In vitro studies of Norwegian Red bovine semen immobilized and cryopreserved in alginate solid gel network

Development of new semen cryopreservation techniques improving sperm survival and ensuring availability of viable spermatozoa for a prolonged time‐period after AI is promising tools to reduce sensitivity of timing of AI and enhance overall fertility. The SpermVital^® technology utilizes immobilization of bull spermatozoa in a solid network of alginate gel prior to freezing, which will provide a gradual release of spermatozoa after AI. The objective of this study was to compare post‐thaw sperm quality and in vitro sperm survival over time of Norwegian Red bull semen processed by the SpermVital^® (SV) technology, the first commercialized production line of SpermVital^® (C) and by conventional procedure applying Biladyl^® extender (B). Post‐thaw sperm motility was not significantly different between SV, C and B semen (p > .05). However, sperm viability and acrosome intactness were higher for SV than C and B semen (p < .05). Small differences in DNA quality were observed (p < .05). Sperm viability after storage in uterus ex vivo was higher for SV than for C semen (p < .05). Furthermore, sperm survival in vitro over time at physiological temperature was significantly higher for SV semen than C semen as well as B semen during the incubation period of 48 hr (p < .05). In conclusion, the SpermVital^® technology is improved and is more efficient in conserving post‐thaw sperm quality and results in higher sperm viability over time in vitro for SV than for C and B semen.     

Effects of five cryoprotectants on proliferation and differentiation‐related gene expression of frozen‐thawed bovine calf testicular tissue

The cryopreservation of testicular tissue is a potential method for preserving male fertility. However, the effect of cryopreservation on bovine calf testicular tissue is scarce. This study investigated the effect of different cryoprotectants on bovine calf testicular tissue at the molecular level. Testicular tissue from ten immature bovine calves (6 months) was collected after slaughter and cryopreserved in an extender containing different concentrations of the following five cryopreservation solutions (CP): bovine serum albumin (BSA) with 5% dimethyl sulfoxide (DMSO), trehalose with 5% DMSO, DMSO and glycerol and ethylene glycol (EG). After 7‐day cryopreservation, the expression levels of three spermatogonial stem cell (SSC)‐related genes, octamer‐4 (OCT4), KIT ligand (MGF/SCF) and kit oncogene (C‐KIT), were investigated by quantitative PCR (qPCR). The cell viability was highest for the tissues preserved with 30 mg/ml BSA (77.82% ± 1.22) and 40 mg/ml trehalose (74.23% ± 1.16) compared with other groups (p < 0.05), and the level of expression of the three genes was highest with 30 mg/ml BSA (p < 0.05). Compared with other CPs, the 30 mg/ml BSA and 40 mg/ml trehalose have the better cryopreserve protection. The 30 mg/ml BSA is the most viable media for the cryopreservation of testicular tissue from cattle.     

Viral skin diseases in the Netherlands

The most common viral skin problems are discussed in the third article in our equine dermatology series.     

Influence of the root endophyte Piriformospora indica on the plant water relations,gas exchange and growth of Chenopodium quinoa at limited water availability

This study aimed to evaluate the ability of Piriformospora indica to colonize the root of Chenopodium quinoa and to verify whether this endosymbiont can improve the growth, performance and drought resistance of this species. The study delivered, for the first time, evidence for successful colonization of P. indica in quinoa. Hence, pot experiment was conducted in the greenhouse, where inoculated and non‐inoculated plants were subjected to ample (40%–50% WHC) and deficit (15%–20%WHC) irrigation treatments. Drought adversely influenced the plant growth, leading to decline the total plant biomass by 74%. This was linked to an impaired photosynthetic activity (caused by lower g_s and C_i/C_a ratio; stomatal limitation of photosynthesis) and a higher risk of ROS production (enhanced ETR/A_gross ratio). P. indica colonization improved quinoa plant growth, with total biomass increased by 8% (controls) and 76% (drought‐stressed plants), confirming the growth‐promoting activity of P. indica. Fungal colonization seems to diminish drought‐induced growth hindrance, likely, through an improved water balance, reflected by the higher leaf ψ_w and g_s. Additionally, stomatal limitation of photosynthesis was alleviated (indicated by enhanced C_i/C_a ratio and A_net), so that the threat of oxidative stress was minimized (decreased ETR/A_gross). These results infer that symbiosis with P. indica could negate some of the detrimental effects of drought on quinoa growth, a highly desired feature, in particular at low water availability.     

Evaluation of Cold Tolerance in NERICAs Compared with Japanese Standard Rice Varieties at the Reproductive Stage

New Rice for Africa (NERICA) is a general name for interspecific rice varieties derived from a cross between the high‐yielding Asian rice (Oryza sativa L.) between locally adapted African rice (Oryza glaberrima Steud.). Eight NERICAs were evaluated for cold tolerance (CT) at the reproductive stage and compared with their O. sativa parents and three Japanese standard rice varieties over 3 years. Cold tolerance was evaluated based on the filled grain ratio (FGR) after cold water irrigation. The FGR was greatly reduced by cold water irrigation. NERICA 1, 2 and 7 had higher FGR (51.9–57.9 %), while NERICA 6, 15 and 16 had lower FGR (6.2–14.5 %). NERICA 1, 2 and 7 were less affected by cold stress, with a 31 % mean reduction in FGR, while NERICA 6, 15 and 16 were greatly affected, with their FGRs being reduced by more than 80 %. NERICA 3 and 4 were moderately affected by cold stress, with about 45 % reduction rate in FGR. FGR significantly influenced the grain weights of the varieties with strong positive correlations (r = 0.83–0.91; P < 0.001), and thus, similar trends in grain weights were observed. Grain weights were reduced by 61.7–96.4 % under cold stress. NERICA 1, 2 and 7 showed significantly better performance than NERICA 3 and 4, while NERICA 6, 15 and 16 performed poorly under cold water irrigation. The Japanese varieties Koshihikari (very tolerant) and Ozora (moderately tolerant) were more affected by cold water irrigation than NERICA 1, 2 and 7. On the basis of the mean reduction rate (%) in FGR under cold stress, the varieties were classified as follows: NERICA 1, 2 and 7 as tolerant; NERICA 3 and 4 as moderately tolerant; and NERICA 6, 15 and 16 as susceptible to cold stress. However, NERICA 7 grain yields were lower under cold stress due to both greatly reduced number of panicles per plant and number of spikelets per panicle. Therefore, NERICA 1 and 2 are suitable candidates for production in the highland regions of East Africa and should be promoted for production.     

Black carbon estimation in French calcareous soils using chemo‐thermal oxidation method

We studied the black carbon (BC) content of ca. 405 samples from French topsoil and artificial soil and carbonate mixtures. Our protocol involved three main steps: (i) decarbonation by HCl, (ii) elimination of non‐pyrogenic organic carbon in a furnace at 375 °C, and (iii) quantification of residual carbon by CHN analysis. BC content increased for calcareous soils according to their carbonates content. Subsequent analyses confirmed the existence of a methodological artefact for BC determination only in calcareous soils. Decarbonation changes the thermal properties of organic matter, creating more recalcitrant carbon than in the initial sample. Higher CaCO₃ and organic carbon content results in a more pronounced artefact. The reversal of the first two steps of the chemo‐thermal oxidation method (i.e. thermal oxidation before soil decarbonation) eliminates this artefact. Overall, our results suggest that BC content may have been overestimated in a large number of studies on calcareous soils.     

Copper‐Induced Oxidative Stress and Responses of the Antioxidant System in Roots of Medicago sativa

The effects of various copper (Cu) concentrations on the antioxidative system in the roots of Medicago sativa were explored. The results indicated that the Cu content of the roots reached a value of 854 μg g^?1 DW at 10 μm Cu and a value of 4415 μg g^?1 DW at 100 μm Cu, suggesting that M. sativa has better ability to tolerate and accumulate Cu than other Cu‐bioaccumulators, and is a potential plant for phytoremediation. Treatment with Cu resulted in a significant increment in the levels of H₂O₂, O₂˙^? and OH˙. The reduced form of ascorbate and glutathione reached a peak at 30 μm Cu, and was followed by a sharp depletion to a lower level than that of the control. In contrast, the levels of the oxidised forms of ascorbate and glutathione showed a progressive increment with increasing Cu concentrations, suggesting that the antioxidant system was unable to cope with Cu stress at higher Cu levels. Under the Cu concentrations tested, the activity of catalase (CAT, EC 1.11.1.6), ascorbate peroxidase (APX, EC 1.11.1.11) and glutathione reductase (GR, EC 1.6.4.2) increased at lower Cu concentrations, and then decreased, reaching a maximum at 30 μm of Cu for APX and GR, at 10 μm for CAT, whereas the activities of guaiacol peroxidase (POD, EC 1.11.1.7) were gradually increased with increasing Cu concentrations. PAGE analysis of superoxide dismutase (SOD, EC 1.1.5.1.1) revealed that one band is a Mn‐SOD and five bands are identified as Cu, Zn‐SOD, whereas Fe‐SOD isoforms were not found in the roots of alfalfa. Cu at 10–100 μm increased the intensity of constitutive isozymes of CAT, APX and POD, whereas it decreased the intensity of isozymes of glucose‐6‐phosphate dehydrogenase (G6PDH, EC 1.1.1.49) significantly. The activities of lipoxygenases (LOX, EC 1.13.11.12) were gradually augmented with increasing Cu concentrations, demonstrating that LOXs are probably involved in production of lipid hydroperoxides and superoxide anion. There was a continuous and pronounced enhancement in the activity of esterase (EST, EC 3.1.1.1) in roots treated with 10–30 Cu μm , whereas EST activity in roots exposed to above 30 μm Cu declined, suggesting that EST plays a protective role under lower Cu concentrations stress.