Composition of lipids, fatty acids and sterols in Okinawan corals

A survey of lipid composition was made for 15 cnidarians from Okinawa, Japan. Eleven zooxanthellate scleractinian corals, an azooxanthellate scleractinian coral Tubastrea sp., a soft coral Lobophytum crassum, a hydroid coral Millepora murrayi and a sea anemone Boloceroides sp. were examined to elucidate the total lipid content, fatty acid composition for each lipid class and sterol composition. All specimens contained monoalkyldiacylglycerol which migrated between the triacylglycerols and esters on thin layer chromatography (TLC). Analysis by high performance thin layer chromatography (HPTLC) and Gas chromatography-mass spectrometry (GC-MS) revealed that these cnidarians were rich in wax ester and triacylglycerol, and that palmitic acid (16:0) was the most abundant fatty acid component of these lipid classes, followed by stearic (18:0) and oleic (18:1, n-9) acid in order of concentration. Of 11 sterols separated, four sterols were identified. It is suggested that sterol composition may be more useful for the biochemical classification of these cnidarians than fatty acid composition.     

Effects of dietary n-3 fatty acids on characteristics and lipid composition of ovine sperm

The fatty acid composition of sperm affects the fertilization rate. The objective was to investigate the effects of dietary fish oil (as a source of n-3 fatty acids) on semen quality and sperm fatty acid composition in sheep. Eight Zandi fat-tailed rams were randomly allocated into two groups and fed either a control diet or a diet supplemented with fish oil. Both diets were isocaloric and isonitrogenous and were fed for 13 weeks, starting in the middle of the breeding season. Semen samples were collected weekly and their characteristics evaluated by standard methods, whereas samples collected at the start and end of the study were assessed (gas chromatography) for sperm lipid composition. Mean (±s.e.m.) sperm concentrations (4.3 × 109 ± 1.3 × 108 v. 3.9 × 109 ± 1.3 × 108 sperm/ml and percentages of motile (77.25 ± 3.34 v. 60.8 ± 3.34) and progressively motile sperm (74.13 ± 1.69 v. 62.69 ± 1.69) were significantly higher in the fish oil group than control. Dietary fish oil increased the proportion of docosahexaenoic acid (DHA, C22:6 n-3) in sperm fatty acid composition. We concluded that feeding fish oil as a source of n-3 fatty acids attenuated seasonal declines in semen quality in rams, perhaps through increased DHA in sperm.     

Roles of antioxidants on prolonged storage of avian spermatozoa in vivo and in vitro

This review focuses on natural and assisted prevention against lipid peroxidation in avian spermatozoa. The presence of high levels of n-6 polyunsaturated fatty acids (PUFAs) in the plasma membrane creates favorable conditions for the formation of peroxidative products, a major cause of membrane damage which may ultimately impair male fertility. However, a complex antioxidant system involving vitamin C, vitamin E and GSH is naturally present in avian semen. Coupled with a battery of enzymatic defenses (e.g., SOD, GSH-Px either Se- or non-Se-dependent), this system acts to prevent or restrict the formation and propagation of peroxides. The presence of specialized sites dedicated to prolonged sperm storage in avian females raises the question of durable protection of sperm membranes against peroxidation. Preliminary observations have revealed the presence of a specific antioxidant system at these sites in which vitamin C could exert a major role. From a practical standpoint, the extensive use of artificial insemination in poultry, along with the emergence in some species of workable techniques to cryopreserve spermatozoa, demand better control of peroxidation occurring in the plasma membrane of spermatozoa before or during storage. Dietary supplementation with vitamin E is effective in limiting lipid peroxidation of sperm plasma membranes, both in chickens and turkeys. In addition, organic Se with or without vitamin E stimulates Se-GSH-Px activity in seminal plasma. Preliminary observations in female chickens have also revealed the effectiveness of dietary supplementation with vitamin E, organic selenium or both to sustain fertility in aging flocks.     

Effects of non-esterified fatty acids on bovine granulosa cells and developmental potential of oocytes in vitro

Cryopreservation of bull semen is sub-optimal, causing cell death of a majority of spermatozoa. Even the surviving cells are affected post-thaw, either structurally or functionally. The aim of this study was to investigate the sequence of events that take place when sperm plasma membrane and acrosome deteriorate during a 4 h incubation period post-thaw, with special attention paid to the acrosome status of dying cells. Frozen-thawed semen of six AI dairy bulls was used. Three straws per batch were pooled and incubated at 37 °C. Sub-samples were taken at 30 min intervals and stained with SYBR 14, propidium iodide (PI) and phycoerythrin-conjugated peanut agglutinin (PE-PNA). Plasma membrane and acrosome integrity were measured by flow cytometry. The experiment was repeated three times. Immediately after thawing, only 3.45% of the dying cells showed acrosomal exocytosis. This number increased dramatically during incubation, reaching 67% after 4 h. Within the intact cell population, the overall decrease in viability and acrosome integrity was kept at five percentage points. Flow cytometry and the triple fluorochrome combination presented a detailed picture of the time course in plasma membrane and acrosome deterioration of frozen-thawed bull semen. The results are expected to be useful for monitoring new cryopreservation protocols.     

Effects of in vitro incubation on a zona binding site found on murine spermatozoa

Murine cauda epididymal sperm possess a site, the acceptor, on the plasma membrane over the apical cap region of the acrosome which recognizes both a proteinase inhibitor of seminal vesicle origin and homologous zonae. The acceptor site may participate in both capacitation and zona binding. This presentation explores the effect of in vitro incubation in a medium known to induce capacitation on the binding capabilities of this site. Approximately 80% of fresh cauda epididymal sperm will bind the seminal inhibitor in vitro. Incubating sperm, pretreated with inhibitor for 2 hr in a medium (M199-M) known to support capacitation, reduces by 60% the number of sperm showing evidence of the inhibitor. No such decrease is seen when sperm are incubated in a medium (M199) that does not support capacitation. During the 2-hr incubation in either medium, 60-70% of the sperm retain two diverse components on the plasma membrane over the acrosome: a receptor for the Fc portion of IgG and an epitope recognized by a monoclonal antibody to the acceptor site. These observations suggest that the plasma membrane in the acrosome region of the cell remains structurally intact during incubation. Furthermore, sperm retain the ability to bind the seminal inhibitor during incubation. After a 2-hr incubation in M199-M, sperm pretreated with heat-solubilized zonae no longer bind the inhibitor. These sperm, however, retain the plasma membrane over the acrosomal cap region. When the sperm are incubated in M199, no decrease in inhibitor binding due to zona treatment is noted.(ABSTRACT TRUNCATED AT 250 WORDS)     

High turnover of fungal hyphae in incubation experiments

Soil biological studies are often conducted on sieved soils without the presence of plants. However, soil fungi build delicate mycelial networks, often symbiotically associated with plant roots (mycorrhizal fungi). We hypothesized that as a result of sieving and incubating without plants, the total fungal biomass decreases. To test this, we conducted three incubation experiments. We expected total and arbuscular mycorrhizal (AM) fungal biomass to be higher in less fertilized soils than in fertilized soils, and thus to decrease more during incubation. Indeed, we found that fungal biomass decreased rapidly in the less fertilized soils. A shift towards thicker hyphae occurred, and the fraction of septate hyphae increased. However, analyses of phospholipid fatty acids (PLFAs) and neutral lipid fatty acids could not clarify which fungal groups were decreasing. We propose that in our soils, there was a fraction of fungal biomass that was sensitive to fertilization and disturbance (sieving, followed by incubation without plants) with a very high turnover (possibly composed of fine hyphae of AM and saprotrophic fungi), and a fraction that was much less vulnerable with a low turnover (composed of saprotrophic fungi and runner hyphae of AMF). Furthermore, PLFAs might not be as sensitive in detecting changes in fungal biomass as previously thought.     

Influence of plasma lipid changes in response to 17β-oestradiol stimulation on plasma growth hormone, somatostatin, and thyroid hormone levels in immature rainbow trout

Plasma total lipids were significantly higher in 17β-oestradiol(E₂)-treated immature rainbow trout Oncorhynchus mykiss at week 4 after implantation, due to increases in polar and neutral lipids. The lipid classes responding were phosphatidylcholine, phosphatidylinositol, phosphatidylethanolamine, sterols and sterol esters, in a proportion that approximately reflected the increase in plasma vitellogenin (VtG) levels as measured by a non-competitive enzyme-linked immunosorbent assay (ELISA). Plasma non-esterified fatty acids and triacylglycerol were not affected by E₂ treatment. Plasma growth hormone GH levels were increased, and plasma somatostatin-14 (SRIF) levels decreased in E₂-treated fish, responses which could be secondary to elevated plasma lipid (VtG) content, although a direct E₂ action on somatotroph function is possible. Plasma T₄ concentrations were not affected by E₂ treatment, but plasma T₃ concentrations were significantly lower than in controls 1 week after implantation when plasma E₂ concentrations were the highest; this is in support of the hypothesis that E₂ has a suppressive action on T₃ production.     

Metabolism of apical versus basolateral sn-2-monoacylglycerol and fatty acids in rodent small intestine

The metabolic fates of radiolabeled sn-2-monoacylglycerol (MG) and oleate (FA) in rat and mouse intestine, added in vivo to the apical (AP) surface in bile salt micelles, or to the basolateral (BL) surface via albumin-bound solution, were examined. Mucosal lipid products were quantified, and the results demonstrate a dramatic difference in the esterification patterns for both MG and FA, depending upon their site of entry into the enterocyte. For both lipids, the ratio of triacylglycerol to phospholipid (TG:PL) formed was approximately 10-fold higher for delivery at the AP relative to the BL surface. Further, a 3-fold higher level of FA oxidation was found for BL compared with AP substrate delivery. Incorporation of FA into individual PL species was also significantly different, with >2-fold greater incorporation into phosphatidylethanolamine (PE) and a 3-fold decrease in the phosphatidylcholine:PE ratio for AP- compared with BL-added lipid. Overnight fasting increased the TG:PL incorporation ratio for both AP and BL lipid addition, suggesting that metabolic compartmentation is a physiologically regulated phenomenon. These results support the existence of separate pools of TG and glycerolipid intermediates in the intestinal epithelial cell, and underscore the importance of substrate trafficking in the regulation of enterocyte lipid metabolism.     

Morphometry of porcine spermatozoa and its functional significance in relation with the motility parameters in fresh semen

Both the study and the relationship between sperm design and sperm function have been a target of several researchers. In our study we have evaluated the relationship between the morphometry of sperm head and midpiece as well as the relationship between morphometry of these two spermatic components and sperm motion characteristics in the boar. Analysis of regression (lineal and multiple) and principal components analysis were used for the study of these relationships. Semen samples from five Iberian boars were taken for analysis. Analysis of morphometry was assessed by CASMA system and motility by CASA system. Sperm midpiece showed a significant relationship (positive or negative, depending on the morphometric parameter evaluated) with sperm head. VSL, LIN, STR, BCF and VAP showed a significant relationship with several head and midpiece morphometric parameters. Finally, through the analysis of multiple lineal regression we obtained several statistical models that predict STR, LIN, VCL, ALH, BCF, PC1 and PC2 (the last two variables have been obtained from a principal components analysis) as a function of one, two or three morphometric parameters. Our results suggest a co-evolution of sperm head and midpiece and in addition that sperm motion characteristics of porcine spermatozoa are influenced by morphometry of head and midpiece.     

Metabolism of lipid and carbohydrate in sea urchin spermatozoa

When the dry sperm of the sea urchin, Hemicentrotus pulcherrimus, were diluted 100 times in artificial sea water at 0°C and at 20°C, they became motile and the levels of ATP and creatine phosphate decreased rapidly. The level of ADP hardly changed, and the AMP level increased after the dilution. After the dilution, the respiratory rate at 2°C was almost one fifth that of 20°C. Both phospholipid and glycogen were used for the energy sources in sea urchin sperm. The level of phospholipid was 10-fold higher than that of glycogen in the dry sperm. The phospholipid level decreased after dilution at 20°C, though the level hardly changed at 0°C, suggesting that phospholipid was hardly metabolized the lower temperature. The level of α -glycerophosphate increased at 20°C after the dilution but did not change at 0°C. The level of glycogen decreased after the dilution, regardless of the temperature. The glycolysis was also activated after the dilution. Of the intermediates of the tricarboxylic acid cycle, the citrate concentration increased at 0°C and the malate concentration also increased at 0°C and especially strongly at 20°C.     

Increased quality of in natura and cryopreserved semen of water buffaloes supplemented with saturated and unsaturated fatty acids from the palm oil industry

Ruminant energy supplementation with vegetable oils or fats has been standing out worldwide and oil palm processing has been receiving growing interest. This study assessed the effect of supplementation with saturated and unsaturated fatty acids from the palm oil industry on the lipid profile of seminal plasma and of the sperm membrane, as well as on the morphological and functional characteristics of raw and cryopreserved buffalo semen. Twelve purebred Murrah bulls (Bubalus bubalis) were assigned to the experimental groups and fed diets for 120 days with no added lipids (CONT, four bulls), or with an extra amount of 3% lipids from crude palm oil (PALM, four bulls), or from palm oil deodorizer distillate (PODD, four bulls). Semen was collected and cryopreserved every 15 days. The lipid composition of membranes and semen quality were determined after collections. Lipid supplementation did not impact feed intake (P>0.05). Diet enrichment with PALM increased the linoleic acid (C18:2,ω6) in seminal plasma. Lipid supplementation did not increase the polyunsaturated fatty acids in the sperm membrane composition, but significantly increased the lignoceric acid (C24:0). Cryopreserved semen of the supplemented bulls presented higher progressive motility (60.2 vs. 67.9 vs. 65.2%; P<0.05) and sperm viability detected by eosin-nigrosin staining (61.1 vs. 69.4 vs. 67.8%; P<0.05). Palm oil reduced major sperm defects in both raw (12.2 vs. 9.3 vs. 13.2%; P<0.0001) and cryopreserved semen (12.4 vs. 9.4 vs. 11.2%; P<0.0001). The lipids added to the diet did not impact the population of spermatozoa with intact plasma and acrosomal membranes (PI-/PSA-), but significantly increased the percentage of spermatozoa with high mitochondrial potential (25.6 vs. 31.5 vs. 32.0%; P=0.008). The results suggest that lipid supplementation based on crude palm oil or palm oil deodorizer distillate can be safely used to feed buffalo bulls and may increase sperm attributes related to male fertility.     

Metabolism of n-6 fatty acids by NIH-3T3 cells transfected with the ras oncogene

N-6 fatty acid metabolism was compared in NIH-3T3 cells and DT cells, which differ only in the presence of the v-Ki-ras oncogene. Non-dividing cells were incubated with [1-¹⁴C]-labelled fatty acids (18:2n-6, 18:3n-6, 20:3n-6 and 20:4n-6) at different time intervals (2–24 h) and concentration (0–120 M). In both cells lines, the uptake of different fatty acids from the medium was similar and reached a maximum at 6–8 h. All fatty acids reached the same maximum level in DT cells, whereas, the relative uptake of added fatty acids by NIH-3T3 cells was different: 20:4n-6>20:2n-6>18:2n-6=18:3n-6. Throughout the incubation (2–24 h), desaturation and elongation of n-6 fatty acids was more active in DT cells than in NIH-3T3 cells. However, in both cell lines, incubated with different n-6 fatty acid precursors, the levels of radiolabelled 20:4n-6 were relatively constant. In DT cells, phosphatidylcholine was found to be the major fraction labelled with n-6 fatty acids precursors and those of endogenous synthesis, whereas, in NIH-3T3 cells the neutral lipid fraction, particularly triglycerides, was also strongly labelled. In concentration dependent studies, phospholipid labelling by fatty acids was saturable. At lower concentrations, especially in DT cells, phospholipids were labelled predominantly. As the concentration increased there was an overflow into the triglyceride fraction. Since the differences in fatty acid metabolism between the two cell lines cannot be related to the growth rate, it is suggested that they were a consequence of the expression of the v-Ki-ras oncogene.Abbreviations BSA bovine serum albumin - CE cholesterol ester - DG diglyceride - DMEM Dulbecco's modification of Eagle's medium - EL ether lipids (glyceryl ether diesters) - FAME fatty acid methyl ester - FCS fetal calf serum - FFA free fatty acids - HEPES N-2-(hydroxyethyl)piperazine-N-2-ethanesulphonic acid - MG monoglyceride - NL neutral lipid - PC phosphatidylcholine - PE phosphatidylethanolamine - PI phosphatidylinositol - PL phospholipid - s.a specific activity - TG triglyceride - TLC thin layer chromatography     

Analysis of preimplantation golden hamster conceptuses resulting from spermatozoa aged in utero

The effects of aging golden hamster spermatozoa in the female reproductive tract on the percentage of ova fertilized, the stage of development, and the chromosome complement of the resulting zygotes were studied. Females were inseminated artificially with cauda epididymal sperm at 6 h (control), 10, 15,18, or 21 h before the estimated time of ovulation. A decrease in the percentage of ova fertilized was found as the time spermatozoa were aged in utero prior to ovulation increased. The zygotes collected at 56 h postovulation from females inseminated 15 or 18 h prior to ovulation were delayed in development, as judged by the number of blastomeres. Although an increase of chromosomally abnormal zygotes was not found, a possibility exists that mosaicism may have been present, as evidenced by zygotes with unequal sized blastomeres, and went undetected.     

Lipid peroxidation in human spermatozoa as relatd to midpiece abnormalities and motility

The formation of malondialdehyde (MDA), a product of lipid peroxidation (LPO), was measured in human spermatozoa from 27 subjects with normal sperm characteristics. Peroxidation of lipids in washed spermatozoa was induced by catalytic amounts of ferrous ions and ascorbate and malondiaidehyde dctermint-d by thiobarbituric method. MDA formation varied considerably from one sample to another. The studied population showed a strong correlation between lipid peroxidation potential (amount of MDA formed by 10⁸ spermatozoa after 1 hour of incubation) and 1) initial motility r = ?0.623, P = 0.001; and 2) morphologic abnormalities of the midpiece r = 0.405, P = 0.05. These results suggest that poor motility is linked with membrane fragility and that spermatozoa with midpiece abnormalities probably have membrane and/or cytoplasmic antiperoxidant system defects. Because LPO potential is related to the two most important characteristics of fertility, it seems possible that it has the potential to become a good biochemical index of semen quality.