Chlorotetracycline and Sodium Chloride Treatment Effects on Some Micro-Organisms and Unsaponifiables of Bolti Fish Fillets

The effects of chlorotetracycline (CTC),brine (NaCI) and combination of them on Bolti unsaponifiables and associated micro-organisms were studied under different storage conditions. Lipolytic and psychrophilic bacterial counts in cold stored fillets with combined employment of NaCl and CTC were much lower than either NaCl or CTC. The data for partial freezing indicated that lipolytic bacteria increased much lower than that of psychrophilic bacteria. NaCl had a synergistic effect on CTC and this phenomenon was superior in decreasing lipolytic bacterial counts to that of psychrophilic bacteria. The hydrocarbons of the fresh Bolti fillets were fractionated by GLC into 10 different components of which 7 were completely characterized. The detected sterols were cholesterol, campesterol and β-sitosterol with cholesterol being the most predominant. With cold storage, slight interconversion might occur between hydrocarbons or sterols and not between these lipid classes. On the contrary, remarkable amounts of sterols were converted to hydrocarbons in partially frozen fillets. The employment of NaCl, CTC and in combination lead to enormous, slight and noticeable conversions from hydrocarbons to sterols of Bolti fillets, respectively.     

Chromatography on silicic acid of the unsaponifiable matter of fats

A method has been described for the separation of unsaponifiables into their major chemical classes by silicie acid adsorption chromatography. Methods are also presented for the isolation of unsaponifiables free of fatty acids. The chromatographic procedure was tested on synthetic mixtures of hydrocarbons, esters, alcohols, and sterols and then applied to the unsaponifiables of extracted and pressed olive oils, soybean, teaseed, and rapeseed oils, lard, and tallow. The major sterol of all the unsaponifiables examined was found to be β-sitosterol. Analytical data such as infrared analysis, carbon-hydrogen analysis, melting points of derivatives, and paper chromatography of the sterol fractions are also presented. Report submitted to the Fourth Congress of the International Society for the Study of Fatty Materials, Graz, September 1–3, 1959.     

Gas chromatographic determination of tocopherols and sterols in soya sludges and residues

A method is described for determining the individual and total sterols and tocopherols of soya sludges and residues. The method involves saponification of the sample followed by gas chromatographic analysis of the unsaponifiables. Data are presented on the reproducibility and accuracy of the method.     

Variations in the composition of sterols,tocopherols and lignans in seed oils from fourSesamum species

Seeds from different collections of cultivatedSesamum indicum Linn and three related wild species [specifically,S. alatum Thonn.,S. radiatum Schum & Thonn. andS. angustifolium (Oliv.) Engl.] were studied for their oil contents and fatty acid composition of the total lipids. The oils from wild seeds were characterized by higher percentages of unsaponifiables (4.9, 2.6 and 3.7%, respectively) compared toS. indicum (1.4–1.8%), mainly due to their high contents of lignans. Total sterols accounted forca. 40, 22, 20 and 16% of the unsaponifiables of the four species, respectively. The four species were different in the relative percentages of the three sterol fractions (the desmethyl, monomethyl and dimethyl sterols) and in the percentage composition of each fraction. Campesterol, stigmasterol, sitosterol and Δ⁵-avenasterol were the major desmethyl sterols, whereas obtusifoliol, gramisterol, cycloeucalenol and citrostandienol were the major monomethyl sterols, and α-amyrin, β-amyrin, cycloartenol and 24-methylene cycloartanol were the main dimethyl sterols in all species. Differences were also observed among the four species in sterol patterns of the free sterols compared to the sterol esters.Sesamum alatum contained less tocopherols (210–320 mg/kg oil), andS. radiatum andS. angustifolium contained more tocopherols (ca. 750 and 800 mg/kg oil, respectively) than didS. indicum (490–680 mg/kg oil). The four species were comparable in tocopherol composition, with γ-tocopherol representing 96–99% of the total tocopherols. The four species varied widely in the identity and levels of the different lignans. The percentages of these lignans in the oils ofS. indicum were sesamin (0.55%) and sesamolin (0.50%).Sesamum alatum showed 1.37% of 2-episesalatin and minor amounts of sesamin and sesamolin (0.01% each).Sesamum radiatum was rich in sesamin (2.40%) and contained minor amounts of sesamolin (0.02%), whereS. angustifolium was rich in sesangolin (3.15%) and also contained considerable amounts of sesamin (0.32%) and sesamolin (0.16%).     

Serum Unsaponifiables Changes as an Indication of Different Pregnancy Periods and Abnormal Cases in Women

Gas chromatographic profiles of serum unsaponifiables were analysed for 70 specimens representing non-pregnant, pregnant at first, second and third trimesters, abortion and pre-eclamptic patients. The analysis showed that the unsaponifiable matter of women's serum consisted of 8 different hydrocarbons and sterols with β-sitosterol the most abundant substance. Quantitative differences in the unsaponifiable constituents of women's serum from nonpregnant, pregnant and abnormal cases were found. Therefore, an examination of unsaponifiable matter appears to provide a rapid and simple laboratory method for the prediction of gestation periods and to define the abnormal cases in women.     

Neutral lipid and fatty acid composition of earthworms (Lumbricus terrestris)

Earthworms (Lumbricus terrestris) were extracted with chloroform-methanol (2∶1) and examined primarily for neutral lipids and fatty acids. TLC showed spots for sterols, hydrocarbons, free fatty acids, phospholipids and pigments but none for glycerides (tri-, di- or mono). Saponification of the crude lipid extract yielded 32% fatty acids, 25% unsaponifiables and 43% unidentified. The lipid contained 3% hydrocarbon and 16% sterols. GLC of the hydrocarbons showed at least 13 components. GLC of the sterol fraction showed peaks corresponding to cholesterol (the major component), γ-sitosterol, β-sitosterol, stigmasterol, campesterol, and ergosterol. GLC showed that at least 38 fatty acids were present, with 18∶1, 18∶2, 18∶0, 20∶1 and 17∶0 predominanting. Abstracted in part from the Ph.D. dissertation of J. Cerbulis, Rutgers, The State University, 1966.     

Cholesterol-lowering activity of plant sterol-egg yolk lipoprotein complex in rats

Free plant sterols cannot be dissolved in oil or water. Using free plant sterols and egg yolks, we developed a plant sterol-egg yolk lipoprotein complex (PSY) that can be dispersed in water and considered suitable for use in processed foods. The cholesterol-lowering activity of PSY was equal to that of free plant sterols and plant sterol esters. Consumption of a freeze-dried PSY-containing omelet reduced serum and hepatic cholesterol concentrations. The results suggest that PSY has cholesterol-lowering activity equivalent to that of free plant sterols and plant sterol esters. Moreover, the cholesterol-lowering activity of PSY was maintained in processed foods.     

Differential uptake of cholesterol and plant sterols by rat erythrocytes in vitro

The in vitro uptake of radioactively labeled cholesterol and the plant sterol β-sitosterol has been examined in rat erythrocytes. From mixed micellar solutions containing egg yolk phospholipid and sodium taurocholate, the erythrocytes showed a nonlinear uptake of the two sterols. The uptake leveled off after about 45 min with the attainment of a 1∶1 total sterol-to-phospholipid ratio within the cell membrane, as determined on a mass basis. From soltuions containing egg yolk phospholipid, or purified egg yolk phosphatidylcholine, a preference for cholesterol over the plant sterol was observed, increasing with time from a cholesterol/β-sitosterol uptake ratio of unity (the media ratio) to a maximum of 2 after a 60-min incubation. Correction of the data for nonspecifically bound sterol increased the ratio to a maximum of 5 at the 30-min time point. The increase in the cholesterol/β-sitosterol uptake ratio with time, following an initial nonspecific association, showed that penetration of the plasma membrane by the sterol was required for the selectivity to be expressed. The presence of lysophosphatidylcholine or bovine serum albumin did not exert any noticeable influence over the extent of selectivity of absorption. Replacement of the egg yolk phospholipid with synthetic dipalmitoyl-phosphatidylcholine led to a loss of the sterol selectivity. No evidence was found to support a selective extraction of sterol from the erythrocyte membrane to account for the observed effects, nor was there any sign of a mass accumulation of phospholipid during the incubation. It is suggested that the media phospholipid influences the membrane permeability toward cholesterol and β-sitosterol. Presented in part at the 72nd annual meeting of the American Oil Chemists' Society, New Orleans, LA, May 1981.     

Cryoprotectants-Free Vitrification and Conventional Freezing of Human Spermatozoa: A Comparative Transcript Profiling

Introduction: Spermatozoa cryopreservation is an important technique to preserve fertility for males. This study aimed at exploring the stability of epigenetics information in human spermatozoa, manipulated by two different technologies, freezing and vitrification. Methods: Spermatozoa samples were distributed into three groups: 1. Fresh spermatozoa (control group), 2. Frozen spermatozoa, 3. Vitrified spermatozoa. Epigenetic differences of fresh and cryopreserved spermatozoa were evaluated using high-throughput RNA sequencing. Results: Differentially expressed genes (DEGs) in frozen (1103 genes) and vitrified (333 genes) spermatozoa were evaluated. The bioinformatical analysis identified 8 and 15 significant pathways in groups of frozen and vitrified spermatozoa, respectively. The majority of these pathways are most relevant to immune and infectious diseases. The DEGs of the fertilization process are not detected during vitrification. The freezing process induces more down-regulation of genes and is relevant to apoptosis changes and immune response. Conclusion: Cryopreservation of human spermatozoa is an epigenetically safe method for male fertility preservation. Cryoprotectant-free vitrification can induce more minor biological changes in human spermatozoa, in comparison with conventional freezing.     

Studies of unsaponifiables in several vegetable oils

The unsaponifiable fractions of soybean, cottonseed, coconut, olive, and avocado oils have been studied in detail. The oils differed in the contents of total unsaponifiables, squalene, tocopherols, and sterols and also in the composition of the tocopherol and sterol fractions. The presence of absence of individual unsaponifiable components may help in establishing the identity of each of the investigated oils and in detecting of admixture by another oil.     

Uneven Distribution of Ceramides,Sphingomyelins and Glycerophospholipids Between Heads and Tails of Rat Spermatozoa

Previous work showed that rat germ cells and spermatozoa contain ceramides and sphingomyelins with high proportions of nonhydroxy and 2-hydroxy (2-OH) polyunsaturated fatty acids (PUFA) with very long chains (VLCPUFA). The aim of this study was to assess how these lipids are distributed between the heads and tails of mature spermatozoa in comparison with other membrane lipid classes. In addition to quantitative differences due to the fact that these gametes have a long, voluminous tail and a minute head, several compositional dissimilarities emerged between these two regions. The total cholesterol/total phospholipid ratio, the choline/ethanolamine glycerophospholipid (ChoGpl/EtnGpl) ratio, and the proportion of plasmalogens within these two classes, were much larger in the head than in the tail. Whereas EtnGpl was rich in 22:5n-6 in both regions, ChoGpl had plenty of 22:4n-9, especially in the heads. An important proportion of the head EtnGpl- 22:5n-6 and ChoGpl 22:4n-9 was in plasmenyl- (rather than in phosphatidyl-) subclasses. The heads concentrated all of the sphingomyelin species with nonhydroxy- and 2-OH VLCPUFA, and the tails most of the saturated fatty acids that are present in total sperm sphingomyelin. Unexpectedly, virtually all of the abundant spermatozoal ceramides, predominantly made up by species with 2-OH VLCPUFA, was located in the tail. The fact that intact rat spermatozoa constitutively have much more VLCPUFA-containing ceramide than sphingomyelin is explained by the present findings, since the former are mostly lipids of the large tail while the latter mostly collect in the small head.     

Immunofluorescence and High-Resolution Microscopy Reveal New Insights in Human Globozoospermia

Globozoospermia is a rare and severe type of teratozoospermia characterized by the presence of round-headed, acrosomeless spermatozoa with cytoskeleton defects. Current data support a negative relationship between globozoospermia and intracytoplasmic sperm injection (ICSI) outcomes, revealing the need to perform exhaustive studies on this type of sperm disorder. The aim of this study was to evaluate different structural, functional and molecular sperm biomarkers in total globozoospermia with proper embryo development after ICSI. The combination of field-emission scanning electron microscopy (FE-SEM) and transmission electron microscopy (TEM) allowed us to identify and correlate eight morphological patterns with both types of microscopy. Additionally, results reported a high percentage of coiled forms, with cytoplasmic retentions around the head and midpiece. By fluorescent microscopy, we detected that most of the sperm showed tubulin in the terminal piece of the flagellum and less than 1% displayed tyrosine phosphorylation in the flagellum. Moreover, we did not detect chaperone Heat shock-related 70 kDa protein 2 (HSPA2) in 85% of the cells. Overall, these findings provide new insights into globozoospermia, which could have potential implications in improving sperm selection methods for assisted reproductive techniques.     

Kisspeptin Receptor on the Sperm Surface Reflects Epididymal Maturation in the Dog

Alongside the well-known central modulatory role, the Kisspeptin system, comprising Kiss1, its cleavage products (Kisspeptins), and Kisspeptin receptor (Kiss1R), was found to regulate gonadal functions in vertebrates; however, its functional role in the male gamete and its localization during maturation have been poorly understood. The present study analyzed Kisspeptin system in dog testis and spermatozoa recovered from different segments of the epididymis, with focus on Kiss1R on sperm surface alongside the maturation during epididymal transit, demonstrated by modification in sperm kinetic, morphology, and protamination. The proteins Kiss1 and Kiss1R were detected in dog testis. The receptor Kiss1R only was detected in total protein extracts from epididymis spermatozoa, whereas dot blot revealed Kiss1 immunoreactivity in the epidydimal fluid. An increase of the Kiss1R protein on sperm surface along the length of the epididymis, with spermatozoa in the tail showing plasma membrane integrity and Kiss1R protein (p < 0.05 vs. epididymis head and body) was observed by flow cytometry and further confirmed by epifluorescence microscopy and Western blot carried on sperm membrane preparations. In parallel, during the transit in the epididymis spermatozoa significantly modified their ability to move and the pattern of motility; a progressive increase in protaminization also occurred. In conclusion, Kisspeptin system was detected in dog testis and spermatozoa. Kiss1R trafficking toward plasma membrane along the length of the epididymis and Kiss1 in epididymal fluid suggested a new functional role of the Kisspeptin system in sperm maturation and storage.     

The Effects of Biopolymer Encapsulation on Total Lipids and Cholesterol in Egg Yolk during in Vitro Human Digestion

The purpose of this study was to examine the effect of biopolymer encapsulation on the digestion of total lipids and cholesterol in egg yolk using an in vitro human digestion model. Egg yolks were encapsulated with 1% cellulose, pectin, or chitosan. The samples were then passed through an in vitro human digestion model that simulated the composition of mouth saliva, stomach acid, and the intestinal juice of the small intestine by using a dialysis tubing system. The change in digestion of total lipids was monitored by confocal fluorescence microscopy. The digestion rate of total lipids and cholesterol in all egg yolk samples dramatically increased after in vitro human digestion. The digestion rate of total lipids and cholesterol in egg yolks encapsulated with chitosan or pectin was reduced compared to the digestion rate of total lipids and cholesterol in other egg yolk samples. Egg yolks encapsulated with pectin or chitosan had lower free fatty acid content, and lipid oxidation values than samples without biopolymer encapsulation. Moreover, the lipase activity decreased, after in vitro digestion, in egg yolks encapsulated with biopolymers. These results improve our understanding of the effects of digestion on total lipids and cholesterol in egg yolk within the gastrointestinal tract.     

Sterols,methylsterols, and triterpene alcohols in threeTheaceae and some other vegetable oils

The unsaponifiables from threeTheaceae (Camellia japonica L.,Camellia Sasanqua Thunb., andThea sinensis L.) oils and alfalfa, garden balsam, and spinach seed oils and shea fat were separated into four fractions: sterols, 4-methylsterols, triterpene alcohols, and less polar compounds by thin layer chromatography. While the sterol fraction was the major one for the unsaponifiables from alfalfa and spinach seed oils, the triterpene alcohol fraction was predominant for the unsaponifiables from all other oils. The sterol, 4-methylsterol, and triterpene alcohol fractions were analyzed by gas chromatography. All the sterol fractions were alike in their compositions, consisting exclusively of Δ⁷-sterols, such as α-spinasterol and Δ⁷-stigmastenol as predominant components together with Δ⁷-avenasterol and 24-methylcholest-7-enol. Obtusifoliol, gramisterol (occasionally accompanied with cycloeucalenol), and citrostadienol, together with several other unidentified components, were found in the 4-methylsterol fractions from all of the oils except shea fat. The 4-methylsterol fraction from shea fat showed a characteristic composition containing a large proportion of unidentified components which had relative retention time greater than that of citrostadienol, while no citrostadienol was detected. β-Amyrin, lupeol, and butyospermol were major components of the triterpene alcohol fractions from most of the oils, but the fraction from spinach seed oil contained cycloartenol and 24-methylene-cycloartanol as predominant components. There is a close similarity in the compositions of unsaponifiables (sterols, 4-methylsterols, and triterpene alcohols) of the threeTheaceae oils. Two sterols, α-spinasterol and Δ⁷-stigmastenol, and five triterpene alcohols were isolated from tea seed oil. Moreover, five unidentified components beside parkeol, butyrospermol, α-amyrin, and lupeol were isolated from the triterpene alcohol fraction of shea fat.     

Changes in Phenotypes and DNA Methylation of In Vitro Aging Sperm in Common Carp Cyprinus carpio

The purpose of the current study was to analyze phenotypic and functional characteristics of common carp (Cyprinus carpio) spermatozoa during in vitro aging and to investigate whether global DNA methylation is affected by sperm aging. Milt was collected from five individual males, stored in vitro on ice in a refrigerator for up to 96 h post stripping (HPS) and used to fertilize eggs with intervals of 1, 24 and 96 h. Computer-assisted sperm analysis and a S3e Cell Sorter was employed to determine the spermatozoa phenotypic characteristics (motility, velocity, concentration and viability). In addition, pH and osmolality of the seminal fluid and the capacity of the spermatozoa to fertilize, hatching rate and health of the resulting embryos were examined at different aging times. Whole-genome bisulfite sequencing was used to compare the global and gene-specific DNA methylation in fresh and aged spermatozoa. The results demonstrated that spermatozoa aging in common carp significantly affects their performance and thus the success of artificial fertilization. The methylation level at the cytosine-phosphate-guanine (CpG) sites increased significantly with 24 HPS spermatozoa compared to the fresh group at 1 HPS and then decreased significantly at 96 HPS. A more detailed investigation of gene specific differences in the DNA methylation was hindered by incomplete annotation of the C. carpio genome in the public databases.     

Extraction of phospholipids from structured dry chicken egg

Chicken egg contains a high level of phospholipids (PL) in the yolk. Recovering yolk total lipids and extracting egg PL with efficiency are important to the availability and utilization of these health‐promoting lipid products. In this study, we prepared two structured dry egg yolk materials and used two common solvents to extract and concentrate the PL. We found that drum‐dried flake‐like yolk is an ideal starting material for lipid extraction. Anhydrous ethanol can extract almost all the neutral lipids and PL. PL with purity over 90% can be prepared by cold acetone precipitation from the total lipids.     

Unsaponifiable lipid constituents of ten indian seed oils

The unsaponifiable lipid constituents, hydrocarbons, triterpene alcohols and sterols of ten seed oils (Catharanthus roseus, Nymphaea nelumbo, Casuari-na equisetifolia, Lagerstroemia therolli, Prosopisjuliflora, Mimusops elengi, Mimusops hexandra, Ponga-miapinnata, Acrocarpus fraxinifolius, and Bauhinia retusa) were investigated by gas liquid chromatography. Total unsaponifiables ran from 4–14%. Some of the seed oils contained large quantities of jβ-amyrin, α-amyrin and cycloartenol. Acrocarpus fraxinifolius was found to contain 84% of lupeol. Stigmasterol (24-ethyl-22ε-dehydrocholesterol), β-sitosterol (24-ethyl-cholesterol) and campesterol (24-methyl-cholesterol) were the common constituents in all the seed oils. Besides these constituents, tirucallol, taraxerol, ψ-taraxasterol, fucosterol, isofucosterol, avenasterol and cholesterol also were detected in small quantities.     

Unusual composition of sterols in a phytophagous insect,Mexican bean beetle reared on soybean plants

Three saturated sterols, cholestanol, campestanol, and stigmastanol, constituted 54, 72, and 77% of the total sterols of the egg, prepupa, and adult, respectively, of the Mexican bean beetle,Epilachna varivestis (Mulsant), reared on soybean plants. Lathosterol (7-cholesten-3β-ol), possibly formed from cholestanol in this insect, constituted 12, 16, and 11.8% of the total sterols isolated from egg, prepupa, and adult, respectively. None of these sterols have been isolated and identified previously as components of the sterols of a phytophagous insect reared on a natural host plant. Cholesterol, a major sterol of most plant feeding insects studied thus far, comprised less than 5% of the total sterols in any of the stages examined. The unique composition of the sterols in this insect in relation to the sterol composition of the host plant is compared to dietary sterol utilization and metabolism in other phytophagous insects. ARS, USDA.