Effect of different mini‐volume colloid centrifugation configurations on flow cytometrically sorted sperm recovery efficiency and quality using a computer‐assisted semen analyzer

Straws of sex‐sorted sperm are usually packaged at a low concentration (e.g., ~2.1 × 10⁶ sperm/ml) and cost significantly more than unsorted conventional semen from the same sire. In order to maximize the efficiency of using sex‐sorted sperm under in vitro fertilization conditions, the selection of an appropriate sperm separation technique is essential. In this study, the effect of using different silane‐coated silica colloid dilutions and layering configurations during centrifugation of sex‐sorted sperm was examined over an extended period of incubation time. Sperm recovery and viability after centrifugation using the colloid separation technique were measured along with several sperm motility parameters using CASA. For this purpose, frozen and thawed sex‐sorted sperm samples were centrifuged using mini‐volume single‐layer (40%, 60% and 80%) and mini‐volume two‐layer (45%/90%, 40%/80% and 30%/60%) separation configurations using PureSperm^®. A single layer of 40% PureSperm^® recovered significantly more sex‐sorted sperm (78.07% ± 2.28%) followed by a single layer of 80% PureSperm^® (68.43% ± 2.33%). The lowest sperm recovery was obtained using a two‐layer PureSperm^® dilution of 45%/90% (47.57% ± 2.33%). Single‐layer centrifugation recovered more sorted sperm (68.67% ± 1.74%) than two layer (53.74% ± 1.74%) (p < .0001). A single layer of 80% PureSperm^® exhibited the highest sorted sperm viability (72.01% ± 2.90%) after centrifugation (p < .05). The mini‐volume single layer of 80% PureSperm^® was determined to be an effective alternative to a two‐layer centrifugation configuration for sex‐sorted sperm selection. In addition, single‐layer colloid dilution of 80% performed either as well as or significantly outperformed the other treatments, as well as the control, with regard to motility (MOT) for all time periods of analysis.     

Altered Expression of Transforming Growth Factor‐Beta Isoforms in Bovine Cystic Ovarian Disease

Cystic ovarian disease (COD) is one of the main causes of infertility in dairy cattle. It has been shown that intra‐ovarian factors may contribute to follicular persistence. Transforming growth factor‐beta (TGFB) isoforms are important paracrine and autocrine signalling molecules that regulate ovarian follicle growth and physiology. Considering the importance of these factors in the ovarian physiology, in this study, we examined the expression of TGFB isoforms (TGFB1, TGFB2 and TGFB3) in the ovary of healthy cows and animals with spontaneous and adrenocorticotrophic hormone (ACTH)‐induced COD. In the oestrous‐synchronized control group, the expression of TGFB1 in granulosa and theca cells was higher in spontaneous cysts than in atretic or tertiary follicles. When we compared TGFB2 expression in granulosa cells from atretic or tertiary follicles from the oestrous‐synchronized control group with that in ACTH‐induced or spontaneous follicular cysts, we found a higher expression in the latter. The expression of the TGFB isoforms studied was also altered during folliculogenesis in both the spontaneous and ACTH‐induced COD groups. As it has been previously shown that TGFB influences steroidogenesis, ovarian follicular proliferation and apoptosis, an alteration in its expression may contribute to the pathogenesis of this disease.     

p-Hydroxybenzoic acid alkyl esters in Andrographis paniculata herbs,commercial extracts,and formulated products

Analysis of two commercial extracts of Andrographis paniculata using high-performance liquid chromatography (HPLC) with photodiode array absorbance detection showed the presence of several unexpected compounds, which were isolated and identified as methyl, ethyl, and propyl esters of p-hydroxybenzoic acid by using high-resolution mass spectrometry and nuclear magnetic resonance. Quantitative analysis using HPLC revealed the presence of 0.22% p-hydroxybenzoic acid methyl ester (methlyparaben) in one commercial extract, and both 0.11% p-hydroxybenzoic acid ethyl ester (ethylparaben) and 0.20% p-hydroxybenzoic acid propyl ester (propylparaben) in a second commercial extract of A. paniculata. Analyses of additional commercial products of A. paniculata in tablet form purchased from Chicago pharmacies also showed the presence of methyl- and ethylparabens. To determine whether these compounds were natural chemical constituents of the plant, pharmacopoeial reference A. paniculata plant powder as well as samples of authenticated A. paniculata plant materials collected from Indonesia, Hong Kong, and mainland China were obtained and analyzed by HPLC-tandem mass spectrometry (LC-MS-MS). LC-MS-MS analyses confirmed the presence of trace concentrations (<0.0008% w/w) of p-hydroxybenzoic acid methyl ester but no p-hydroxybenzoic acid ethyl or propyl esters in these plant samples. The limits of detection of the LC-MS-MS assay for these compounds were 5 pg on-column and 5 ppb in the plant material. The levels of these p-hydroxybenzoic acid esters measured in the commercial products of A. paniculata suggest that they were introduced inadvertently during processing or as artificial additives.     

Effect of plant growth regulators on paddy rice

The effect of 4 plant growth regulators (FOR), BAS 111, DCiB, EL 500, Terpal C, on growth and yield of paddy rice was studied in field experiments in Southern China. PGR-application was practiced by seed soaking, root dipping at transplanting and leaf spraying at elongation stage respectively, using a 2, 5 × 10^?5 M concentration of the particular PGR. There was only a minor stem reducing effect of the PGRs at the early stage of tillering, most pronounced after seed soaking. But at harvest time all trials reached the same plant height. In spite of this, seed soaking with EL 500, BAS 111 and Terpal C increased grain yield between 10 and 13 % in the first crop. The results are discussed in connection with a direct PGR-effect on the generative phase of rice.     

小豆的生长发育规律研究——Ⅴ.分期播种条件下小豆生长后期的反应

笔者通过分期播种的手段,研究小豆的生长后期在不同自然温光条件下的反应,进一步揭示小豆的生物学规律。结果表明:北京地区种植小豆最迟不应逾6月底。     

Quantification of bovine milk oligosaccharides using liquid chromatography-selected reaction monitoring-mass spectrometry

Oligosaccharides are important components of milk with bioefficacy as prebiotics, anti-infectives, and immune system modulators and as a possible source of sialic acid for neural function. Bovine milk oligosaccharides are lower in concentration and lack the diversity of human milk oligosaccharides but could be a commercial source of milk oligosaccharides for pediatric foods. For this development, an ability to quantify the oligosaccharides is required. This study validated a hydrophilic interaction chromatography high-performance liquid chromatography-high-resolution selected reaction monitoring-mass spectrometry (HILIC HPLC-HRSRM-MS) method for measuring six different oligosaccharides in bovine milk, bovine colostrum, and infant formulas. The extraction resulted in a high recovery (90-103%) with a repeatability coefficient of variation ranging from 2 to 9% for the two dominant oligosaccharides, 3'-sialyllactose and 6'-sialyllactose, and ranging from 1 to 17% for the much lower concentration oligosaccharides, 6'-sialyllactosamine, disialyllactose, and N-acetylgalactosaminyllactose. The sixth oligosaccharide, 3'-sialyllactosamine, was not detected in any of the samples.     

Isotope labeling studies on the formation of multiple addition products of alanine in the pyrolysis residue of glucose/alanine mixtures by high-resolution ESI-TOF-MS

Pyrolysis was used as a microscale sample preparation tool to generate glucose/alanine reaction products to minimize the use of expensive labeled precursors in isotope labeling studies. The residue remaining after the pyrolysis at 250 °C was analyzed by electrospray time-of-flight mass spectrometry (ESI-TOF-MS). It was observed that a peak at m/z 199.1445 in the ESI-TOF-MS spectrum appeared only when the model system contained at least 2-fold excess alanine. The accurate mass determination indeed indicated the presence of two nitrogen atoms in the molecular formula (C(10)H(18)N(2)O(2)). To verify the origin of the carbon atoms in this unknown compound, model studies with [(13)U(6)]glucose, [(13)C-1]alanine, [(13)C-2]alanine, [(13)C-3]alanine, and [(15)N]alanine were also performed. Glucose furnished six carbon atoms, and alanine provides four carbon (2 × C-2 and 2 × C-3) and two nitrogen atoms. When commercially available fructosylalanine (N-attached to C-1) was reacted with only 1 mol of alanine, a peak at m/z 199.1445 was once again observed. In addition, when 3-deoxyglucosone (3-DG) was reacted with a 2-fold excess of alanine, a peak at m/z 199.1433 was also generated, confirming the points of attachment of the two amino acids at C-1 and C-2 atoms of 3-DG. These studies have indicated that amino acids can undergo multiple addition reactions with 1,2-dicarbonyl compounds such as 3-deoxyglucosone and eventually form a tetrahydropyrazine moiety.     

Malondialdehyde-altered protein occurs in atheroma of Watanabe heritable hyperlipidemic rabbits

It has been proposed that chemically reactive lipids released during lipid peroxidation convert low density lipoprotein (LDL), the major carrier of plasma cholesterol, to an abnormal form and that receptor-mediated clearance of this altered LDL produces cholesteryl ester deposition in macrophage-derived foam cells of atheroma. Immuno-cytochemical analyses now reveal the presence of protein modified by malondialdehyde, a peroxidative end product, which colocalizes with the extracellular deposition of apolipoprotein B-100 protein of LDL in atheroma from Watanabe heritable hyperlipidemic rabbits. These findings provide direct evidence for the existence in vivo of protein modified by a physiological product of lipid peroxidation within arterial lesions.     

Assessing the diet of the endangered Beale’s eyed turtle (Sacalia bealei) using faecal content and stable isotope analyses: Implications for conservation

1. Turtles from Asia are on the brink of extinction with 53% of species considered endangered or critically endangered. Unfortunately, the ecology of many threatened species remains largely unknown.
2. In this study, the diet of the endangered Beale’s eyed turtle (Sacalia bealei) was investigated using two methods, visual faecal content analysis and stable isotope analysis.
3. Results from both methods indicated that S. bealei is highly reliant on riparian resources, especially fruits and terrestrial insects. Stable isotope data indicated that terrestrial resources made up around half (47–53%) of all assimilated food resources. These findings suggest that S. bealei facilitates energy flow from riparian forests to stream ecosystems. Moreover, S. bealei is likely to be a seed disperser for riparian plants.
4. This study represents the first application of stable isotope methods to examine the diet of Asian freshwater turtles and their resource use. We stress the importance of similar studies to improve our understanding of remnant turtle populations before they disappear as a result of human activities.

     

Analytical challenges: determination of tetrodotoxin in human urine and plasma by LC-MS/MS

Tetrodotoxin (TTX) is a powerful sodium channel blocker found in puffer fish and some marine animals. Cases of TTX poisoning most often result from puffer fish ingestion. Diagnosis is mainly from patient's signs and symptoms or the detection of TTX in the leftover food. If leftover food is unavailable, the determination of TTX in the patient's urine and/or plasma is essential to confirm the diagnosis. Although various methods for the determination of TTX have been published, most of them are for food tissue samples. Dealing with human urine and blood samples is much more challenging. Unlike in food, the amount of toxin in the urine and blood of a patient is generally extremely low; therefore a very sensitive method is required to detect it. In this regard, mass spectrometry (MS) methods are the best choice. Since TTX is a very polar compound, there will be lack of retention on conventional reverse-phase columns; use of ion pair reagent or hydrophilic interaction liquid chromatography (HILIC) can help solve this problem. The problem of ion suppression is another challenge in analyzing polar compound in biological samples. This review will discuss different MS methods and their pros and cons.