Studies on the Intracellular Ca2+ Concentration of Frozen–Thawed Dog Spermatozoa: Influence of Equex from Different Sources, Two Thawing Diluents and Post-thaw Incubation in Capacitating Conditions

The addition of 0.5% (v/v) of Equex STM Paste (Nova Chemical Sales, Scituate Inc., MA, USA), whose active ingredient is sodium dodecyl sulphate (SDS), to a Tris–egg yolk extender was demonstrated to improve the longevity of frozen–thawed dog spermatozoa during in vitro incubation at 38°C. The aim of the first experiment was to compare the effects of two SDS‐containing compounds, Equex STM Paste and Equex Pasta (Minitüb, Tiefenbach, Germany), when added to a Tris–egg yolk based extender, on the post‐thaw longevity of dog spermatozoa, as well as on the intracellular Ca²⁺ concentration of spermatozoa, during post‐thaw incubation at 38°C. The post‐thaw sperm survival and longevity, as well as the quality of the sperm movement, were significantly better when using Equex STM Paste. Such prolonged sperm longevity, however, was associated to a higher intracellular Ca²⁺ concentration in a large subpopulation of the live spermatozoa. A second experiment was aimed to evaluate the effects of sperm dilution immediately post‐thaw with a Tris buffer containing glucose or fructose. The two Tris buffers were no different for any of the sperm parameters studied. The aim of a third experiment was to evaluate the sperm longevity, motility patterns and intracellular Ca²⁺ concentration of cryopreserved dog spermatozoa during post‐thaw incubation in capacitating conditions [canine capacitating medium (CCM) with or without 5 μg/ml of heparin]. Heparin had no significant effects on any of the sperm parameters evaluated. During the first 8 h of incubation, the majority of the live spermatozoa had a high intracellular Ca²⁺ content. However, after 8–10 h of incubation, it had significantly declined. The highest proportion of fast motile sperm, and the highest curvilinear velocity, average path velocity and amplitude of lateral head displacement for the total motile sperm were observed during the 2–4‐h incubation period. It was concluded that: (a) the addition of 0.5% (v/v) of Equex STM Paste to a Tris–egg yolk based extender significantly improved the post‐thaw longevity of dog spermatozoa, but the same concentration of Equex Pasta had no significant beneficial effects; (b) sperm dilution after thawing with a Tris buffer containing glucose or fructose made no difference in post‐thaw sperm longevity; (c) the addition of 5 μg/ml of heparin to CCM had no significant capacitating effects on frozen–thawed dog spermatozoa.     

Stereospecific analysis of phospholipid classes in skeletal muscle from rats fed different fat sources

The fatty acid (FA) and dimethylacetal profiles of the sn-1 and sn-2 positions of different phospholipid (PL) classes from skeletal muscle of rats as affected by dietary FA profiles were studied. Rats were fed either a control diet, an olive oil-enriched diet, or a sunflower oil-enriched diet. The FA composition of both positions of the studied PL classes was affected by diet to different extents. The FA composition of the sn-2 position of phosphatidylserine was the most influenced by diet, while phosphatidylinositol was less affected by dietary modification. The FA profile of phosphatidylcholine reflected consumed FA better than any other studied PL. Thus, olive oil rats showed higher oleic acid (C18:1 n-9) contents in both positions of phosphatidylcholine, and sunflower oil rats had higher proportions of arachidonic acid (C20:4 n-6) in the sn-1 position of this PL class. Dimethylacetals were scarcely affected by diet, and only the dimethylacetal composition of phosphatidylethanolamine showed significant modifications.     

DNA Status on Thawed Semen from Fighting Bull: A Comparison Between the SCD and the SCSA Tests

The assessment of sperm chromatin status is compulsory in a complete spermiogram. Here we applied the sperm chromatin structure assay (SCSA) and the sperm chromatin dispersion (SCD) test to assess the chromatin status of three fighting bulls. Cryopreserved semen (two straws/bull) were analysed by duplicate after thawing and after 6 h at 37°C with and without oxidative stress (1 m m FE²⁺). Results (SCD: percentage of spermatozoa with halo; SCSA: SD-DFI, %DFI and HDS) were analysed for differences between bulls and treatments, sensitivity and specificity (receiver operating characteristic curves) and repeatability (repeatability coefficients as 2SD of duplicate differences).%DFI for the three bulls was below 2% at 0 h, indicating no risk for fertility according to previous reports. It increased slightly for two of the bulls after FE²⁺ treatment (%DFI < 5%) and more pronouncedly for the other bull (C, %DFI∼10%), which merits further investigation. SCD rendered higher percentage of halos for bull C, but could not discriminate between samples with and without oxidizing treatment (AUC: 0.52). SCSA (%DFI) showed a high discriminating ability between treatments (AUC: 0.96). The repeatability coefficient was also higher for SCD (5.9) than for %DFI (1.8), indicating lower repeatability for SCD. Overall, %DFI might be the most useful parameter for assessing sperm chromatin on fighting bull. SCD might yield different information than SCSA, hence further research is warranted.     

Photopolymerization and Mass-Independent Sulfur Isotope Fractionations in Carbon Disulfide

Irradiation of gaseous carbon disulfide [CS2(g)] at 313 nanometers produces a dark brown aerosol of (CS2)x. Its thermal decomposition products include disulfur (S2), carbon monosulfide (CS), and (CS)x. The photopolymerization process is accompanied by a large mass-independent isotopic fractionation of sulfur (a 5 to 10 per mil sulfur-33 excess and a 61 to 84 per mil sulfur-36 deficit). Excess sulfur-33 has been observed in several classes of meteorites. Photochemical production of (CS2)x may be important in the origin and evolution of cosmochemical environments such as the presolar nebula, meteorites, asteroids, and planetary atmospheres.     

Comparison of the TBARS Assay and BODIPY C11 Probes for Assessing Lipid Peroxidation in Red Deer Spermatozoa

Several methods are used to measure lipid peroxidation (LPO) in spermatozoa. The objective of this study was comparing the thiobarbituric acid reactive species (TBARS) method and the BODIPY 581/591 C₁₁ (B581) and BODIPY 665/676 C₁₁ (B665) fluorescent probes to measure induced peroxidative damage in thawed epididymal spermatozoa from Iberian red deer. Samples from three males were thawed, pooled, diluted in PBS, incubated at room temperature and assessed at 0, 3, 6 and 24 h under different experimental conditions: Control, hydrogen peroxide (H₂O₂) 0.1 mm or 1 mm , or tert‐butyl hydroperoxide (TBH) 0.1 mm or 1 mm . LPO was assessed by the TBARS assay [malondialdehyde (MDA) detection] and by the fluorescence probes B581 and B665 (microplate fluorimeter and flow cytometry). Increasing MDA levels were only detectable at 1 mm of TBH or H₂O₂. Both fluorescence probes, measured with fluorometer, detected significant increases of LPO with time in all treatments, except Control. Flow cytometry allowed for higher sensitivity, with both probes showing a significant linear relationship of increasing LPO with time for all oxidizing treatments (p < 0.001). All methods showed a good agreement, except TBARS, and flow cytometry showed the highest repeatability. Our results show that both B581 and B665 might be used for LPO analysis in Iberian red deer epididymal spermatozoa, together with fluorometry or flow cytometry. Yet, the TBARS method offered comparatively limited sensitivity, and further research must determine the source of that limitation.     

Community engagement in biobanking: Experiences from the eMERGE Network

Advances in genomic technologies and the promise of "personalised medicine" have spurred the interest of researchers, healthcare systems, and the general public. However, the success of population-based genetic studies depends on the willingness of large numbers of individuals and diverse communities to grant researchers access to detailed medical and genetic information. Certain features of this kind of research - such as the establishment of biobanks and prospective data collection from participants' electronic medical records - make the potential risks and benefits to participants difficult to specify in advance. Therefore, community input into biobank processes is essential. In this report, we describe community engagement efforts undertaken by six United States biobanks, various outcomes from these engagements, and lessons learned. Our aim is to provide useful insights and potential strategies for the various disciplines that work with communities involved in biobank-based genomic research.     

Efficacy of intramammary treatment in unbred and primigravid dairy heifers

A total of 73 breeding-age and primigravid Jersey heifers in 4 herds was randomly allotted to treatment and control groups according to expected calving date. Thirty-five heifers were injected intramammarily with a nonlactating cow product containing penicillin/streptomycin. Thirty-eight heifers served as untreated controls. Of the 35 treated heifers, 34 (97.1%) were infected at time of treatment. In the untreated control group, all 38 heifers (100%) were infected at treatment time. At parturition, prevalence of intramammary infection in treated heifers decreased to 40%, whereas in the control group, prevalence remained about the same (97.4% of heifers). Prevalence of Staphylococcus aureus mastitis in treated heifers was reduced from 17.1% to 2.9% after treatment. In the control group, prevalence of S aureus mastitis decreased from 26.3% to 15.8%. Heifers treated during the second trimester of pregnancy had the greatest reduction in prevalence of mastitis and in somatic cell count at parturition, compared with controls. Findings indicated that intramammary treatment during pregnancy in primigravid heifers was effective in reducing prevalence of mastitis and somatic cell counts at parturition.     

Effects of Cryopreservation on Bull Spermatozoa Distribution in Morphometrically Distinct Subpopulations

Assisted sperm morphometry analysis (ASMA) was used in this study to determine the effects of cryopreservation on bull spermatozoa distribution in morphometrically distinct subpopulations. Ejaculates were collected from five bulls and were divided. One portion was diluted at 30 degrees C in a skim milk-egg yolk medium, containing 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 a minimum of 200 sperm heads were analysed from each sample by means of the Sperm-Class Analyser (SCA), and the mean measurements recorded. Our results showed that applying the ASMA technology and multivariate cluster analyses, it was possible to determine that three separate subpopulations of spermatozoa with different morphometric characteristics coexist in bull ejaculates (large, average and small spermatozoa). The mean values of each sperm head dimension among the three subpopulations of spermatozoa were significantly different (p < 0.001). Besides, there were significant (p < 0.001) differences in the distribution of these three sperm subpopulations between fresh and thawed samples. Thus, the percentage of representation of the subpopulation that includes those spermatozoa whose dimensions are the biggest, decreased from 52.06% in extended fresh samples to 15.51% in the thawed ones. Contrarily, the percent of representation of the subpopulation containing the smallest spermatozoa, increased from 8.70% in extended fresh samples to 34.04% in the thawed ones. In conclusion, the present study confirms the heterogeneity of sperm head dimensions in bull semen, heterogeneity that vary through the cryopreservation procedure.     

Bacterial Isolates Associated With Dystocia And Retained Placenta In Iraqi Buffaloes

The present study was conducted on 50 recently calved Iraqi Buffalo cows. Depending on the kind of parturition, buffalo cows were divided into two main groups, the first group had normal unassisted parturition (NP) (26 animals) and the second group with certain periparturent complications (PPC) (24 animals). After 24 h of parturition, these two groups were further subdivided into two groups as cows expel their foetal membranes in <24 h postpartum and referred as non‐retained placenta (NRP) while cows that did not expel their foetal membrane after 24 h referred as retained placenta (RP). Sampling for bacteriology, uterine discharge for polymorphonuclear cells per cent and blood samples for polymorphonuclear neutrophil (PMN) and the enzyme creatine kinase activity were performed at 6, 24 and 48 h postpartum. In PPC group, the most prevalent bacteria after 6 h of calving were Escherichia coli, β‐haemolytic Streptococci and Lactobacillus acidophilus. Total bacterial isolates in the uterus of buffaloes with RP in PPC group after 24 and 48 h were 129 and 183 respectively. Among the isolates, Archanobacterium pyogenes, Fusobacterium necrophorum, Prevotella melaninogenicus and Staphylococcus aureus were the most prevalent isolates after 48 h of RP buffaloes in PPC group. Polymorphonuclear neutrophil were significantly (p < 0.01) increased in the uterine discharge than in blood in buffaloes with RP in both PPC and NP groups. In conclusion, uterine contamination occurs as a result of postpartum ascending contamination by non‐specific environmental organisms. The presence of Lactobacillus sp. in the uterus indicated a healthy uterus. Peripartum complications followed by retention of foetal membranes with the dominance of E. coli in the uterine lumen might favour the colonization of other bacteria including facultative anaerobic and strictly anaerobic in the uterine wall of buffaloes.