Reproductive period affects lipid composition and quality of fresh and stored spermatozoa in Turkeys

Semen of Turkeys between 31 and 52 weeks of age was analyzed to investigate the cause of reduction in Turkey fertility at the end of the reproductive period. Sperm motility and viability, lipid concentration, fatty acid composition and lipid peroxides were evaluated on fresh spermatozoa or spermatozoa stored for 48h at 4 degrees C. Fertility of fresh semen was also evaluated.Fertility obtained with fresh semen decreased at 44-47 weeks of age. Ageing was also accompanied by a decrease in sperm viability (at 47 weeks) and later by a decrease in motility of spermatozoa (at 52 weeks). Polyunsaturated fatty acids (PUFAs) were the first lipids of fresh spermatozoa affected by age, especially n-3 and n-9 PUFAs. Changes in these PUFAs were followed by a 30% increase in lipid peroxidation at 47 and 52 weeks of age and a reduction in phospholipid content at 52 weeks.In vitro storage did not cause lipid peroxidation in sperm obtained during the first half of the reproductive period but malondialdehyde (MDA) levels significantly increased in sperm obtained during the second half of this period. In vitro storage also decreased phospholipid content of spermatozoa from 41 weeks of age, and viability and motility regardless of age.In conclusion, lipid alteration mainly originating from PUFAs peroxidation could partly explain the decrease in semen quality and fertility observed with ageing. In addition, lipid peroxidation was increased during in vitro storage of spermatozoa from older Turkeys.     

Liquid storage of turkey semen: changes in quality parameters, lipid composition and susceptibility to induced in vitro peroxidation in control, n-3 fatty acids and alpha-tocopherol rich spermatozoa

The study considered two major aims: (a) to measure the changes in quality parameters, lipid composition and antioxidant activity occurring in turkey spermatozoa during liquid storage; (b) to determine if the enrichment of sperm in n-3 fatty acids and alpha-tocopherol affect sperm survival during storage. Turkey breeders were fed a control diet or an Omega3 diet enriched with fish oil and alpha-tocopheryl-acetate. Ejaculates were pooled (5ejaculates/pool; 4pools/treatment) and stored in vitro for 48h at 4 degrees C. Viability, motility, susceptibility to induced peroxidation and alpha-tocopherol content were measured in spermatozoa; lipid and phospholipid fatty acid composition were measured in spermatozoa and seminal plasma. The proportion of motile and viable spermatozoa significantly decreased, and the proportion of dead spermatozoa significantly increased. The susceptibility of turkey spermatozoa to induced peroxidation also significantly increased during storage. The enrichment of turkey spermatozoa with n-3 long chain PUFA and vitamin E by dietary treatment did not prevent the negative effect of storage on sperm quality and sensitivity to induced in vitro peroxidation; however, it was efficient in partially prevent the increase of sperm death, therefore the proportion of dead spermatozoa was higher in control (37.4%) compared to treated spermatozoa (31.7%) after 48h liquid storage. Major changes were recorded in the lipid composition of turkey spermatozoa during liquid storage in both experimental dietary groups, whereas no significant changes were measured in seminal plasma. In spermatozoa, a great loss in the phospholipid and free cholesterol content was measured. Moreover, the loss in total sperm phospholipid was associated to a peculiar and selective decrease in the bounded fatty acids: saturates and monounsaturates were greatly reduced and polyunsaturates did not change. As a consequence, the polyunsaturated to saturated fatty acid ratio increased during 48h liquid storage. The observed changes in the lipid and phospholipid-bound fatty acid composition of turkey spermatozoa occurring during liquid storage might be related to different events and have been discussed.     

Phospholipid fatty acid composition, vitamin E content and susceptibility to lipid peroxidation of duck spermatozoa

Recent studies on chicken semen have suggested that the lipid and fatty acid composition of spermatozoa may be important determinants of fertility. Phospholipid fatty acid composition, vitamin E content and in vitro susceptibility to lipid peroxidation of duck spermatozoa were investigated using GC-MS and HPLC based methods. The total phospholipid fraction of duck spermatozoa was characterized by high proportions of the n-6 polyunsaturated fatty acids arachidonic (20:4n-6), docosatetraenoic (22:4n-6) and docosapentaenoic (22:5n-6) acids but a substantial proportion of the n-3 fatty acid docosahexaenoic (22:6n-3) acid was also present. Palmitic (16:0) and stearic (18:0) fatty acids were the major saturates in sperm phospholipids. Among the phospholipid classes, phosphatidylserine (PS) had the highest degree of unsaturation due to very high proportions of 22:6n-3, 22:5n-6, 22:4n-6 and 20:4n-6, comprising together more than 75% of total fatty acids in this fraction. Phosphatidylethanolamine (PE) also contained high proportions of these four C(20-22) polyunsaturates, which together formed 60% of total fatty acids in this phospholipid. Spermatozoa and seminal plasma of duck semen were characterized by unexpectedly low content of vitamin E, being more than 4-fold lower than in chicken semen. In duck semen the major proportion of the vitamin E (>70%) was located in the spermatozoa. The very high proportion of 22:6n-3 in PS and PE fractions of duck sperm lipids and the comparatively low levels of vitamin E could predispose semen to lipid peroxidation. Nevertheless the in vitro susceptibilities to Fe2+-stimulated lipid peroxidation of duck and chicken spermatozoa were very similar. The results of the study suggest that increased superoxide dismutase and glutathione peroxidase activity and increased antioxidant activity of seminal plasma may compensate for the low levels of vitamin E to help protect the membranes of duck spermatozoa, which exhibit a high degree of unsaturation from oxidative stress.     

Changes in turkey semen lipids during liquid in vitro storage

The changes in lipid composition of spermatozoa and seminal plasma and changes in motility, viability, and morphological integrity of spermatozoa were measured in turkey semen diluted in Beltsville poultry semen extender and stored for 48 h (4 degrees C). The total phospholipid content of spermatozoa decreased during storage, while no quantitative decrease was observed in seminal plasma. More precisely, significant decreases in phosphatidylcholine, and to a lesser extent in sphingomyeline, phosphatidylserine, and phosphatidylinositol were observed in spermatozoa. The fatty acid profile of turkey spermatozoa partly reflected diet composition and had a high level of n-9 polyunsaturated fatty acids. Neither fatty acid profile nor free cholesterol were affected by storage. The lipid composition of seminal plasma was quite different from that observed in spermatozoa and was similar to the high density lipoprotein composition of chicken seminal plasma. In vitro storage did not significantly affect lipid classes and only small changes were observed in phospholipid classes of seminal plasma. The motility, viability, and morphological integrity of spermatozoa decreased during storage. These changes in phospholipid content may be explained by membrane phospholipid lysis followed by endogenous metabolism or by a complex combination of lysis, metabolism, and peroxidation. They are likely to affect semen quality and the success of in vitro storage severely.     

Effect of long-term supplementation with arachidonic or docosahexaenoic acids on sperm production in the broiler chicken

The possibility was investigated that dietary supplementation of the male chicken with long-chain polyunsaturated fatty acids of the n-6 and n-3 series may prevent the decrease in sperm output that normally occurs by 60 weeks of age. From 26 weeks of age, birds were raised on wheat-based diets supplemented with either maize oil (rich in linoleic acid, 18:2n-6), arasco oil (rich in arachidonic acid, 20:4n-6) or tuna orbital oil (rich in docosahexaenoic acid, 22:6n-3). The effects of the last two oils were investigated at two levels of vitamin E supplementation (40 and 200 mg kg(-1) feed). By 60 weeks of age, there was a small increase in the proportion of the main polyunsaturate of chicken sperm phospholipid, docosatetraenoic acid 22:4n-6, in chickens fed arasco oil diet compared with chickens given the maize oil diet, an effect that was potentiated at the higher dietary intake of vitamin E. Supplementation with tuna orbital oil significantly reduced the proportions of 20:4n-6 and 22:4n-6 in the sperm phospholipid and increased the proportion of 22:6n-3. The diet supplemented with tuna orbital oil and the lower level of vitamin E markedly depleted vitamin E from the tissues of the birds and decreased the concentration of vitamin E in the semen; these effects were largely prevented by the higher level of vitamin E in the diet. The susceptibility of semen to lipid peroxidation in vitro was increased in chickens fed arasco and tuna orbital oils with 40 mg vitamin E kg(-1) feed, but was reduced when 200 mg vitamin E kg(-1) feed was provided in the diet. The number of spermatozoa per ejaculate decreased by 50% between 26 weeks and 60 weeks of age in the birds fed the maize oil diet. This age-related decrease in the number of spermatozoa was almost completely prevented by feeding the birds with the oils enriched in either 20:4n-6 or 22:6n-3. Testis mass at 60 weeks of age was approximately 1.5 times greater in birds given of the arasco and tuna orbital oil diets compared with those given the maize oil diet.     

Changes in sperm quality and lipid composition during cryopreservation of boar semen

Egg yolks are commonly used in diluents in order to improve the freezability of semen. Two aspects of the role of lipids in boar semen freezability are reported in this article. The first one concerns the eventual exchanges of lipid components between the spermatozoa and the yolk-based diluent during cryopreservation. Two types of yolk have been considered as ingredients in diluents for cryopreservation: yolks with a standard fatty acid composition and yolks enriched in docosahexaenoic acid (DHA). The relation between lipid exchanges and the quality of fresh semen is considered. The other aspect concerns the possibility to enhance the freezability of boar spermatozoa by altering the plasma membranes under the influence of dietary fatty acids. Spermatozoa were damaged significantly by the cryopreservation cycle in all experiments. Spermatozoa with the best fresh quality had accumulated the largest quantity of lipids upon thawing. A general decrease in the proportion of polyunsaturated fatty acids was observed after thawing. The yolks enriched in n-3 fatty acids failed to improve the quality of sperm following cryopreservation. The proportion of DHA was significantly higher in spermatozoan phospholipids from thawed cells that had been in contact with n-3 yolks. A significant reduction in cholesterol was observed in spermatozoa after the cryopreservation cycle, which correlated with an increased number of acrosome-reacted cells and changes in the parameters of motility. The addition of 3% fish oil to the daily boar ration significantly increased the content of DHA (from 33 to 45% of the total fatty acids) in the spermatozoa. Ejaculate concentrations were significantly increased in the experimental group. DHA-enriched semen did not show improved freezability, at least not as assessed by in vitro parameters.     

Effect of docosahexaenoic acid and alpha-tocopherol enrichment in chicken sperm on semen quality, sperm lipid composition and susceptibility to peroxidation

The aim of the present experiment was to study the effect of fish oil and Vitamin E rich diets on semen production, sperm functions and composition in broiler breeders. The following parameters were measured: semen volume and concentration, sperm motility and viability, sperm susceptibility to induced peroxidation, sperm lipid and alpha-tocopherol contents. Dietary n-3 PUFA were successfully transferred into spermatozoan phospholipid by fish oil feeding according to the following main features: (a) the C22:6n-3 and C22:5n - 3 contents were increased, but C22:4n-6 remained the peculiar and major polyunsaturate; (b) the content and proportion of total PUFA did not change; (c) the proportional increase of n-3 PUFA was compensated by the decrease of n-6 PUFA, an increase in the proportion of n-9 fatty acids was also found. The sperm content of alpha-tocopherol was doubled increasing the dietary availability of the vitamin to 300 mg/kg of feed. The specific n-3 PUFA and Vitamin E enrichment of chicken sperm affected cell functions. Significant interactions between the two treatments were also found for some parameters. The best sperm quality condition in control sperm (rich mainly in n-6 PUFA) was found supplying 200mg Vitamin E/kg of feed to the male breeders, and in contrast in n-3 rich sperm supplying 300 mg Vitamin E/kg.     

Deficit of didocosahexaenoyl phospholipid in the eyes of larval sea bass fed an essential fatty acid deficient diet

Larval sea bass Dicentrarchus labrax of 27 days old were reared on Artemia enriched with Super Selco©, Tuna Orbital Oil or Yeast. The first diet is commonly used in mariculture for larval rearing, the second diet was designed to deliver an optimal docosahexaenoic acid (22: 6n-3) to eicosapentaenoic acid (20: 5n-3) ratio, and the third diet was deficient in docosahexaenoic acid (22: 6n-3). The eyes of these larvae were analysed after 28 days and the molecular species of the three main phospholipid classes, phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylserine (PS) determined. Eyes from larvae fed Artemia enriched with yeast showed large decreases in molecular species containing 22: 6n-3 compared to those supplemented with tuna orbital oil, most notably in 16: 0/22: 6n-3 PC which fell from 10.6 to 0.4%, in 22: 6n-3/22: 6n-3 and 18: 1/22: 6n-3 PE which fell from 29.6 to 0.3% and from 10.8 to 1.1% respectively, and in 22: 6n-3/22: 6n-3 PS which fell from 34.3 to 1.7%. Molecular species containing all other fatty acids, and especially 20: 5n-3, were elevated in eyes from the yeast-supplemented fish. In larvae fed Artemia enriched with Super Selco, amounts of eye 22: 6n-3/22: 6n-3 phospholipid were slightly lower in all three phospholipid classes compared to eyes from the tuna orbital oil-supplemented larvae. There was also a trend of decreased saturated fatty acid/22: 6n-3 and monounsaturated fatty acid/22: 6n-3 molecular species in all classes from the Super Selco-supplemented fish, the deficits being made up with molecular species containing 20: 5n-3 and 22: 5n-3. These results are discussed in relation to larval viability with particular respect to visual function.     

Dietary omega-3 fatty acids (fish oils) have limited effects on boar semen stored at 17 °C or cryopreserved

To evaluate the influence of dietary supplementation of omega-3 polyunsaturated fatty acids (n-3 PUFA) on storage of boar semen, three experiments were conducted: two involved long-term, fresh semen storage (Exp. 1 and Exp. 2), whereas the other involved cryopreservation (Exp. 3). Boars were allocated randomly to three dietary treatments (for 6-7 mo). In addition to a daily allowance of 2.5 kg of a basal diet, they received: 1) 62 g of hydrogenated animal fat (AF); 2) 60 g of menhaden oil (MO), containing 18% docosahexanoic acid (DHA) and 15% eicosapentanoic acid (EPA); or 3) 60 g of tuna oil (TO), containing 33% DHA and 6.5% EPA. In Experiment 1 (n = 26) and Experiment 2 (n = 18), semen was cooled and stored in vitro for several days at 17 °C before assessment, whereas in Experiment 3 (n = 18), viability, motility, acrosomal integrity, susceptibility to peroxidation (LPO), and DNA fragmentation were determined in fresh and frozen-thawed sperm. In Experiment 1, sperm from boars fed TO had better resistance to fresh storage; even after 7 or 9 d of storage at 17 °C, there were more (P = 0.03) motile sperm in boars fed TO (>60%) than in those fed AF or MO. In Experiment 2, fish oil supplementation did not influence any aspect of sperm quality during semen storage (P > 0.10). In Experiment 3, cryopreservation decreased the proportion of motile and viable frozen-thawed sperm as well as acrosomal integrity and increased DNA fragmentation and LPO (P < 0.01) relative to fresh semen, although sperm quality was unaffected by treatments (P > 0.09). In conclusion, although adding fish oil to the diet failed to significantly improve the quality of cryopreserved boar sperm, inconsistent responses of long-term storage of cooled sperm to dietary n-3 PUFA supplementation warrant further investigation.     

Effects of n-3 polyunsaturated dietary supplementation on the reproductive capacity of male turkeys

To measure the effects of dietary n-3 polyunsaturated fatty acid (PUFA) supplementation on the reproductive capacity of adult male turkeys in industrial flocks, the males of 22 commercial farms were fed either a standard diet or a fish oil diet enriched in n-3 PUFAs. The fatty acid composition of the spermatozoa and reproductive performance were measured throughout the reproductive period. The fish oil diet very effectively increased the percentage of n-3 fatty acids (FA) (22:5n-3 and 22:6n-3) in spermatozoa and correspondingly decreased the percentage of n-6 PUFAs (20:4-6 and 22:4n-6): the n-3/n-6 ratio in spermatozoa fatty acids were 0.04-0.07 with the standard diet and 0.32-0.4 with the fish oil diet. These changes did not affect the spermatozoa content of n-9 PUFAs, particularly of 22:3n-9 which is abundant in turkey spermatozoa (9-12% of the total fatty acids). The supplementation was effective in the middle as at the end of the reproductive period. The reproductive capacity of males was modified by the diet and the positive effect of the n-3 supplemented diet increased with age (increase in hatching rates of nearly 2 points at 48-58 weeks for males fed fish oil diet). These results indicate that an increase in the dietary ratio of n-3/n-6 PUFAs is valuable to sustain the reproductive capacity of male turkeys especially when they are getting older.     

Differential involvement of rat sperm choline glycerophospholipids and sphingomyelin in capacitation and the acrosomal reaction

Rat spermatozoa main lipid classes and their fatty acids were studied to assess their possible changes in capacitation and the acrosomal reaction (AR), induced in vitro. Capacitation-associated protein tyrosine phosphorylation, and the efflux of 30% of the total cholesterol from gametes to the medium, took place concomitantly with the release of a similar percentage, i.e., a larger amount, of the total phospholipid, mostly after hydrolysis of the major choline glycerophospholipids (CGP). Main medium lipid metabolites after capacitation were lyso-CGP and polyenoic fatty acids typical of CGP (22:4n-9, 22:5n-6), as free fatty acids (FFA). The AR, induced by a calcium ionophore, resulted in further phospholipid loss, but the produced metabolites remained in the gametes. CGP decrease in AR accounted for some additional FFA and lyso-CGP, but mostly for (22:5n-6-rich) diglycerides. Hydrolysis of sphingomyelins (SM) to ceramides also occurred, mostly affecting species with very long chain polyenoic fatty acids. Quantitatively, CGP and SM were the lipid classes decreasing the most after capacitation and AR, respectively. The massive cholesterol and phospholipid loss from the gametes during capacitation is thus associated with protein phosphorylation, a function that has been located to the sperm tail. The lipid metabolites produced during AR, by accumulating in the gamete heads, could be implicated in sperm–oocyte interactions.     

Quality and lipid composition of spermatozoa in rabbits fed DHA and vitamin E rich diets

The effects of fish oil (FO) and vitamin E (vE) dietary supplementation on semen quality, sperm susceptibility to lipid peroxidation, tocopherols content and fatty acid profiles were studied in rabbits. Fifty-two rabbit bucks randomly divided in four groups received a control diet and enriched diets containing either FO (1.5%, w/w), vE (200 mg/kg) or both. Semen volume, concentration, motility and viability were analysed at various time-points and the lipid composition was assessed on sperm cells. The phospholipid fatty acid profile was determined: n-6 PUFA were the major fatty acids found, with a proportion of 42%, whereas the n-3 PUFA accounted for nearly 1%, mainly represented by C22:6n-3 (docosahexaenoic acid, DHA). FO supplementation produced a seven-fold increase in the content of DHA in sperm phospholipids and a comprehensive rearrangement of the phospholipid fatty acid composition, while an unexpected negative effect of feeding high level of vE on the proportion of total PUFA was found. Despite the remarkable changes observed in sperm lipid composition, semen quality parameters were not affected by the dietary treatments and the interaction between the two dietary supplements had a significant effect only on sperm concentration. An increase in semen production by ageing and a concomitant rise in sperm susceptibility to in vitro peroxidation was found. α- and δ-tocopherol, present in rabbit sperm in similar amount, were not affected by dietary treatment. δ-tocopherol content had a significant linear negative regression with age and showed a significant negative correlation with the susceptibility to peroxidation values.     

Phosphatidylinositol 3-kinase pathway regulates sperm viability but not capacitation on boar spermatozoa

Phosphatidylinositol 3-kinase (PI3-K) plays an important role in cell survival in somatic cells and recent data pointed out a role for this kinase in sperm capacitation and acrosome reaction (AR). This study was undertaken to evaluate the role of PI3-K pathway on porcine spermatozoa capacitation, AR, and viability using two unrelated PI3-K inhibitors, LY294002 and wortmannin. In boar spermatozoa, we have identified the presence of PDK1, PKB/Akt, and PTEN, three of the main key components of the PI3-K pathway. Incubation of boar sperm in a capacitating medium (TCM) caused a significant increase in the percentage of capacitated (25 +/- 2 to 34 +/- 1% P < 0.05, n = 6) and acrosome reacted (1 +/- 1 to 11 +/- 1% P < 0.01, n = 6) spermatozoa compared with sperm in basal medium (TBM). Inhibition of PI3-K did affect neither the capacitation status nor AR nor protein p32 tyrosine phosphorylation of boar spermatozoa incubated in TBM or TCM. Boar sperm viability in TBM was significantly decreased by 40 and 20% after pretreatment with LY294002 or wortmannin, respectively. Similar results were observed after incubation of boar spermatozoa in TCM. Treatment of boar spermatozoa with the analog of cAMP, 8Br-cAMP significantly prevented the reduction on sperm viability. Our results provide evidence for an important role of the PI3-K pathway in the regulation of boar sperm viability and suggests that other signaling pathways different from PI3-K must be activated downstream of cAMP to contribute to regulation of sperm viability. Finally, in our conditions the PI3-K pathway seems not related with boar sperm capacitation or AR.     

Influence of storage time on functional capacity of flow cytometrically sex-sorted boar spermatozoa

Sex-sorting of boar spermatozoa is an emerging biotechnology, still considered suboptimal owing to the slowness of the process, which requires long sorting periods to obtain an adequate number of spermatozoa to perform a non-surgical insemination. This period involves storage of sorted cells that could impair their functional capacity. Here, we have studied how the storage of sex-sorted boar spermatozoa affects their functional capacity. Sorted spermatozoa were assessed at various times (0, 2, 5h or 10h) during storage after sorting and compared with diluted and unsorted spermatozoa for sperm motility patterns, plasma membrane and acrosomal integrity and their ability to penetrate homologous IVM oocytes. Sex-sorted sperm motility and membrane integrity only decreased significantly (p<0.05) by the end of the storage period (10h) compared to unsorted spermatozoa. Sperm velocity, ALH and Dance increased significantly (p<0.05), immediately post-sorting, returning to unsorted sperm values during storage. Acrosome integrity was not seriously affected by the sorting process, but decreased (p<0.05) during storage after sorting. Sorted spermatozoa stored 2h after sorting did not differ from unsorted in penetration rates and numbers of spermatozoa per oocyte, reaching the highest (p<0.05) penetration rates and sperm numbers per oocyte, when co-cultured for 6 or more hours. Non-storage or storage for 5h or 10h negatively (p<0.05) affected sperm penetration ability. In conclusion, although flow cytometrically sex-sorted spermatozoa are able to maintain motility, viability and acrosomal integrity at optimal levels until 10h of storage after sorting, fertilizing ability is maintained only over shorter storage times (<5h).     

Age-associated changes in central nervous system glycerolipid composition and metabolism

In this review, changes in brain lipid composition and metabolism due to aging are outlined. The most striking changes in cerebral cortex and cerebellum lipid composition involve an increase in acidic phospholipid synthesis. The most important changes with respect to fatty acyl composition involve a decreased content in polyunsaturated fatty acids (20:4n-6, 22:4n-6, 22:6n-3) and an increased content in monounsaturated fatty acids (18:1n-9 and 20:1n-9), mainly in ethanolamine and serineglycerophospholipids. Changes in the activity of the enzymes modifying the phospholipid headgroup occur during aging. Serine incorporation into phosphatidylserine through base-exchange reactions and phosphatidylcholine synthesis through phosphatidylethanolamine methylation increases in the aged brain. Phosphatidate phosphohydrolase and phospholipase D activities are also altered in the aged brain thus producing changes in the lipid second messengers diacylglycerol and phosphatidic acid.     

Combined effect of DHA and alpha-tocopherol enrichment on sperm quality and fertility in the turkey

The aim of the present study was to characterize the dietary effects of n-3 LC-PUFA and alpha-tocopheryl acetate (vE) on the quality, phospholipid fatty acid composition, alpha-tocopherol content (alpha-T) and in vitro susceptibility to lipid peroxidation in turkey semen. Fertility of fresh semen was also evaluated. Male turkeys were randomly divided and fed either a control diet or a fish oil and vE rich diet (FO diet) from 40 to 60 weeks of age. The FO diet increased the proportion of n-3 fatty acids in spermatozoa and as a consequence the (n-3)/(n-6) ratio also increased. These changes did not affect the proportion of n-9 PUFAs, particularly of C22:3n-9, in semen. The sperm content of alpha-T was dependent by the dietary supplementation of the vitamin and the sperm content was more than doubled supplying 120 mg kg(-1) of feed to the males compared to the 60 mg kg(-1) of feed in the control diet. In agreement with the major content of alpha-T in spermatozoa collected from the FO group were significantly less susceptible to in vitro induced oxidation. The reproductive capacity of the male breeders was not affected by the diet; however the result is considered of some relevance for field conditions where even very small changes have economic interest being applied to large bird population.     

Determination of acrosin activity of boar spermatozoa by the clinical method: optimization of the assay and changes during short-term storage of semen

A clinical assay to evaluate total acrosin activity developed for human semen has been optimized for use in boar spermatozoa. The main modifications included a decrease of sperm number per assay from 1.0 to 10.0 x 10(6) to 12.5 to 75.0 x 10(3) spermatozoa, and the time of incubation from 180 to 60 min. Linearity of response for differing quantities of spermatozoa was maintained. Extensive washing of spermatozoa was necessary to eliminate seminal plasma, the source of acrosin inhibitors. Seminal plasma that was diluted 1000 times inhibited acrosin activity by about 50%. To abolish the inhibitory effect of seminal plasma it was necessary to use 25,000-fold dilution. Total acrosin activity of boar spermatozoa was about 100 times higher than that of human spermatozoa. Acrosin activity of boar spermatozoa in extended semen decreased during 7 d of storage. These results indicate that the clinical assay of acrosin activity can be used for boar spermatozoa to evaluate the quality of boar semen.     

Enrichment of platelet phospholipids with eicosapentaenoic acid and docosahexaenoic acid inhibits thromboxane A2/prostaglandin H2 receptor binding and function

Human platelet lipids were enriched in vitro with different amounts of either docosahexaenoic acid (22:6n-3), eicosapentaenoic acid (20:5n-3) or linoleic acid (18:2n-6). Of the total fatty acid incorporated, between 82 and 95% was associated with the phospholipid (PL) fraction, with the remainder as either neutral lipid or hydroxy fatty acid. Within the PL fraction, the majority (64% of total) of each fatty acid was incorporated into phosphatidylcholine. It was found that platelet aggregation induced by the thromboxane A2/prostaglandin H2 mimetic (15S)-hydroxy-11,9-(epoxymethano)prosta-5Z,13E-dienoic acid (U46619) was inhibited after PL enrichment with 22:6n-3 or 20:5n-3, but not after 18:2n-6 enrichment. The specificity of 22:6n-3 and 20:5n-3 for U46619 activation was demonstrated by the finding that neither fatty acid significantly inhibited thromboxane A2/prostaglandin H2-independent aggregation induced by A23187 or thrombin. Furthermore, enrichment with 22:6n-3 or 20:5n-3 resulted in inhibition of [3H]U46619 specific binding, while enrichment with 18:2n-6 did not affect binding. Scatchard analysis revealed that thromboxane A2/prostaglandin H2 receptor affinity for [3H]U46619 decreased 4.8-fold following 22:6n-3 incorporation. These results demonstrate that platelet phospholipid enrichment with 22:6n-3 or 20:5n-3 results in a selective inhibition of thromboxane A2/prostaglandin H2 receptor function.     

Decrease of Brain Phospholipid Synthesis in Free-Moving n-3 Fatty Acid Deficient Rats

Abstract: The autoradiographic method with [¹⁴C]-docosahexaenoic acid ([¹⁴C]22:6 n-3) was used to determine whether a diet deficient in n-3 fatty acids, inducing a decrease in 22:6 n-3 circulating level, was associated with changes in local rates of phospholipid synthesis in the rat brain. As compared with rats fed a normal diet (peanut plus rapeseed oil), a n-3 fatty acid deficiency [peanut oil group (P group)] induced a generalized decrease (?35 to ?76%) of 22:6 n-3 incorporation rates into phospholipids in all the regions examined. This effect was confirmed by using [³H]22:6 n-3 infusion by biochemical analysis and quantifications corrected for the contribution of docosahexaenoate derived from lipid store recycling to the unesterified pool, taken as the precursor pool for phospholipid synthesis in the whole brain. In normal or n-3 fatty acid-deficient rats, the values of the brain-to-plasma 22:6 n-3 specific activity ratio (Ψ) were similar (0.03), indicating that a considerable endogenous source of 22:6 n-3 (97%), likely derived from phospholipid degradation, dilutes the specific activity of the tracer coming from plasma. Using the specific activity of 22:6 n-3 in plasma instead of brain would thus lead to a gross underestimation of the rate of phospholipid synthesis. The results also demonstrate that the pattern of ¹⁴C or ³H distribution in brain lipids was not modified by the n-3 fatty acid-deficient diet. The major lipids labeled were phospholipids, particularly phosphatidylethanolamine. Nevertheless, the unesterified 22:6 n-3 concentrations in plasma and brain were significantly reduced (eight- and threefold, respectively) in the P group. In addition, the proportion of 22:6 n-3 in the brain total lipid fraction, total phospholipids, and phosphatidylcholine, -ethanolamine, and -serine was significantly decreased in n-3 fatty acid-deficient rats. This was partially compensated for by an increase in the 22:5 n-6 level. These results are discussed in relation to the limitation of 22:6 n-3 use to quantify, by the quantitative autoradiographic method, changes in local rates of phospholipid synthesis in rat brain.     

Seasonal changes in lipid classes and fatty acid composition in the digestive gland of Pecten maximus

Seasonal variations in lipid classes and fatty acid composition of triacylglycerols and phospholipids in the digestive gland of Pecten maximus were studied over a period of 16 months. Acylglycerols predominated (19-77% of total lipids), in accordance with the role of the digestive gland as an organ for lipid storage in scallops. Seasonal variations were mainly seen in the acylglycerol content, while phospholipids (2.5-10.0% of total lipids) and sterols (1.9-7.4% of total lipids) showed only minor changes. The most abundant fatty acids were 14:0, 16:0, 18:0, 16:1(n-7), 18:1(n-9), 18:1(n-7), 18:4(n-3), 20:5(n-3) and 22:6(n-3) and these showed similar seasonal profiles in both, triacylglycerol and phospholipid fractions. In contrast to the phospholipid fraction, the triacylglycerol fraction contained more 20:5(n-3) than 22:6(n-3). In three phospholipid samples we noted a high percentage of a 22-2-non-methylene-interrupted fatty acid, previously described to have a structural role in several bivalve species. The main polyunsaturated fatty acids displayed important seasonal variations parallel to those of the acylglycerols, suggesting good nutritional conditions. A positive correlation existed between the level of saturated fatty acids and temperature, whereas the levels of polyunsaturated fatty acids correlated negatively with temperature.