Effects of α-tocopherol and freezing rates on the quality and heterologous in vitro fertilization capacity of stallion sperm after cryopreservation.

The effects of supplementation of α-tocopherol and different freezing rates (FRs) on the ability of stallion sperm to fertilize bovine oocytes with intact zona pellucida were investigated, in an attempt to develop a model to assess cryopreserved sperm function. Semen was obtained from four purebred Lusitano stallions (n = 4). Each ejaculate was subjected to cryopreservation with a commercial extender (Ghent, Minitub Iberia, Spain), without any supplementation (control) or supplemented with 2-mM α-tocopherol. The semen was exposed to two different FRs between 5 °C and -15 °C: slow (5 °C/min) and moderate (10 °C/min). After thawing, the viability (SYBR®-14 and propidium iodide [PI]), mitochondrial membrane potential (JC-1, 5,5',6,6'-tetrachloro-1,1',3,3'tetraethylbenzimidazolyl carbocyanine iodine) and membrane lipid peroxidation (C11-BODIPY(581/591)) of each sample were determined by flow cytometry. Moreover, the heterologous IVF rate was measured to evaluate the fertilization capacity of postthaw semen in the four different treatments. For both extenders, the viability was higher for spermatozoa cooled slowly (39.40 ± 2.17 vs. 17.59 ± 2.25-control; 31.96 ± 2.19 vs. 11.46 ± 1.34-Tocopherol; P < 0.05). The α-tocopherol extender improved (P < 0.05) postthaw lipid peroxidation (10.28 ± 0.70 vs. 15.40 ± 0.95-slow FR; 10.14 ± 0.40 vs. 13.48 ± 0.34-moderate FR); however, it did not improve viability and mitochondrial membrane potential. Regarding the IVF rate, in the moderate FR, α-tocopherol supplementation reported a higher percentage of IVF (20.50 ± 2.11; P < 0.05), comparing with the control (14.00 ± 1.84). Regarding the slow FR, no significance differences were observed for percentage of IVF between the two extenders and the FRs. However, it seems that the α-tocopherol supplementation improved the IVF rate. In conclusion, this research reported that bovine oocytes intact zona pellucida can be used to evaluate the quality of postthaw stallion semen and α-tocopherol supplementation in the stallion freezing extender might exert a protective effect against oxidative damage during heterologous IVF.

8,8''-Biapigeninyl stimulates osteoblast functions and inhibits osteoclast and adipocyte functions: Osteoprotective action of 8,8''-biapigeninyl in ovariectomized mice.

8,8''-Biapigeninyl (BA), a condensation product of two apigenin molecules, is found abundantly in the nuts of Cupressus sempervirens. We investigated the effects of BA on murine bone cells in vitro and in ovariectomized (OVx) mice. BA at 10(-10)M and 10(-8)M, inhibited osteoclastogenesis of bone marrow cells (BMCs) and displayed concentration dependence. BA at 10(-8) M and 10(-6) M inhibited differentiation of 3T3-L1 and BMCs to mature adipocytes. BA (10(-10)M) stimulated osteoblast proliferation, differentiation and mineralization. In stimulating osteoblast function, BA was found to be 10(4)-fold more potent than apigenin. The effect of BA in osteoblasts appeared to be mediated via estrogen receptors (ER) as antiestrogen, ICI-182780 abolished BA-stimulated osteoblast differentiation. In OVx mice BA treatment (at 1.0-, 5.0- and 10.0 mg kg(-1) day(-1) doses) given orally for 30 days dose-dependently inhibited mRNA levels of osteoclastic genes including tartrate-resistant acid phosphatase, receptor activator of nuclear factor (RANK), tumor necrosis factor alpha, interleukin-6 and the ratio of RANK ligand/osteoprotegerin ratio in bones compared with OVx mice treated with vehicle. In addition, BA treatment to OVx mice dose-dependently stimulated production of osteoprogenitor cells in the bone marrow and increased mRNA levels of osteogenic genes core binding factor alpha-1, type I collagen and bone morphogenic protein-2 in bones compared with OVx+vehicle group. Microcomputed tomography revealed that BA treatment to OVx mice improved parameters of trabecular and cortical architecture. BA exhibited no uterine estrogenicity. From these data, we conclude that BA exerts osteoprotective effect in OVx mice by multiple beneficial effects on bone cells.