Redox cycling induces spermptosis and necrosis in stallion spermatozoa while the hydroxyl radical (OH•) only induces spermptosis

Oxidative stress is a major factor explaining sperm dysfunction of spermatozoa surviving freezing and thawing and is also considered a major inducer of a special form of apoptosis, visible after thawing, in cryopreserved spermatozoa. To obtain further insights into the link between oxidative stress and the induction of apoptotic changes, stallion spermatozoa were induced to oxidative stress through redox cycling after exposure to 2‐methyl‐1,4‐naphthoquinone (menadione), or hydroxyl radical formation after FeSO₄ exposure. Either exposure induced significant increases (p < 0.05) in two markers of lipid peroxidation: 8‐iso‐PGF_2α and 4‐hydroxynonenal (4‐HNE). While both treatments induced changes indicative of spermptosis (caspase‐3 activation and decreased mitochondrial membrane potential) (p < 0.01), menadione induced sperm necrosis and a dramatic reduction in motility and thiol content in stallion spermatozoa. Thus, we provided evidence that oxidative stress underlies spermptosis, and thiol content is a key factor for stallion sperm function.     

Seed longevity and dormancy of four summer annual grass weeds in turf

Digitaria sanguinalis, Eleusine indica, Setaria glauca and S. viridis are troublesome summer annual weeds in turf. For taking rational decisions on the necessity for the level and type of weed management, it is important to know when weeds are ready to emerge (dormancy status) and also how long weed seeds can survive in the soil. Seeds of these four species were buried 4.0–4.5 cm deep in steel mesh net bags placed under permanent turf and periodically exhumed for 3 years to evaluate viability and determine the dormancy/non‐dormancy cycle. D. sanguinalis, S. glauca and S. viridis showed the typical dormancy cycle of summer annual species, and their seed viability declined completely after 3 years of burial. In contrast, E. indica demonstrated unusual behaviour, with long persistence and no dormancy.     

In situ adherence of Vibrio spp. to cryosections of Atlantic salmon, Salmo salar L., tissue.

Pathogenic and presumed non-pathogenic bacteria isolated from fish were tested for their adhesion to cryosections from different mucosal surfaces of Atlantic salmon, Salmo salar L. Adhered bacteria were detected by immunohistochemistry. Mucus was stained and fixed with Alcian blue after incubation of bacteria. The majority of the bacteria tested, i.e. Vibrio anguillarum serotype O1 , Vibrio salmonicida , Vibrio viscosus, Flexibacter maritimus and 'gut vibrios', i.e. Vibrio iliopiscarius and intestinal isolates of V. salmonicida , all adhered to mucus on all salmon epithelial surfaces tested, including sections from the foregut, hindgut, pyloric caeca, gills and skin. In contrast, V. anguillarum serotype O2, including both serotypes O2a and O2b, did not adhere to mucus, but did adhere to all other components of the tissues. As a positive control for adhesion of bacteria on cryosections, Escherichia coli was bound to piglet ileal mucosal lining, and as a negative control for adhesion, Staphylococcus aureus was found not to bind to any of the tissues tested. The present study shows that adhesion to mucus was not restricted to pathogenic bacteria, and furthermore, that not all pathogenic bacteria studied adhered to mucus. Hence, on the basis of these findings, the present authors suggest that V. anguillarum O2 may have an invasion strategy which does not involve adhesion to mucus, and thus, differs from the other pathogenic bacteria in the present study, which all bound to salmon mucus.     

Flow cytometry in Spermatology: A bright future ahead

Techniques such as mass spectrometry have led to unprecedented knowledge of the proteins that are present in the spermatozoa of humans and other mammals. However, in spite of their high‐throughput and fractioning techniques, most of the techniques in use only offer average values for the entire sperm population. Yet, ejaculate is very heterogeneous, and average values may mask relevant biological information.The application of flow cytometry may overcome this disadvantage, allowing proteomic analysis at the single‐cell level. Moreover, recent advances in cytometry, allowing multiple analyses within a single cell combined with powerful statistical tools, as an expanding subfield in spermatology, are described. The increased use of advanced flow cytometers in andrology laboratories will allow the rapid development of multiparametric, multicolour flow cytometry in andrology that will expand the clinical applications and research possibilities of flow cytometry‐based proteomic approaches, especially in the subfields of clinical andrology and sperm biotechnology.     

Maya Blue Paint: An Ancient Nanostructured Material

Maya blue paint was often used in Mesoamerica. The origin of its color and its resistance to acids and biocorrosion have not been fully understood. High-resolution transmission electron microscopy, electron energy loss spectroscopy, and x-ray microanalysis studies of authentic samples show that palygorskite crystals in the paint form a superlattice that probably occurs as a result of mixing with indigo molecules. An amorphous silicate substrate contains inclusions of metal nanoparticles encapsulated in the substrate and oxide nanoparticles on the surface. The beautiful tone of the color is obtained only when both the particles and the superlattice are present.     

The reversal of xylazine/ketamine immobilisation of fallow deer with yohimbine

Fallow deer were immobilised using a combination of xylazine and ketamine. Adult males (n = 10) and adult females (n = 10) received 4 mg/kg of each drug intramuscularly. Juveniles (n = 11) received 2 mg/kg of each drug, intravenously. Times to recumbency were as follows: adult males 4.9 +/- 2.9 min, adult females 4.1 +/- 1.9 min, juveniles 2.3 +/- 1.1 min. After 30 min each deer received 0.2 mg/kg of yohimbine, or an equal volume of sterile diluent intravenously. Yohimbine substantially reduced the recovery times of treated deer. Adults males were releasable 7.2 +/- 4.3 min after yohimbine administration, whereas control males were not releasable until 165 +/- 18 min. Treated adult females were releasable after 6.6 +/- 4.3 min, while control females were not releasable until 84 +/- 29 min. Juveniles were releasable 2.1 +/- 0.8 min after administration of yohimbine but control juveniles were not releasable until 62 +/- 16 min. Xylazine/ketamine administration produced statistically significant changes in packed cell volume, total plasma protein, albumin, sodium, glucose, creatine phosphokinase and inorganic phosphate values after 30 min. Yohimbine administration had no effect on these changes.     

Secondary Corpora Lutea Induced by hCG Treatment Enhanced Demi‐Embryo Survival in Lactating High‐Yielding Dairy Cows

Using a novel in vivo model considering a low developmental competence embryo (demi‐embryo) and a subnormal fertility recipient (lactating high‐yielding dairy cow), this experiment evaluated the effect of human chorionic gonadotrophin (hCG) treatment at embryo transfer (ET) on embryonic size at implantation, embryonic survival and recipient plasma progesterone (P₄) and bovine pregnancy‐specific protein B (PSPB) concentrations until day 63 of pregnancy. Embryos were bisected and each pair of demi‐embryos was bilaterally transferred to recipients (n = 61) on day 7 of the oestrous cycle. At ET recipients were randomly assigned to treatment with 1500 IU hCG or to untreated controls. Higher (p < 0.01) pregnancy rates on days 25, 42 and 63, and embryo survival rate on day 63 were observed in hCG‐treated cows with secondary CL than in hCG‐treated cows without secondary CL and in untreated cows. Pregnancy rates and embryo survival rate were similar in hCG‐treated cows without secondary CL and untreated cows. Embryonic size on day 42 was not affected by treatment with hCG, presence of secondary CL and type of pregnancy (single vs twin). Presence of secondary CL increased (p < 0.05) plasma P₄ concentrations of pregnant cows on days 14, 19 and 25 but not thereafter and of non‐pregnant cows on days 14–21. Treatment with hCG and presence of secondary CL had no effect on plasma PSPB concentrations, which were higher (p < 0.05) in twin than in single pregnancies. In conclusion, secondary CL induced by hCG treatment at ET significantly increased plasma P₄ concentrations, the survival rate of demi‐embryos and the pregnancy rate of high‐yielding lactating dairy cows. Embryos were rescued beyond maternal recognition of pregnancy, but later embryonic survival, growth until implantation and placental PSPB secretion until day 63 of pregnancy were not affected by treatment or presence of secondary CL.     

The Effects of Cryopreservation on the Morphometric Dimensions of Iberian Red Deer (Cervus elaphus hispanicus) Epididymal Sperm Heads

Computer-automated sperm-head morphometry was used in this study to determine the effects of cryopreservation on red deer sperm-head morphometry. Epididymal sperm samples were collected from 40 mature stags and were divided. One portion was diluted at room temperature in a Tris-citrate egg yolk medium, containing 6% glycerol. A microscope slide was prepared from single extended sperm samples prior to freezing. The remainder of each sample was frozen in nitrogen vapours. After thawing, sperm smears were prepared as described above. All slides were air dried and stained with Hemacolor. The sperm-head dimensions for length, width, area, perimeter and shape factor (length/width), for a minimum of 135 spermatozoa were determined for each slide by means of the Sperm-Class Analyser (SCA). Firstly, our results show that cryopreservation substantially reduced (p < 0.001) sperm motility and plasma membrane and acrosome integrities. In addition, sperm heads were significantly smaller in cryopreserved spermatozoa than in the companion extended samples for area (32.05 microm2 vs 32.56 microm2; p < 0.05), length (8.46 microm vs 8.53 microm; p < 0.0001) and shape factor (1.833 vs 1.849; p < 0.0001) for all stags. These differences were found within 29 of 40 stags (75%) for at least three of the morphometric parameters. The individual variability (CV) of sperm head measurements from extended samples was negatively correlated (p < 0.005) with the per cent of change in sperm head measurements after cryopreservation for area (r = -0.465), width (r = -0.483) and perimeter (r = -0.375). Thus, the lower the sperm head variability in the extended samples, the greater the sperm change as a consequence of the cryopreservation. These results suggest that the variability (heterogeneity) in sperm head dimensions of individual stags may be a good indicator of sperm freezability.