Flying-fox (Pteropus spp.) sperm membrane fatty acid composition,its relationship to cold shock injury and implications for cryopreservation success

The very large acrosome of Pteropus species spermatozoa is prone to damage during cooling procedures. Cryogenic succuss has been linked to membrane composition, therefore the lipid composition of five Pteropus species sperm acrosomal and plasma membranes were investigated to provide insight into reasons for cold shock susceptibility. Rapid chilling and re-warming of spermatozoa from three Pteropus species resulted in a decrease (P < 0.05) in acrosomal integrity. Biochemical analysis of lipids revealed that stearic acid (18:0) was the predominant saturated fatty acid and oleic acid (18:1, n-9) the predominant unsaturated fatty acid in both acrosomal and plasma membranes. Linolenic acid (18:3, n-3) was only detected in plasma membranes of Pteropus hypomelanus and was detected in acrosomal membranes of all Pteropus spp. studied (except Pteropus giganteus). Although detected in both plasma and acrosomal membranes of Pteropus vampyrus, docosahexaenoic acid (22:6) was not detected at all in Pteropus poliocephalus, only in trace levels in the acrosomal and plasma membranes of P. giganteus and P. hypomelanus and not in acrosomal membranes of Pteropus rodricensis. No difference was seen in the levels of polyunsaturated fatty acids (PUFAs) within plasma membranes, however PUFAs were lower (P < 0.05) in acrosomal membranes of P. giganteus compared with P. vampyrus. Pteropus spp. spermatozoa have a very low ratio of unsaturated/saturated membrane fatty acids (<0.5). Membranes containing more PUFAs are more fluid, so the use of cryogenic media which improves membrane fluidity should improve Pteropus spp. spermatozoal viability post-thaw.     

Sperm membrane fatty acid composition in the Eastern grey kangaroo (Macropus giganteus), koala (Phascolarctos cinereus), and common wombat (Vombatus ursinus) and its relationship to cold shock injury and cryopreservation success

Marsupial spermatozoa tolerate cold shock well, but differ in cryopreservation tolerance. In an attempt to explain these phenomena, the fatty acid composition of the sperm membrane from caput and cauda epididymides of the Eastern grey kangaroo, koala, and common wombat was measured and membrane sterol levels were measured in cauda epididymidal spermatozoa. While species-related differences in the levels of linolenic acid (18:3, n-6) and arachidonic acid (20:4, n-6) were observed in caput epididymal spermatozoa, these differences failed to significantly alter the ratio of unsaturated/saturated membrane fatty acids. However in cauda epididymidal spermatozoa, the ratio of unsaturated/saturated membrane fatty acids in koala and kangaroo spermatozoa was approximately 7.6 and 5.2, respectively; substantially higher than any other mammalian species so far described. Koala spermatozoal membranes had a higher ratio of unsaturated/saturated membrane fatty acids than that of wombat spermatozoa (t = 3.81; df = 4; p < or = 0.02); however, there was no significant difference between wombat and kangaroo spermatozoa. The highest proportions of DHA (22:6, n-3), the predominant membrane fatty acid in cauda epididymidal spermatozoa, were found in wombat and koala spermatozoa. While species-related differences in membrane sterol levels (cholesterol and desmosterol) were observed in cauda epididymidal spermatozoa, marsupial membrane sterol levels are very low. Marsupial spermatozoal membrane analyses do not support the hypothesis that a high ratio of saturated/unsaturated membrane fatty acids and low membrane sterol levels predisposes spermatozoa to cold shock damage. Instead, cryogenic tolerance appears related to DHA levels.     

Comparative aspects of sperm membrane fatty acid composition in silver (Vulpes vulpes) and blue (Alopex lagopus) foxes, and their relationship to cell cryopreservation

Cryogenic protocols have been developed for the storage of farmed silver fox (Vulpes vulpes) spermatozoa. However, these same protocols and modifications of these protocols have failed to satisfactorily preserve spermatozoa collected from farmed blue foxes (Alopex lagopus). Because cryogenic success has been linked to membrane composition, the plasma membrane lipid composition of farmed blue fox and silver fox spermatozoa was studied. Silver fox spermatozoal membranes have significantly higher levels of docosapentaenoic acid (DPA; 22:5, n-6) compared to blue fox spermatozoa, and blue fox spermatozoal membranes have significantly higher levels of stearic acid (18:0). Silver fox spermatozoal membranes not only have a higher ratio of unsaturated/saturated membrane fatty acids, but also higher levels of membrane desmosterol and cholesterol.     

Effect of growth temperature on membrane fatty acid composition and susceptibility to cold shock of Bacillus amyloliquefaciens.

We investigated the fatty acid composition of the membrane of Bacillus amyloliquefaciens grown at different temperatures. A decrease in growth temperature was accompanied by an increase in the ratio of branched- to straight-chain fatty acids and a marked increase in the level of unsaturation of branched-chain fatty acids. When cells of this organism grown at 30 degrees C were cold shocked, viability and ability to secrete extracellular protease were lost. Growth of this organism at lower temperatures or addition of Tween 80 to cells caused the critical temperature zone for cold shocking to be lowered significantly. These results suggest a direct correlation between membrane fluidity and the susceptibility to cold shock.     

Physical activity and its relationship to rodeo injury and success

The purpose of this study was to determine the interrelationship among physical activity, injury, and success of rodeo athletes. Seventy-two male Professional Rodeo Cowboys' Association members served as participants and were grouped into timed, steer-wrestling, saddle-bronc, bareback, bull-riding, and multiple rough-stock events. Participants completed demographic and overall physical activity questionnaires. Sixty-two percent of the subjects participated in regular exercise for a minimum of 2 days per week. Pearson bivariate correlation coefficients revealed weak correlations (r = 0.151, -0.188, and -0.074; p > 0.05) among metabolic (MET) level, earnings rate, and injury rate. A 1-way between-subjects analysis of variance indicated no differences in MET level among groups. Physical activity did not affect performance or injury rate, and competitors in various events did not have different levels of energy expenditure. Therefore, rodeo sports-medicine personnel should encourage athletes to engage in other sport-specific training activities to help prevent rodeo injuries.     

Modification of fatty acid composition of membrane phospholipid in hepatocyte monolayer with n-3, n-6 and n-9 fatty acids and its relationship to triacylglycerol production

The objective of these studies with rat hepatocytes in primary culture was to establish that: (a) membrane phospholipids would become enriched with the specific fatty acid supplemented to the media and (b) hepatocyte monolayer triacylglycerol synthetic rates were dependent on the type of fatty acid enrichment of the membrane phospholipids. Hepatocytes cultured in the absence of media lipid developed a phospholipid fatty acid composition which is indicative of an essential fatty acid deficiency. The extensive rise in 18:1(n - 9) content indicated that delta 9-desaturase was active. The fatty acid composition of phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol in the microsomal- and mitochondrial-enriched fractions was highly dependent upon the type of fatty acid supplemented to the medium. Incorporation of fatty acids into phospholipids was rapid, and a new steady-state in fatty acid composition was achieved within approx. 36 h. Changes in the fatty acid composition of these hepatocyte phospholipid subclasses resulting from media supplementation with 18:2/20:4(n-6) or 20:5(n-3) were similar, but not identical, to changes which occurred in vivo as a result of consuming diets rich in 18:2(n-6) or 20:5(n-3). Hepatocyte lipogenesis was highly dependent upon the type of fatty acid supplemented to the medium. Prior conditioning with 16:0 increased triacylglycerol synthesis and secretion. Secretion of triacylglycerol was reduced by polyenoic fatty acid enrichment with 20:5(n-3) greater than 20:4/18:2(n-6). The suppression of triacylglycerol synthesis by 20:5(n-3) was due to an increased (P less than 0.05) diacylglycerol specific activity, which indicates that 20:5(n-3) suppression of hepatic triacylglycerol production may be caused in part by the inhibition of diacylglycerol acyltransferase.     

Drought acclimation and lipid composition in Folsomia candida: implications for cold shock,heat shock and acute desiccation stress

Many of the physiological adaptations evolved in terrestrial invertebrates to resist desiccation have also been shown to enhance the survival of low temperatures. In this study we have examined temporal changes in the physiology of the collembolan Folsomia candida during acclimation to mild desiccation stress (98.2% RH), and how physiological changes correlate with resistance to subsequent cold shock, heat shock and acute desiccation stress. Drought-acclimation increased the resistance to cold and acute drought but reduced the resistance to heat shock. The composition of membrane phospholipid fatty acids (PLFA) changed during acclimation resulting in a higher degree of unsaturation by the end of the 192-h acclimation period. This resembles typical membrane alterations seen in ectothermic animals exposed to cold. Only small changes were seen in the neutral lipid fraction. The temporal changes in cold resistance and drought resistance correlated well with changes in PLFA composition and accumulation of sugars and polyols (’cryoprotectives’). It is proposed that the drought-induced PLFA desaturation, combined with the membrane protecting accumulation of cryoprotectives, are important physiological adaptations providing tolerance to both desiccation and cold.     

Ethanol-induced changes in the fatty acid composition of Lactobacillus hilgardii, its effects on plasma membrane fluidity and relationship with ethanol tolerance

The effect of environmental ethanol concentration on the fatty acid composition of strains of Lactobacillus hilgardii, differing in their tolerance to ethanol, was determined. A marked increase in the proportion of lactobacillic acid (a cyclopropane fatty acid) and a decrease in oleic and vaccenic acids with increasing ethanol concentration was observed. The amount of lactobacillic acid determined at standard conditions (25°C, 0% ethanol) was found to be proportional to the ethanol tolerance of the strains studied. The effect of this alcohol on plasma membrane fluidity was studied by differential scanning calorimetry. The adaptive response to growth in the presence of high concentrations of ethanol produced membranes which, within the limits of ethanol tolerance, maintained the fluidity and integrity in an environment which tends to increase membrane rigidity. When pre-adapted cells are analysed in the absence of environmental ethanol there is a measurabie increase in fluidity. It is proposed that this phenomenon may be correlated with the increase in the proportion of lactobacillic acid. The existence of a relationship between membrane fluidity and ethanol tolerance is discussed.     

Change in sugar, sterol and fatty acid composition in banana meristems caused by sucrose-induced acclimation and its effects on cryopreservation

To understand the mechanisms of sucrose‐induced acclimation in relation to plant cryopreservation, sugars, sterols, fatty acids of different lipid fractions (neutral lipids, glycolipids and sphingolipids and phospholipids), as well as free fatty acids were analyzed in proliferating meristem cultures of different banana varieties. The four banana varieties that were selected show different post‐thaw shoot regeneration rates (0–53.4%). All mentioned parameters were analyzed using (1) control meristems that were cultured on a normal sucrose concentration (0.09 M), which resulted in low survival after cryopreservation; and (2) 2‐week sucrose precultured meristems (0.4 M). This sucrose preculture, essential for regeneration after cryopreservation, resulted in a significant increase of each of seven sugars detected. The ratio of stigmasterol/sitosterol (St/Si) in sucrose‐pretreated meristems significantly increased. The sucrose pretreatment also resulted in a significant increase of total fatty acid content of the neutral lipid fraction and of the glycolipid and sphingolipid fraction, as well as the total free fatty acid content. The individual fatty acid content of the phospholipids was differently changed by the sucrose pretreatment for the given varieties studied. In most cases, sucrose pretreatment resulted in an increase of the double bond index (DBI) in the neutral lipids and a decrease of DBI in the glycolipids and sphingolipids, in phospholipids as well as in free fatty acids. Principal component analysis of all collected data revealed that (1) for the control material, sucrose and total sugar contents were closely linked to the post‐thaw shoot regeneration, suggesting that sucrose and total sugar may be main limiting factors to survive cryopreservation; (2) accumulation of large quantities of sugars (glucose, fructose, sucrose and total sugar) in sucrose‐pretreated material cannot explain the differences in survival after cryopreservation of the four banana varieties. We assume that a minimal amount of sugars is needed in meristem cultures to survive cryopreservation. Still, other limiting factors do influence the survival following the sucrose pretreatment. We observed that the parameters which are closely linked to the post‐thaw shoot regeneration are a minimal change in the ratios of St/Si, the minimal change of the DBI of phospholipids and free fatty acids, as well as linoleic acid content (C18:2); and (3) inositol, raffinose, myristic acid (C14:0) and oleic acid (C18:1) were present in small quantities; however, they could be correlated to survival after cryopreservation, suggesting that they may be also involved in cryopreservation process.     

Stratification and intra- and inter-specific differences in fatty acid composition of common dolphin (Delphinus sp.) blubber: implications for dietary analysis

Sixty-five fatty acids were quantified in the blubber of common dolphins (Delphinus delphis, D. capensis) incidentally caught off the coast of southern California. Dolphins were grouped by sex, reproductive status and species, and a blubber sample was collected at a mid-lateral site located caudal to the trailing edge of the dorsal fin. Samples were divided horizontally into inner, middle and outer layers and gradients in fatty acid content (mass percent) were observed across the depth of the blubber. Levels of monounsaturated fatty acids were greatest in the outer layer, whereas levels of saturated and polyunsaturated fatty acids were greatest in the inner layer. Degree of stratification was greatest in sexually mature dolphins. Blubber of sexually immature, but physically mature, male dolphins was also highly stratified, suggesting that this difference may be attributed to differences in diet. Classification and regression tree analysis resulted in the fewest misclassifications when dolphins were grouped by species, possibly indicating that these closely related animals forage on different prey species. Dietary-derived fatty acids were typically selected as splitting criteria in classification and regression tree analyses, suggesting that the observed differences in fatty acid composition between the various groups of dolphins may be attributed to differences in diet.     

Effect of different carbon sources on membrane permeability,membrane fluidity,and fatty acid composition of a psychrotrophic Acinetobacter sp. HH1-1 during growth at low temperatures and after cold shock

The effect of different carbon sources on the ability of a psychrotrophic Acinetobacter sp., strain HH1-1, to grow at low temperatures and respond to cold shock was investigated by monitoring cell membrane permeability, membrane fluidity and fatty acid composition. Cells were grown in batch cultures with acetate, Tween 80 or olive oil as the sole source of carbon and incubated at 25, 5^°C or subjected to a 25 to 5 ^°C decrease in growth temperature (cold shock). Cell membrane changes were observed following cold shock for all carbon sources. Cells became leaky and membranes less fluid immediately after cold shock. The fatty acid composition of cells also varied significantly with carbon source. A higher content of oleic acid (cis-9-octadecenoic acid – 18:1) was observed in cells grown in the presence of Tween 80 and olive oil compared to cells grown in the presence of acetate. Increased content of palmitoleic acid (cis-9-hexadecenoic acid – 16:1) observed during growth at 5^°C and following cold shock indicated that this fatty acid may be important for growth at low temperatures. Acetate-grown cells responded more quickly to cold shock than did Tween 80 or olive oil-grown cells by restoring membrane fluidity and by taking K+ back into the cells. In addition, acetate-grown cells modified the content of fatty acid cis-9-hexadecenoic acid at 2h post cold shock as opposed to 24h post cold shock in cells grown in the presence of Tween 80 or olive oil. This research indicated that cells are most affected by rapid decreases in growth temperature and growth at low temperatures when cells utilized olive oil as the sole source of carbon.     

Collection, seminal characteristics and chilled storage of spermatozoa from three species of free-range flying fox (Pteropus spp.)

This study reports observations on the collection and characteristics of semen from free-range populations of flying fox in Brisbane, Australia. Semen was successfully recovered by electroejaculation from 107 of 115 wild flying foxes (Pteropus alecto, Pteropus poliocephalus and Pteropus scapulatus). A proportion of ejaculates collected from all three species contained seminal vesicle secretions, the incidence of which appeared related to breeding season. Ejaculate volume was small (5--160 microL), requiring a specialised collection vessel and immediate extension to avoid desiccation. Sperm morphological abnormalities and characteristics are described for the first time. In two species (P. scapulatus and P. alecto), sperm quality varied with breeding season. Dilution in Tris-citrate-fructose buffer and subsequent incubation (37 degrees C) of Pteropus semen for 2-3h appeared to have a negative impact on sperm motility and the percentage of sperm with intact plasma membranes and acrosomes and represents a concern for the potential development and use of assisted breeding technology in these species. Preliminary attempts to develop a short-term chilled preservation protocol for flying fox semen revealed that sperm viability (percentage motility and percentage live sperm with intact acrosomes) was significantly reduced after 102 h chilled storage at 5 degrees C; nevertheless, approximately 40% of the spermatozoa were still motile and contained intact acrosomes. Glycerol was neither protective nor detrimental to sperm survival during chilled storage. Microbial flora of the prepuce, urethra and semen of all species were isolated and their antibiotic susceptibility tested. Tetracycline, penicillin, ciprofloxacin, and ceftazidime were the most effective antibiotics in preventing growth of all identified bacteria; however, their effects on sperm survival were not investigated.     

Effect of exogenous fatty acids on growth,membrane fluidity,and phospholipid fatty acid composition in yeast

The growth response of a double-mutant fatty acid auxotroph of yeast Saccharomyces cerevisiae to exogenous saturated fatty acids of a homologous series from 12:0 to 16:0, each supplied with oleate, linoleate, linolenate, or cis-Δ¹¹- eicosenoate, cannot be explained in terms of the efficiency of incorporation of the fatty acids into phospholipids or alteration of membrane fluidity. There is, however, a negative correlation between growth and levels of 12:0 plus 13:0 in phospholipids, as well as a positive correlation between growth and levels of 14:0, 1 5:0, and 1 6:0. We, therefore, conclude that the predominant factor in these phospholipid fatty acyl chain modifications is maintenance of an optimal concentration of C14:0 through C16:0 in phospholipids of this organism.     

Changes in sugar content and fatty acid composition of in vitro sugar beet shoots after cold acclimation: influence on survival after cryopreservation

To cryopreserve sugar beet shoot tips using an encapsulation-dehydration technique, cold hardening of in vitro plants was needed to obtain high survival rates after freezing. Cold acclimation not only enhanced dehydration and freezing tolerance, but also induced several changes in sugar beet shoots. Plants contained greater amounts of sucrose, D-glucose and D-fructose and the fatty acid composition of lipids changed. Furthermore, the unsaturation level of membrane lipids, estimated by the (C_18:2 + C_18:1)/C_16:0 ratio, increased after cold hardening. These changes were correlated with better survival rates after cryopreservation.     

Mevinolinic acid biosynthesis by Aspergillus terreus and its relationship to fatty acid biosynthesis.

Mevinolinic acid, the open acid form of mevinolin, which is a metabolite of Aspergillus terreus, has been shown to be a competitive inhibitor of the enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase (Alberts et al., Proc. Natl. Acad. Sci. U.S.A. 77:3957-3961, 1980). The biosynthesis of mevinolinic acid was studied by examining the incorporation of [1-14C]acetate and [methyl-14C]methionine into the molecule. These isotopes were rapidly incorporated into mevinolinic acid, with [1-14C]acetate and [methyl-14C]methionine incorporation being linear for at least 10 and 30 min, respectively. A comparison of acetate incorporation into mevinolinic acid and fatty acids indicated that mevinolinic acid biosynthesis increased with a maximum between days 3 and 5 of growth; at this time cell growth had ceased and fatty acid biosynthesis was negligible. Hydrolysis of the mevinolinic acid and isolation of the products showed that [1-14C]acetate and [methyl-14C]methionine were incorporated into the 2-methylbutyric acid side chain as well as into the main (alcohol) portion of the molecule.     

Chemical and fatty acid composition of male mouflon (Ovis ammon musimon Pal.) meat

The aim of this research was to determine the effect of age on chemical and fatty acid composition of the male mouflon (Ovis ammon musimon Pal.) meat. The male mouflons included in the study originated from the North Adriatic island Rab, Croatia. Three age groups were included (young, sub-adult, adult). An effect of age on chemical composition was not found. However, higher percentage of total fat, dry matter and ash was determined in the meat of young mouflons. Predominating saturated fatty acids in mouflon meat were myristic, palmitic and stearic acids. Age significantly (p < 0.05) affected only the percentage of palmitic acid being the highest in the meat of young mouflons. Percentage of the predominant monounsaturated fatty acid, oleic acid, did not change significantly with age. A higher (p < 0.05) percentage of the predominant polyunsaturated fatty acid, linoleic acid, was found in the meat of sub-adult mouflons. The meat of the young mouflons had a higher (p < 0.05) percentage of conjugated linoleic acid. The meat of the sub-adult and adult mouflons was found to have a higher (p < 0.05) percentage of total saturated fatty acids. A lower (p < 0.05) ratio of polyunsaturated and saturated fatty acids was found in the meat of young than in the meat of sub-adult mouflons. The ratio of n-6 and n-3 was lower (p < 0.05) in the adult than in the sub-adult mouflons. Higher (p < 0.05) atherogenicity and thrombogenicity indices were found in the young rather than in the adult mouflons. Low fat content (<1 %) and favourable ratios of polyunsaturated, saturated, n-6 and n-3 fatty acids make the mouflon meat a good choice for human diet.