Flow Cytometric Chromosomal Sex Sorting of Stallion Spermatozoa Induces Oxidative Stress on Mitochondria and Genomic DNA

To date, the only repeatable method to select spermatozoa for chromosomal sex is the Beltsville sorting technology using flow cytometry. Improvement of this technology in the equine species requires increasing awareness of the modifications that the sorting procedure induces on sperm intactness. Oxidative stress is regarded as the major damaging phenomenon, and increasing evidence regards handling of spermatozoa – including sex sorting – as basic ground for oxidative damage. The aim of this study was to disclose whether the flow cytometric sorting procedure increases the production of reactive oxygen species (ROS), and to identify if ROS production relates to DNA damage in sorted spermatozoa using specific flow cytometry‐based assays. After sorting, oxidative stress increased from 26% to 33% in pre‐ and post‐incubation controls, to 46% after sex sorting (p < 0.05). Proportions of DNA fragmentation index post‐sorting were approximately 10% higher (31.3%); an effect apparently conduced via oxidative DNA damage as revealed by the oxyDNA assay. The probable origin of this increased oxidative stress owes the removal of enough seminal plasma due to the unphysiological sperm extension, alongside a deleterious effect of high pressure on mitochondria during the sorting procedure.     

Characterization of chickpea differentials for pathogenicity assay of ascochyta blight and identification of chickpea accessions resistant to Didymella rabiei

Forty-eight chickpea germplasm lines, including 22 differentials used in previous studies, were characterized for disease phenotypes following inoculation with six isolates of Didymella (anamorph Ascochyta ) rabiei , representing a wide spectrum of pathogenic variation. Representative isolates were also directly compared with six previously identified races on eight chickpea genotypes. Many of the chickpea differentials reacted similarly to inoculation with each isolate of D. rabiei , and several previously identified races caused similar levels of disease on the differentials. This indicates that the number of differentials can be reduced significantly without sacrificing accuracy in describing pathogenic variation of D. rabiei on chickpea. Pathogenic variation among samples of US isolates allowed classification of the isolates into two pathotypes. The distribution of disease phenotypes of the 48 germplasm lines was bimodal after inoculation with pathotype I isolates, whereas the distribution of disease phenotypes was continuous after inoculation with pathotype II isolates. Such distinct distribution patterns suggest that chickpea plants employ different resistance mechanisms to each pathotype and that the two pathotypes may have different genetic mechanisms controlling pathogenicity. The advantages of using the two-pathotype system in assaying pathogenicity of the pathogen and in studying resistance mechanisms of the host are discussed. Three chickpea accessions, PI 559361, PI 559363 and W6 22589, showed a high level of resistance to both pathotypes, and can be employed as resistance sources in chickpea breeding programmes for resistance to ascochyta blight.     

Epigallocatechin‐3‐Gallate (EGCG) Reduces Rotenone Effect on Stallion Sperm–Zona Pellucida Heterologous Binding

Stallion spermatozoa are highly dependent on oxidative phosphorylation for ATP production to achieve normal sperm function and to fuel the motility. The aim of this study was to evaluate the response of equine sperm under capacitating conditions to the inhibition of mitochondrial complex I by rotenone and to test whether epigallocatechin‐3‐gallate (EGCG), a natural polyphenol component of green tea, could counteract this effect. After 2‐h incubation of stallion spermatozoa in modified Tyrode's medium, rotenone (100 nm , 500 nm and 5 μm ) and EGCG (10, 20 and 60 μm ), alone or in combination, did not induce any significant difference on the percentage of viable cells, live sperm with active mitochondria and spermatozoa with intact acrosome. The inhibition of complex I of mitochondrial respiratory chain of stallion sperm with rotenone exerted a negative effect on heterologous ZP binding ability. EGCG at the concentrations of 10 and 20 μm (but not of 60 μm ) induced a significant increase in the number of sperm bound to the ZP compared with that for control. Moreover, when stallion sperm were treated with rotenone 100 nm , the presence of EGCG at all the concentrations tested (10, 20 and 60 μm ) significantly increased the number of sperm bound to the ZP up to control levels, suggesting that this green tea polyphenol is able to reduce the toxicity of rotenone.     

Mining wild barley for powdery mildew resistance

Powdery mildew, caused by Blumeria graminis f. sp. hordei (Bgh), is a worldwide disease problem on barley (Hordeum vulgare) with potentially severe impact on yield. Historically, resistance genes have been identified chiefly from cultivated lines and landraces; however, wild barley (H. vulgare subsp. spontaneum) accessions have proven to be extraordinarily rich sources of powdery mildew resistance. This study describes the characterization of a collection of 316 wild barley accessions, known as the Wild Barley Diversity Collection (WBDC), for resistance to powdery mildew and the genetic location of powdery mildew resistance loci. The WBDC was phenotyped for reaction to 40 different Bgh isolates at the seedling stage and genotyped with 10 508 molecular markers. Accessions resistant to all 40 isolates of Bgh were not found; however, three accessions (WBDC 053, 085 and 089) exhibited resistance to 38 of the isolates. Gene postulation analyses revealed that many accessions, while resistant, contained none of the 12 genes present in the Pallas near‐isogenic lines Mla1, Mla3, Mla6, Mla7, Mla9, Mla12, Mla13, Mlk1, MlLa, Mlg, Mlat and Ml(Ru2), suggesting that the accessions carry novel genes or gene combinations. A genome‐wide association study of powdery mildew resistance in the WBDC identified 21 significant marker‐trait associations that resolved into 15 quantitative trait loci. Seven of these loci have not been previously associated with powdery mildew resistance. Taken together, these results demonstrate that the WBDC is a rich source of powdery mildew resistance, and provide genetic tools for incorporating the resistance into barley breeding programmes.     

Dimethylformamide Improves the In vitro Characteristics of Thawed Stallion Spermatozoa Reducing Sublethal Damage

A total of 42 ejaculates were used in the experiment; six ejaculates per stallion, obtained from seven Pure Spanish stallions (PRE), were split and frozen in freezing media with different concentrations and combinations of cryoprotectant (CPA): (i) Cáceres (skim milk based extender) containing 2.5% glycerol (2.5GL), (ii) Cáceres containing 1.5% glycerol and 1.5% dimethylformamide (1.5%GL–1.5%DMFA), (iii) Cáceres extender supplemented with 1.5% glycerol and 2.5% dimethylformamide (1.5%GL–2.5%DMFA) and (iv) Cáceres extender supplemented with 4% dimethylformamide (4%DMFA). After at least 4 weeks of storage in liquid nitrogen (LN), straws were thawed and semen analysed by computer‐assisted sperm analysis and flow cytometry (membrane lipid architecture (Merocyanine 540), integrity and sublethal damage (YoPro‐1) and mitochondrial membrane potential (JC‐1)). After thawing, better results were observed in samples frozen in 4%DMFA or in combinations of 1.5%GL–2.5%DMFA, in fact total motility increased by 16% in the 4%DMFA group compared to 2.5%GL (P < 0.05). Also, there was an increment in the percentage of progressive motile sperm in the 1.5%GL–2.5%DMFA group (9.8% 2.5GL vs 19% in the 1.5%GL–2.5%DMFA group p < 0.05); also, samples frozen in the 4%DMFA group had more intact (YoPro‐1 negative) sperm post‐thawing, 29.3% in 2.5%GL vs 36.7% in 4%DMFA group (p < 0.05). Membrane lipid architecture was not affected by any of the cryoprotectants tested, while samples frozen in 4%DFMA had a lower percentage of mitochondria with lower membrane potential. It is concluded that DMFA improves the outcome of cryopreservation of stallion spermatozoa mainly reducing sublethal cryodamage.     

Membrane Lipids of the Stallion Spermatozoon in Relation to Sperm Quality and Susceptibility to Lipid Peroxidation

Lipids were extracted from ejaculated spermatozoa from seven individual stallions to distinguish neutral lipids (NL) and polar lipids (PL) and determine their variation among stallions and their relationship with sperm quality and sperm susceptibility to lipid peroxidation. The isolated fatty acids were correlated with sperm quality (membrane integrity, mitochondrial membrane potential (ΔΨm) and expression of active caspases) and the sensitivity of the sperm plasma membrane to LPO. The miristic (C14: 0), palmitic (C16: 0), stearic (C18: 0) and oleic (C18: 1n9) acids were predominant among the NLs. Within the phospholipid fraction, the docosapentanoic acid (C22: 5n6) was dominant, albeit varying among stallions. Surprisingly, the percentage of polyunsaturated fatty acids was positively correlated with sperm quality and a low propensity for LPO, probably because these particular fatty acids provide a higher fluidity of the plasma membrane. The stallion showing the poorest sperm membrane integrity plus a high level of LPO in his ejaculate had a lower percentage (p < 0.05) of this fatty acid in his sperm plasma membranes.     

Urethane anesthesia in adult female rats: preliminary observations

Urethane is widely used as a rodent anesthetic in the laboratory setting, and is characterized as producing long‐lasting anesthesia. The purpose of this study was to evaluate the quality of anesthesia provided by a single dose of urethane based on the response to a noxious stimulus. If the quality of anesthesia was insufficient to prevent gross purposeful movement (GPM), isoflurane was also administered until no response to noxious stimulation occurred. Five adult Harlan Sprague Dawley rats (6 months of age, 250–300 g) were given urethane (1.4 g kg^?1 IP) and evaluated for 120 minutes post‐injection. If the rats became laterally recumbent by 20 minutes post‐injection, a large hemostat was positioned around the tail and the response to tail clamping was assessed. If no GPM occurred, an additional 20 minutes was allowed to elapse. If the rats were not laterally recumbent or GPM was present, they were placed in a chamber and isoflurane in oxygen was administered. Inspired isoflurane concentrations (ISO) were measured using a S/5 anesthetic gas analyzer (Datex‐Ohmeda Division, Helsinki, Finland) calibrated before each experiment with a standardized calibration gas mixture (DOT‐34 NRC 300/375 m 1014, Datex‐Ohmeda Division, Helsinki, Finland). A period of 20 minutes was allowed for equilibration to inspired ISO. The tail‐clamp stimulus was then re‐applied and the animal's response recorded. If GPM was absent, ISO was lowered by 10–20% and an additional 20 minute interval elapsed. In contrast, if GPM was present, ISO was increased by 10–20%. This procedure was repeated until the ISO required to prevent GPM was determined in duplicate. The position within the estrus cycle influenced pain thresholds in the rats. As such, a vaginal smear was prepared from each rat and the position in the estrus cycle was determined based on vaginal cytology. Rats were euthanatized at the end of the study period. All values were mean ± SD. Four rats became recumbent after urethane injection (time to recumbency: 45 ± 17 seconds). Of these, two rats (one estrus, one metestrus) did not require isoflurane supplementation for the duration of the study. The three remaining rats (two metestrus, one estrus) required isoflurane supplementation. The mean ISO required to prevent GPM was 0.26 ± 0.16%. Position within the estrus cycle did not appear to affect the animal's response to urethane. These results indicate that urethane anesthesia is not long lasting in all rats and provides variable quality of anesthesia. This is of particular concern in the laboratory setting where muscle relaxants are often administered to rats shortly after urethane injection.     

The Impact of Reproductive Technologies on Stallion Mitochondrial Function

The traditional assessment of stallion sperm comprises evaluation of sperm motility and membrane integrity and identification of abnormal morphology of the spermatozoa. More recently, the progressive introduction of flow cytometry is increasing the number of tests available. However, compared with other sperm structures and functions, the evaluation of mitochondria has received less attention in stallion andrology. Recent research indicates that sperm mitochondria are key structures in sperm function suffering major changes during biotechnological procedures such as cryopreservation. In this paper, mitochondrial structure and function will be reviewed in the stallion, when possible specific stallion studies will be discussed, and general findings on mammalian mitochondrial function will be argued when relevant. Especial emphasis will be put on their role as source of reactive oxygen species and in their role regulating sperm lifespan, a possible target to investigate with the aim to improve the quality of frozen–thawed stallion sperm. Later on, the impact of current sperm technologies, principally cryopreservation, on mitochondrial function will be discussed pointing out novel areas of research interest with high potential to improve current sperm technologies.     

Inheritance and linkage of a gene for resistance to race 4 of fusarium wilt and RAPD markers in chickpea

Several races of Fusarium oxysporum Schlechtend.:Fr f. sp. ciceris (Padwick) Matuo and K. Sato cause economic losses from wilting disease of chickpea ( Cicer arietinum L.). While the genetics of resistance to race 1 have been reported, little is known of the genetics of resistance to race 4. We undertook a study to determine the inheritance of resistance and identified random amplified polymorphic DNA markers (RAPDs) linked to the gene for resistance. For the investigation, we used 100 F₅ derived F₇ recombinant inbred lines (RILs) that had been developed from the cross of breeding lines C-104 x WR-315. Results indicated that resistance is controlled by a single recessive gene. The RAPD markers previously shown to amplify fragments linked to race 1 resistance also amplified fragments associated with race 4 resistance. The RAPD loci, CS-27₇₀₀, UBC-170₅₅₀ and the gene for resistance to race 4 segregated in 1:1 ratios expected for single genes. Both RAPD markers were located 9 map units from the race 4 resistance locus and were on the same side of the resistance gene. Our results indicated that the genes for resistance to race 1 and 4 are 5 map units apart. The need to determine the genomic locations of race specific resistance genes and the possibility that these genes are clustered to the same genomic region should be investigated. This revised version was published online in July 2006 with corrections to the Cover Date.     

Multi-atom resonant photoemission: A method for determining near-neighbor atomic identities and bonding

Measurements and theoretical calculations are reported for an interatomic multi-atom resonant photoemission (MARPE) effect that permits direct determination of near-neighbor atomic identities (atomic numbers). MARPE occurs when the photon energy is tuned to a core-level absorption edge of an atom neighboring the emitting atom, with the emitting level having a lower binding energy than the resonant level. Large peak-intensity enhancements of 33 to 105 percent and energy-integrated effects of 11 to 29 percent were seen in three metal oxides. MARPE should also be sensitive to bond distance, bonding type, and magnetic order, and be observable via the secondary processes of x-ray fluorescence and Auger decay.