Cleaved PARP‐1, an Apoptotic Marker,can be Detected in Ram Spermatozoa

The presence of apoptotic features in spermatozoa has been related to lower quality and functional impairment. Members of the poly‐ADP‐ribose polymerases (PARP) familyare involved in both DNA repair and apoptosis, playing important roles in spermatogenesis. Poly‐ADP‐ribose polymerase can be cleaved by caspases, and the presence of its cleavage product (cPARP) in spermatozoa has been related to chromatin remodelling during spermatogenesis and to the activation of apoptotic pathways. There are no reports on immunodetection of cPARP in ram spermatozoa; thus, we have tested a commercially available antibody for this purpose. cPARP was microscopically detected in the acrosomal ridge of some spermatozoa (indirect immunofluorescence). A preliminary study was carried out by flow cytometry (direct immunofluorescence, FITC). Ram semen was extended in TALP and incubated for 4 h with apoptosis inducers staurosporine (10 μm ) or betulinic acid (200 μm ). Both inducers and incubation caused a significant increase in cPARP spermatozoa (0 h, control: 21.4±3.3%, inducers: 44.3±1.4%; 4 h, control: 44.3±2.4%, inducers: 53.3±1.4%). In a second experiment, we compared the sperm fractions after density gradient separation (pellet and interface). The pellet yielded a slightly lower proportion of cPARP spermatozoa (28.5±1.2% vs 36.2±2.0% in the interface; p < 0.001), and a 12‐h incubation increased cPARP similarly in both fractions (p < 0.001). cPARP seems to be an early marker of apoptosis in ram semen, although its presence in untreated samples was weakly related to worse quality (pellet/interface). We suggest to study the relationship of PARP and cPARP levels with between‐male differences on sperm fertility.     

Effects of Serum Deprivation and Cycloheximide on Cell Cycle of Low and High Passage Porcine Fetal Fibroblasts

Arrest of cells in G0/G1 cell cycle phase is desired for nuclear transfer procedures. Serum starvation and cell cycle inhibitors are different ways to induce synchronization of the cell cycle. This study investigated the effects of serum starvation and cycloheximide (CHX) on the cell cycle of low (5th) and high (15th) passages fetal porcine fibroblasts. Cell cycle phases were determined using fluorescent activated cell sorting. Fifth passage fibroblast cultures had higher (p < 0.05) proportion of cells in G0/G1 only after 72 h of serum starvation (77.60 ± 0.65) when compared with non‐starved cells (71.44 ± 1.88). Serum starvation for all periods tested induced an increase (p < 0.05) on proportion of cells in G0/G1 on the 15th passage. No significant differences were observed on the 5th passage cultures exposed to CHX, although, on the 15th passage an increase on proportion of cells was observed after all periods of exposure (p < 0.05). These data indicates that high passage cells in vitro are more susceptible to serum starvation and CHX G0/G1 synchronization.     

Influence of Ascorbic Acid and Glutathione Antioxidants on Frozen-Thawed Canine Semen

Poor sperm viability post-thaw has resulted in constant research into methods of cryopreservation of canine semen. One factor that may be involved in poor viability is sperm oxidative stress caused by excessive formation of reactive oxygen species. The present study was performed in order to evaluate the effect of different concentrations of ascorbic acid (AA) and glutathione (Glu) added to an extender for the freeze-thawing of dog sperm. Semen from five mature dogs was collected and frozen in two studies. Prior to and after freezing, sperm motility, morphology and membrane status were examined. In addition, sperm motility was examined up to 120 min after thawing to evaluate thermo-resistance. In study I, semen was collected twice from each dog. On both occasions, semen was divided into three aliquots: control, Glu 1 m m and Glu 5 m m . In study II, semen was collected twice and divided into three aliquots; control, AA 50 μ m and AA 250 μ m . Initial sperm motility was significantly higher in sperm diluted with AA 50 μ m ; sperm longevity, however, measured by a thermal-resistance test (TRT), was higher for Glu treatments. Higher concentration of Glu produced significant improvement in TRT and membrane status, whereas higher concentration of AA had a negative impact in sperm longevity. Antioxidant supplementation to semen freezing extenders improved semen quality post-thaw. Moreover, Glu had the most beneficial effect when supplemented at 5 m m .     

GONG Observations of Solar Surface Flows

Doppler velocity observations obtained by the Global Oscillation Network Group (GONG) instruments directly measure the nearly steady flows in the solar photosphere. The sun's differential rotation is accurately determined from single observations. The rotation profile with respect to latitude agrees well with previous measures, but it also shows a slight north-south asymmetry. Rotation profiles averaged over 27-day rotations of the sun reveal the torsional oscillation signal-weak, jetlike features, with amplitudes of 5 meters per second, that are associated with the sunspot latitude activity belts. A meridional circulation with a poleward flow of about 20 meters per second is also evident. Several characteristics of the surface flows suggest the presence of large convection cells.     

Effects of stocking on the genetic structure of brown trout,Salmo trutta,in Central Europe inferred from mitochondrial and nuclear DNA markers

Abstract Stocking has had a considerable effect on wild brown trout, Salmo trutta L., populations throughout Europe. To elucidate this impact and to outline further management strategies, the genetic structure of 25 wild populations and five hatchery stocks from Czech Republic and Slovakia were analysed using mitochondrial (control region) and nuclear DNA (microsatellites, LDH‐C1*) markers. Stocking practices have caused massive hybridisation between the Atlantic and Danube brown trout strains in the central Danube basin and have lead to a loss of among‐population divergence in Slovakia and the eastern part of Czech Republic. Comparison with studies from neighbouring countries revealed substantial differences in haplotype, allele frequencies and genetic diversity across Central Europe. Differences in stocking management and origin of breeding stocks appear to be crucial factors for the spatial variability of the genetic structure of brown trout.     

Some Factors Affecting the Abortion Rate in Dairy Herds with High Incidence of Neospora‐Associated Abortions are Different in Cows and Heifers

The aim of this study was to determine if the factors affecting the abortion rate in dairy herds with high incidence of Neospora‐associated abortions are different in pregnancies of cows and heifers chronically infected with Neospora caninum. In heifers (n = 229), an increase in the cumulative number of days with a mean relative humidity (RH) lower than 60% during the second trimester of gestation increases the risk of abortion. Yet, the likelihood of abortion was 7.6 times lower for pregnant heifers inseminated with Limousin bull semen, compared with those inseminated with Holstein‐Friesian bull semen. In pregnancies of parous cows (n = 521), an increase in rainfall and in the cumulative number of days with a mean RH lower than 60% during the second trimester of gestation increased the abortion rate. However, in contrast, an increase in the lactation number produced a decrease in the abortion rate, with a likelihood of abortion 4.8 times lower for pregnant cows inseminated with Limousin bull semen, and three times lower for those inseminated with Belgian Blue bull semen, compared with dairy cows inseminated with Holstein‐Friesian bull semen. Finally, the likelihood of abortion was 3.2 times lower for pregnancies of parous cows with low antibody titres against N. caninum (6–30 units) as compared to those with high antibody titres (≥30 units), whereas in heifers this variable had no effect. The practical recommendations of the present study include the control of the cow environment during the second trimester of gestation, the priority of culling for parous cows with higher antibody titres against N. caninum and the insemination of Neospora‐seropositive cows with semen from the Limousin breed.     

Single layer centrifugation of fresh dromedary camel semen improves sperm quality and in vitro fertilization capacity compared with simple sperm washing

Single layer centrifugation (SLC) through a colloid is a tool for selecting viable mammalian spermatozoa but has not been used previously for fresh dromedary camel sperm. Semen from six camels (2 ejaculates/male) was diluted 1:5 (v:v) or 1:10 (v:v) in a Tris–citrate–fructose buffer for mechanical liquefaction by gentle pipetting. Following liquefaction, semen was processed either by SLC or by centrifugation without a colloid (control). Total and progressive motilities, CASA kinematics, vitality and acrosome integrity (eosin–nigrosin) and plasma membrane integrity (Hypo‐osmotic swelling test; HOST), and fertilizing ability in a heterologous assay (zona‐free goat oocytes) were evaluated. Both total (p = .003) and progressive motilities (p = .003) were higher in SLC‐processed than in control semen samples, irrespective of dilution. Positive HOST values increased when using colloid in 1:5 (p = .001) and 1:10 dilution (p = .010). Colloid‐selected sperm had higher penetration rates than controls (p < .001 and p = .02 for 1:5 and 1:10 dilutions, respectively). However, only the SLC sperm at 1:5 dilution showed higher percentages of pronuclear formation (p = .02) than controls. Dilution effect was only significant for total motility before in vitro fertilization, with higher values for the 1:5 dilution (p = .033). The recovery rates of motile sperm between dilutions were similar (26.1% vs 35.4%; p = .226). In conclusion, SLC is a promising tool for selecting functional dromedary camel sperm and warrants more research.