motility and litter size during the process of freezing-thawing. The optimal concentration of SMP for the frozen extenders in this study was determined to be 0.4mg/mL.

The advantages of low-density lipoproteins in the cryopreservation of bull semen.

Egg low-density lipoprotein (LDL) was added at concentrations (w/v) of 7%, 8% or 9% to the extenders used to freeze bull semen and its effects on seminal parameters and anti-oxidant activities of frozen-thawed sperm were assessed. Analysis of data showed that sperm exposed to 8% LDL exhibited the greatest percentages of sperm motility, acrosome integrity and membrane integrity, compared to the control which differed from the treatment groups by replacing LDL with 20% egg yolk (P<0.05). No difference was observed for membrane integrity between 8% and 9% LDL groups (P>0.05). The extender supplemented with LDL did not exhibit improvement in SOD levels. However, 8% LDL group favored the highest anti-oxidant activities of CAT, GSH-Px and GSH in comparison to other groups (7%, 9% LDL and the control) (P<0.05). No difference was observed for CAT activity between 9% LDL and the control group. In conclusion, sperm cryopreserved in the extender containing 8% LDL in place of egg yolk exhibited the greatest percentages of post-thaw sperm motility, acrosome integrity and membrane integrity, in comparison with the control, and favored the highest anti-oxidant activities of CAT, GSH-Px and GSH in comparison with other groups. The replacement of egg yolk by LDL in the composition of extenders was beneficial for bull sperm cryopreservation.

Effect of Gynostemma Pentaphyllum polysaccharide on boar spermatozoa quality following freezing-thawing.

Gynostemma Pentaphyllum Polysaccharide (GPP) was added at concentrations of 0.25, 0.5, 1.0, 1.5 and 2.0 mg/ml to the extenders used to freeze boar semen and its effects on the quality of frozen-thawed sperm were assessed. The sperm motility was significantly higher in the extenders containing 0.25 and 0.5 mg/ml GPP, as compared to other groups (P<0.05). The extender supplemented with 0.5 mg/ml GPP favored the highest intact membrane and intact acrosome percentages in comparison with other groups (P<0.05), respectively. The mitochondrial activity was significantly higher at the concentrations of 0.25, 0.5 and 1.0 mg/ml GPP than that of other treatments, and the control group (P<0.05). In biochemical assays, the extender supplemented with 0.25 and 0.5 mg/ml GPP significantly improved SOD levels, compared to other groups (P>0.05). However, the extenders supplemented with GPP did not cause significant differences in levels of CAT and GSH-Px, compared to the control (P>0.05). In summary, GPP exhibited a dose-related response and the lower concentration produced greater protective effect. According to the standard semen quality parameters and antioxidant activities measured in this study, the concentration of 0.5 mg/ml GPP caused a beneficial cryoprotective effects on the quality of frozen-thawed boar semen. It is proposed that an extender containing 0.5 mg/ml GPP could be used as cryoprotective medium of better efficiency.