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Association between membrane lipid composition and progressive motility in bovine spermatozoa

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In the dairy breeding industry, prediction of bull fertility in artificial insemination (AI) is important for efficient and economically sustainable production. However, it is challenging to identify bulls with superior fertility applying conventional in vitro sperm assays. In the present study, sperm functionality was investigated to identify a multivariate model that could predict fertility. Two groups of young Norwegian Red bulls were selected, one with inferior fertility (18 bulls) and one with superior fertility (19 bulls) based on non-return rate after 56 days (NR56). Frozen-thawed semen doses were analysed for sperm chromatin integrity, viability, acrosome integrity, motility, and ATP content. A targeted approach was used to study intracellular concentrations of amino acids and trace elements in viable sperm cells. Significant differences between the two groups of bulls were observed, both for sperm functional attributes and intracellular concentrations of metabolites. Pearson correlation analyses indicated a negative relationship between NR56 and chromatin integrity parameters, DNA fragmentation index (DFI) and high DNA stainability (HDS). Several motility parameters correlated positively with NR56. The concentrations of cysteine and glutamic acid in sperm cells correlated negatively with NR56, while the concentrations of aspartic acid, leucine and serine showed a positive NR56-correlation. The sperm intracellular concentrations of the trace elements Fe, Al and Zn, correlated negatively with NR56. Correlations were observed between several sperm parameters and metabolites. Stepwise multiple regression analysis indicated that the best predictor of NR56 was a model containing %DFI, together with the intracellular sperm concentration of aspartic acid, Fe and Zn. This model explained 59% of the variability in NR56.
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Background: Application of frozen-thawed semen is an important tool for improving the vivo fertility, but the process of freezing and thawing causes significant damage to spermatozoa. Objective: The aim of this study was to evaluate the effect of cryopreservation on CASA characteristics, mitochondrial transmembrane potential, plasma, and acrosome integrities, morphology and in vivo fertility of buffalo bull spermatozoa. Materials and methods: Semen was collected from four mature buffalo bulls with artificial vagina at 42 °C. Ejaculates having > 1 mL volume, > 60 % sperm visual motility and > 0.5 x 109 sperm/mL concentration from each bull were diluted in Tris-citric acid egg yolk glycerol extender (TCA) making two aliquots per bull for analysis at post dilution and cryopreserved respectively. Results: Analysis of variance (ANOVA) showed that the process of freezing and thawing significantly reduced (P < 0.05) CASA characteristics including total motility (TM, %), progressive motility (PM, %), rapid velocity (RV, %), average path velocity (VAP, µm/sec), straight line velocity (VSL, µm/sec), curvilinear velocity (VCL, µm/sec), beat cross frequency (BCF, Hz), straightness (STR, %) and linearity (LIN, %). Furthermore, the process of freezing and thawing significantly reduced (P < 0.05) subjective motility (SM, %), Supra-vital plasma membrane integrity (SVPMI, %), high mitochondrial membrane potential (HMMP, %), viable spermatozoa with intact acrosome (V/IACR, %). Moreover, it was observed that the freezing thawing process significantly decreased the in vivo fertility (%, 50.35 % vs. 61.39 %; P < 0.05) as compared to post diluted semen. Conclusion: It is concluded that the process of freezing and thawing significantly reduced semen quality and in vivo fertility of buffalo bull in terms of various functional parameters.
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Biological membranes are key elements for the maintenance of cell architecture and physiology. Beyond a pure barrier separating the inner space of the cell from the outer, the plasma membrane is a scaffold and player in cell-to-cell communication and the initiation of intracellular signals among other functions. Critical to this function is the plasma membrane compartmentalization in lipid microdomains that control the localization and productive interactions of proteins involved in cell signal propagation. In addition, cells are divided into compartments limited by other membranes whose integrity and homeostasis are finely controlled, and which determine the identity and function of the different organelles. Here, we review current knowledge on membrane lipid composition in the plasma membrane and endomembrane compartments, emphasizing its role in sustaining organelle structure and function. The correct composition and structure of cell membranes define key pathophysiological aspects of cells. Therefore, we explore the therapeutic potential of manipulating membrane lipid composition with approaches like membrane lipid therapy, aiming to normalize cell functions through the modification of membrane lipid bilayers.
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Background There is speculation that beef bull semen quality is inferior to that of dairy bulls although few scientific studies are available in the literature. The aim of this study was to evaluate sperm quality in beef bull semen and to determine which parameters could be indicative of fertility after insemination. Sperm quality, assessed by computer assisted sperm motility analysis and flow cytometric evaluation of membrane integrity, levels of reactive oxygen species, mitochondrial membrane potential, acrosome status and DNA fragmentation index, was evaluated in beef and dairy bull semen. ResultsFor beef bulls, normal morphology (r = 0.62, P < 0.05) and WOBBLE (r = 0.57, P < 0.05) were significantly correlated with 56-day non-return rate, whereas sperm quality was not significantly correlated with the fertility index score for dairy bulls. Membrane integrity (46 ± 8.0% versus 40 ± 11%, P < 0.05), normal morphology (87 ± 6% versus 76 ± 8%; P < 0.05), and high respiratory activity (52 ± 13 versus 12 ± 4%; P < 0.001) were higher for dairy bulls than for beef bulls. The DNA fragmentation index was lower for dairy bull spermatozoa than beef (3.8 ± 1.1% versus 6.1 ± 2.9%; P < 0.01), whereas some sperm kinematics were higher. Multivariate analysis indicated that type of bull (beef versus dairy) had an impact on sperm quality. Conclusions Different assays of sperm quality may be needed for appropriate analysis of beef and dairy bull semen. These finding could be important for cattle breeding stations when evaluating semen quality.
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Artificial insemination with cryopreserved semen enables affordable, large-scale dissemination of gametes with superior genetics. However, cryopreservation can cause functional and structural damage to spermatozoa that is associated with reactive oxygen species (ROS) production, impairment of sperm motility and decreased fertilizing potential, but little attention has been paid to protein changes. The goal of this study was to investigate the oxidative modifications (measured as carbonylation level changes) of bull spermatozoa proteins triggered by the cryopreservation process. Flow cytometry and computer-assisted sperm analysis were used to evaluate changes in viability, ROS level and motility of spermatozoa. Western blotting, in conjunction with two-dimensional electrophoresis (2D-oxyblot) and matrix-assisted laser desorption/ionization time-of-flight/time-of-flight spectrometry, was employed to identify and quantify the specifically carbonylated spermatozoa proteins. Cryopreservation decreased motility and viability but increased the number of ROS-positive cells. We identified 11 proteins (ropporin-1, outer dense fiber protein 2, glutathione S-transferase, triosephosphate isomerase, capping protein beta 3 isoform, actin-related protein M1, actin-related protein T2, NADH dehydrogenase, isocitrate dehydrogenase, cilia- and flagella-associated protein 161, phosphatidylethanolamine-binding protein 4) showing differences in protein carbonylation in response to cryopreservation. The identified proteins are associated with cytoskeleton and flagella organization, detoxification and energy metabolism. Moreover, almost all of the identified carbonylated proteins are involved in capacitation. Our results indicate for the first time that cryopreservation induces oxidation of selected sperm proteins via carbonylation. We suggest that carbonylation of sperm proteins could be a direct result of oxidative stress and potentially lead to disturbances of capacitation-involved proteins or could indicate cryopreservation-induced premature capacitation.
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The fatty acid composition of the sperm membrane is an important factor involved in the overall sperm quality, including motility. However, in the canine species, the exact composition of the plasma membrane is still unknown. Therefore, the purpose of this study was to evaluate the plasma membrane lipid composition of motile sperm cells and to compare it with asthenospermic samples, as an attempt to determine possible involvements of membrane lipids in dog sperm cell motility. The sperm-rich fraction of ten mature dogs was collected, and samples were subjected to density gradient centrifugation by Percoll(®) , in order to separate motile and asthenospermic samples. Processed semen samples were evaluated for sperm motility, plasma and acrosome membrane integrity, mitochondrial activity and susceptibility to oxidative stress. Lipid plasma membrane composition was identified by mass spectrometry (MALDI-MS). The motile sperm samples presented the following phospholipids in a high frequency in the plasma membrane: phosphatidylcholine 38:4 (composed of stearic and arachidonic fatty acids), phosphatidylcholine 36:1 (stearic and oleic fatty acids), phosphatidylethanolamine 34:4 (myristic and arachidonic fatty acids), glycerophosphatidic acid 36:4 (palmitic and arachidonic fatty acids), phosphatidylcholine 40:4 plasmanyl and phosphatidylcholine 40:5 plasmenyl. Furthermore, no lipid markers were found in the asthenospermic samples. Results also indicate that differences on plasma membrane composition between motile and asthenospermic samples are crucial factors for determining sperm motility, sperm functionality and susceptibility to oxidative stress. In conclusion, plasma membrane lipid composition varies considerable between motile and asthenospermic samples. Therefore, lipid markers of sperm motility can be considered, such as phosphatidylcholine, phosphatidylethanolamine, phosphatidylcholine plasmanyl, phosphatidylcholine plasmenyl and phosphatidic acid.
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Revue selective de la pathologie subcellulaire des spermatozoides humains et analyse de son action limitante sur la fertilite. On insiste sur l'importance clinique de l'examen ultrastructural du sperme
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Since the inception of bovine semen cryopreservation, egg yolk and milk based extenders have been used to protect sperm from the detrimental effects of cooling and freezing. In recent years, demand for alternatives to conventional commercial extenders has arisen as the risk of introducing exotic diseases through transporting egg yolk based products has been recognized. Egg yolk can also interfere with sperm evaluation and the presence of particulate material in the extender may reduce fertility. Soybeans contain lecithin, a phospholipid fraction that can substitute for high molecular weight lipoprotein and phospholipids from egg yolk and prevent or ameliorate damage to the sperm plasma membrane that occurs during extension, cooling, and cryopreservation. Soy lecithin based extenders have been evaluated for processing and freezing bovine semen, although extender from soybean milk has not been studied as extensively. Commercially available soy lecithin based extenders are used increasingly but remain under scrutiny and are not universally accepted. With these observations in mind, this review is intended to examine effects of conventional cryopreservation procedures, methods of assessment, and potential for developing soybean extract as an acceptable alternative to traditional egg yolk and milk based extenders for bull sperm cryopreservation.
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Summary We examined the association between progressive motility of spermatozoa and in vitro fertilization (IVF) competence of bovine ejaculates. Fresh semen was evaluated using a computerized sperm quality analyzer for bulls using progressive motility as the primary parameter. Ejaculates with high progressive motility (HPM; >81%) were compared with those with low progressive motility (LPM; <62%). Semen concentration and sperm velocity were lower (P < 0.05) in HPM versus LPM ejaculates. Volume and motile sperm concentration did not differ between groups (P > 0.05). Examination of sperm morphology revealed a higher proportion of spermatozoa with abnormal morphology (P < 0.01) in LPM versus HPM ejaculates, the predominant abnormal feature being a bent tail (P < 0.05). Sperm viability, acrosome integrity and DNA fragmentation did not differ between HPM and LPM samples. Mitochondrial membrane potential was higher (P < 0.01) in HPM versus LPM semen. Zinc concentrations in the seminal plasma correlated with progressive motility (R2 = 0.463, P = 0.03). In addition, representative ejaculates from HPM and LPM groups were cryopreserved in straws and used for IVF. The proportions of embryos cleaved to 2- and 4-cell stages (88.1 ± 1.1 versus 80.5 ± 1.7, P = 0.001) and developed to blastocysts (33.5 ± 1.6 versus 23.5 ± 2.2, P = 0.026) were higher for HPM than LPM semen. The total cell number of embryos and blastocyst apoptotic index did not differ between groups. Although sperm progressive motility is associated with IVF competence, further examination is required to determine whether progressive motility can serve as a predictor of semen fertilization capacity in vivo.
Plasma membranes were isolated from rat and mouse livers, a transplanted rat hepatoma, two transplanted mouse hepatomas and spontaneous mouse hepatomas.Phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, lysophosphatidylcholine, free fatty acids and triglycerides were separated and their fatty acid profiles determined.The various lipid classes of rat and mouse liver plasma membranes each demonstrated more or less specific fatty acid profiles. The number of double bonds decreased in the order: phosphatidylserine ⩾ phosphatidylethanolamine > phosphatidylinositol > phosphatidylcholine ⪢ sphingomyelin and lysophosphatidylcholine. Small species differences were noted in most lipid classes. A marked sex difference was observed in sphingomyelin of mouse liver membranes but in none of the phospholipids of rat liver membranes.The increased cholesterol content of all hepatoma versus liver plasma membranes was accompanied by a decrease of fatty acyl poly-unsaturation in most lipid classes of the rat hepatoma but not of the mouse hepatoma membranes. The fatty acid profiles of the mouse hepatoma membranes deviated much less from those of mouse liver than did the pattern of rat hepatoma versus rat liver.The results were discussed in relation to lipid fluidity of which fatty acyl unsaturation and cholesterol are the main parameters.
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Fertility is a multiparametric phenomenon that relies on the use of semen of sufficient quality and quantity, accurate timing and method of insemination, and appropriate herd management. When using artificial insemination (AI), the dairy producer must manage a range of these factors, including heat detection, timing of insemination in relation to estrus, and correct handling of the frozen straws. Manual semen analysis using a light microscope has been the standard method for analysis in most semen production centers (SPCs). Quality control (QC) is the assurance that each batch of straws has undergone semen analysis to verify that the sample is likely to be fertile. Computer-assisted sperm analysis (CASA) is a powerful tool for the objective assessment of sperm motility and is used for evaluating semen quality. Flow cytometry analyzes cells suspended in a stream of fluid passing at high velocity in front of one or several lasers.
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Season-induced variation in fatty acid and cholesterol composition in bovine semen has been associated with semen quality. Given the specific roles of the various semen compartments (seminal fluids, sperm head, and sperm tail) in fertilization, we hypothesized that environmental-stress-induced alterations in the lipid composition of a specific compartment might impair semen quality and sperm function. Semen samples were collected from five mature Holstein-Friesian bulls during the summer (August to September) and winter (December to January). Semen was evaluated by computerized sperm-quality analyzer, calibrated for bulls' semen, and centrifuged to separate the spermatozoa from the seminal fluids. The spermatozoal fraction was sonicated to separate the sperm head and tail compartments. Cold lipid extraction was performed with chloroform:methanol (2:1, vol/vol). Lipids were identified and quantified by gas chromatography. Seasonal variation was found in both physiological and structural parameters. The proportion of spermatozoa defined as morphologically normal was higher in the winter, with higher motility, progressive motility, and velocity relative to summer samples. Lipid composition within fractions varied between seasons with prominent impairment in the tail compartment, characterized by high saturated fatty acid, low polyunsaturated fatty acid, and low cholesterol concentrations during the summer. Given the association between alterations in lipid composition and reduced sperm motility and velocity during the summer, it is suggested that lipid composition might serve to predict sperm quality.
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Der Gehalt an mehrfach ungesättigten Fettsäuren in Relation zur Motilität der Spermatozoen Die absoluten Mengen der in den Phospholipiden gebundenen Fettsäuren wurden bei Fertilitätsstörungen gaschromatographisch analysiert. Unsere Ergebnisse zeigen, daß zwischen dem Gehalt an Docosahexaensäure (22:6) und der Spermatozoendichte sowie der Motilität eine signifikante Beziehung besteht. Diese Erkenntnisse lassen vermuten, daß die Peroxidbildung eine Ursache für den niedrigen Gehalt an Docosahexaensäure in Spermatozoen mit einer schlechten Motilität ist.
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Decreased conception rate of dairy cows in the summer is mainly associated with the deleterious effects of environmental thermal stress on the female reproductive tract. Here, we suggest that decreased reproductive performance might be partially due to inferior-quality semen. Semen from five representative bulls was collected in summer (August to September) and winter (December to January) and evaluated with a computerized sperm-quality analyzer for bulls (SQA-Vb). No seasonal effect was found in fresh ejaculate, but sperm examined post-thawing showed lower velocity, motility and progressive motility (P<0.04) in summer vs. winter samples. Element concentrations in the seminal plasma, determined by inductively coupled plasma-atomic emission spectrometry, differed between seasons, with higher (P<0.01) concentration values of K, Mg, Na and S elements in winter vs. summer samples. Therefore, season-induced alterations in seminal plasma element concentration should be taken into account when using an extender for cryopreservation. Acrosome integrity was assessed by a triple-fluorescence test using Hoechst 33342, fluorescein isothiocyanate-conjugated Pisum sativum agglutinin (FITC-PSA) and propidium iodide. Acrosome reaction was examined by a one-step staining method using FITC-PSA. The proportion of sperm cells with a damaged acrosome post-thawing tended to be higher (P<0.07) in semen collected during the summer vs. winter. Such alterations suggest that seasonal reductions in sperm function might also be involved in the decreased conception rate of dairy cows in summer.
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An increasing number of cancer patients can now hope to have a full and normal life due to significant improvements in treatment outcomes and survival rates. The application of cryobiology to store fertile gametes before sterilizing treatments has been a natural progression. Greater awareness has markedly increased the worldwide demand for long-term storage of sperm, and has prompted the UK Human Fertilization and Embryology Authority to extend the period of storage permitted by their regulations to 55 years. Other patients undergoing sterilizing chemotherapy and/or radiotherapy such as haemoglobinopathies requiring bone marrow transplantation and autoimmune disorders such as rheumatoid arthritis may further increase the indications for sperm storage. Most adult and adolescent patients and their relatives/spouses/parents/guardians value this service even though very few eventually use the sperm. There is an urgent need to develop national and international guidelines for the provision, organization, maintenance and management of the cryopreservation services.
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The lipid composition of the sperm membrane has a significant effect upon the functional characteristics of spermatozoa. In the present study we investigated the fatty acid (FA) composition of subpopulations of spermatozoa separated on a discontinuous Percoll gradient (47:90%) and the FA composition of phospholipids (PL) of sperm heads and tails in both normal and abnormal semen samples. In normozoospermic samples, polyunsaturated fatty acids (PUFA) represented 34.0 +/- 1.3 (mean +/- SE, mole %) and 25.6 +/- 1.2% of total FA of PL of the 47 and 90% Percoll fractions respectively. Docosahexaenoic acid (22:6omega3, DHA) contributed to more than 60% of total PUFA. DHA was significantly lower in both the 47% (P < 0.05) and the 90% (P < 0.01) Percoll fractions of oligozoospermic samples and in the 90% Percoll layer of asthenozoospermic samples (P < 0.01), compared with normozoospermic samples. The omega6/omega3 ratio was significantly increased in both Percoll fractions of samples with oligozoospermia (47%, P < 0.001 and 90%, P < 0.001) or with asthenozoospermia (47%, P < 0.05 and 90%, P < 0.001) compared with normozoospermic samples. The oxidative potential index (OPI) of spermatozoa recovered from the 47% Percoll layer was significantly higher (P < 0.0001) than of those recovered from the 90% Percoll. Mean melting point (MMP), an index of membrane fluidity, was significantly lower in head than in tails (P < 0.01) of spermatozoa, and also in both the 47% (P < 0.01) and 90% (P < 0.001) Percoll fractions of normozoospermic samples in comparison with oligozoospermic samples. The MMP was significantly higher (P < 0.05) in samples of patients with idiopathic oligo/asthenozoospermia, varicocele, and male accessory gland infection (MAGI). These differences in FA composition of PL in subpopulations of human spermatozoa, and in their heads and tails may be related to sperm maturity and to differences in physiological function.
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A total number of 900 semen ejaculates were collected by means of artificial vagina from 10 rams (five local and five crossbreed 'local x chios'). Each ram was scheduled for semen collection three times per week at equal intervals for approximately two months in each season. At each collection, two separate successive ejaculates were obtained. Semen ejaculates were tested for volume, pH, mass motility, methylene blue reduction time (MBRT) and sperm-cell concentration per milliliter. Sex drive of rams was estimated by measuring the reaction time (RT). The time from the first to the second ejaculate was also recorded (TBE). Data of the two breeds show that the overall means for seminal attributes were 0.75 ml, 6.82, 4.2, 216.1 s and 5102.26 x 10(6) sperm/ml, respectively. In both breeds the overall means for RT and TBE were 43.7 and 152.3 s, respectively. Excluding ejaculate volume and MBRT, breed of ram was without significant effect on the investigated seminal characters, and sex drive parameters. Semen of the second ejaculate was significantly (P < 0.01) less in volume, greater in motility, closer to neutrality, less in sperm-cell concentration. MBRT of the two ejaculates differed nonsignificantly. However, characteristics of the second ejaculate fell within the normal range of high quality semen. Although the tested rams are continuous breeding animals, yet seasonal variations in semen characteristics were observed. The best-quality semen occurred in winter (volume, 0.77 ml; pH, 6.95; motility, 4.53; MBRT, 3.24 min. and sperm concentration, 4932.72 x 10(6)/ml). According to the known indices of semen quality of the ram, semen of the other seasons is judged to be of good quality. The mean RT for both breeds was less than one minute. Breed effect on RT and BET was insignificant. Season of the year exerted a significant (P < 0.01) effect on RT. The shortest time was recorded in summer, whereas the longest one was that of autumn. This indicates that the high ambient temperature of summer, prevailing in this locality, did not decrease the sexual activity of the tested rams.
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Sperm plasma membrane lipids have an important role to play in determining membrane fluidity and sperm motility. The objective of the present study was to determine whether there are differences in the lipid and fatty acid (FA) composition of boar sperm and seminal plasma in the ejaculates of boars having different sperm motilities. Semen was collected from two groups of boars having normal (> 60%; n = 53) or low (< 60%; n = 53) motility sperm and the semen was evaluated for motility, morphology and vitality. The semen was then centrifuged to separate the sperm from the seminal plasma and both were kept at -20 °C until analyzed for lipid content and FA profile by gas chromatography. Total antioxidant status (TAS) of seminal plasma was determined using a commercial kit. There were differences (P ≤ 0.05) in sperm total lipids, cholesterol, saturated fatty acids (SFA), phospholipids, n-3 polyunsaturated fatty acids (PUFA), docosahexaenoic acid (DHA) and the ratio of n-6:n-3 PUFA between boars with normal and low motility sperm. Total lipids, cholesterol, phospholipids, PUFA, DHA and n-3 PUFA were positively correlated with sperm motility, viability, normal morphology and normal plasma membrane. In contrast, SFA and the ratio of n-6: n-3 PUFA were negatively correlated (P ≤ 0.05) with sperm motility, viability, normal morphology and normal plasma membranes. The TAS of seminal plasma from boars having normal motility sperm was higher (P ≤ 0.05) than that of boars having low motility sperm and TAS was positively correlated (P = 0.0001) with sperm motility, viability, normal morphology and normal plasma membranes. In summary, differences in sperm motility were related to n-3 PUFA content in the sperm plasma membrane and extracellular antioxidants in seminal plasma which protect sperm plasma membranes from lipid peroxidation during periods of oxidative stress.
Article
The Sperm Quality Analyzer (SQA-Vp) was evaluated for assessing concentration and motility of porcine semen. Both fresh and diluted semen from 50 different boars from a commercial artificial insemination (AI) centre were investigated. For the fresh ejaculate, the concentration obtained with SQA-Vp was compared with a photometer and a haemocytometer. For the diluted samples, the concentration and motility were compared with computer assisted semen analysis (CASA) and visual sperm analysis. The agreement between methods was studied with Bland-Altman plots and the repeatability with coefficient of variation (CV) as well as Bland-Altman plots. The sperm concentration (x10(6)/ml) obtained with SQA-Vp (379.3 ± 134.9) for fresh ejaculates agreed well with concentration by the photometer (447.2 ± 154.2; difference= -67.9 x 10(6)/ml; difference + 2SD = 55.3 x 10(6)/ml; difference - 2SD = -191.1 x 10(6)/ml) and with the haemocytometer (332.8 ± 141.11; d = 92.8; d + 2SD = 448.6; d - 2SD = -263). For diluted semen, the agreement between the concentration (x10(6)/ml) assessed with SQA-Vp (20.4 ± 4.3) was good with CASA (23.2 ± 5.8; d = -2.8; d + 2SD = 6.2; d - 2 SD = -11.8) but poor with the haemocytometer (18.8 ± 5.0; d = 1.6; d+ 2SD = 12.2; d - 2SD = -9). The % motile spermatozoa assessed by SQA-Vp (65.8 ± 10.0) in diluted semen agreed well with CASA (72.2 ± 13.7; d = -6.4; d+ 2SD = 20; d - 2SD = -32.8) and with visual assessment (64.1 ± 11.6; d = 1.7; d+ 2SD = 30.9; d - 2SD = -27.5). The SQA-Vp showed a good repeatability (CV; repeatability coefficient) for measuring the concentration of both fresh (3.9%; d = 10.7; d + 2SD = 30.9; d - 2SD = -9.5) and diluted semen (2.6%; d = 1.0; d + 2SD = 2.38; d - 2SD = -0.42) and for motility (3.2%; d = 0.9; d + 2SD = 8.5; d - 2SD = -6.7). The mean SQA-Vp values fell between the other methods' results for both fresh and diluted semen. Moreover the repeatability was acceptable. Therefore SQA-Vp can be used as a valid device for sperm quality analysis in pigs.
Article
Motility is one of the most important characteristics associated with the fertilizing ability of spermatozoa and is an expression of their viability and structural integrity. Computer-assisted semen analyser (CASA) provides precise and accurate information on different sperm motion characteristics. This article reviews various aspects of computer-aided motility analysis of bull sperm like sample preparation, standardization of instrument settings, importance of various motility parameters evaluated by the system and its impact on basic functional studies of spermatozoa. It gives special emphasis to various aspects of bull sperm motion analysis especially sub-populations of spermatozoa, hyper-activation, motion characteristic in different genetic and age groups, etc. and their utility in predicting the fertility of dairy bulls. The need to fill the gap in research and the necessity of universal standardization of the equipment has been discussed.
Article
Environmental factors, especially temperature and light exposure, influence the health and productivity of dairy cows during lactation, possibly via similar physiological mechanisms. For example, heat stress is a critical component of decreased milk yield during summer. However, less is known about the effect of heat stress during the dry period. The objective of this study was to evaluate the effects of heat stress prepartum under a controlled photoperiod on lactation performance and hepatic metabolic gene expression of periparturient multiparous Holstein cows (n = 16). Cows were dried off approximately 46 d before expected calving date and assigned to treatment randomly after blocking by mature equivalent milk production and parity. Treatments consisted of either heat stress (HT) or cooling (CL) with fans and sprinklers, both under a photoperiod of 14L:10D. Rectal temperature was measured twice daily during the dry period. After calving, cows were housed in a freestall barn with cooling devices, and milk yield was recorded daily up to 210 d in milk. Blood samples were taken from dry off until +42 d relative to calving for metabolites and from -2 until +2 d relative to calving for hormone analysis. Daily dry matter intake was measured from -35 to +42 d relative to calving. Liver biopsies were collected at dry off, -20, +2, and +20 d relative to calving for cows on HT (n = 5) and CL (n = 4) to measure mRNA expression of suppressors of cytokine signaling-2 (SOCS-2), insulin-like growth factor binding protein-5 (IGFBP-5), a key transcription factor in lipid biosynthesis (SREBP-1c), and enzymes of lipid metabolism (FASN, ACACA, and ACADVL) by real-time quantitative PCR. Heat stress increased rectal temperatures (39.2 vs. 38.8 degrees C), plasma prolactin concentrations at -1 (171 vs. 79 ng/mL) and 0 d (210 vs. 115 ng/mL) relative to calving, and decreased dry matter intake at 0 and +14 d relative to calving and 3.5% fat-corrected milk postpartum (26.1 vs. 35.4 kg/d) compared with CL cows. Relative to CL cows, hepatic mRNA expression of SOCS-2 and IGFBP-5 was downregulated in HT cows. Expression of ACADVL was upregulated in CL cows at d +2 but downregulated at d +20 relative to HT cows. Concentrations of C16:0 and cis C18:1 were greater in the milk and liver of CL cows compared with HT cows, which reflects greater lipid mobilization. These results suggest that heat-stress abatement in the dry period improves subsequent lactation, possibly via suppression of plasma prolactin surge around calving, SOCS-2 expression, and regulation of hepatic lipid metabolism.
Article
Fatty acid (FA) composition of the spermatozoa may be an important determinant of fertility. The aim was to evaluate polyunsaturated fatty acid (PUFA) composition of the blood plasma and spermatozoa in infertile men with idiopathic oligoasthenoteratozoospermia (OAT). Eighty-two infertile men with idiopathic OAT and seventy-eight fertile men defined according to semen concentration and proven fertility were enrolled in the study. The semen parameters were assessed according to World Health Organization criteria; three omega-3 fatty acids--alpha-linolenic acid (ALA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), and two omega-6 fatty acids--linoleic acid (LA) and arachidonic acid (AA) concentrations were measured in blood plasma and spermatozoa; and the seminal plasma enzymatic antioxidant levels of catalase, and superoxide dismutase (SOD) were also assessed. Proven fertile men had higher blood and spermatozoa levels of omega-3 FAs compared with the infertile patients. The ratio of serum omega-6/omega-3 fatty acids was significantly higher in infertile (14.8+/-4.3) patients compared to fertile controls (6.3+/-2.2) (P=0.001). Additionally, levels of AA were higher and the omega-3 index (EPA+DHA) was lower in infertile subjects than in fertile controls (all P values<0.05). Infertile men had higher mean AA:DHA ratio and AA:EPA (6.4+/-2.9 and 12.0+/-4.9, respectively) than fertile men (3.3+/-1.8 and 6.7+/-2.6, respectively) (both P=0.001). A strong negative correlation was found between the AA:DHA and AA:EPA ratios and total sperm count (r=-0.62, P=0.001 and r=-0.64, P=0.001, respectively), sperm motility (r=-0.63, P=0.001 and r=-0.61, P=0.001, respectively), and sperm morphology (r=-0.61, P=0.001, and r=-0.59, P=0.002, respectively). Infertile men had lower concentrations of omega-3 FAs in spermatozoa than fertile men. These results suggest that research should be performed to assess the potential benefits of omega-3 FA supplementation as a therapeutic approach in infertile men with idiopathic OAT.
Article
Ejaculated spermatozoa must undergo physiological priming as they traverse the female reproductive tract before they can bind to the egg's extracellular coat, the zona pellucida (ZP), undergo the acrosome reaction, and fertilize the egg. The preparatory changes are the net result of a series of biochemical and functional modifications collectively referred to as capacitation. Accumulated evidence suggests that the event that initiates capacitation is the efflux of cholesterol from the sperm plasma membrane (PM). The efflux increases permeability and fluidity of the sperm PM and causes influx of Ca(2+) ions that starts a signaling cascade and result in sperm capacitation. The binding of capacitated spermatozoa to ZP further elevates intrasperm Ca(2+) and starts a new signaling cascade which open up Ca(2+) channels in the sperm PM and outer acrosomal membrane (OAM) and cause the sperm to undergo acrosomal exocytosis. The hydrolytic action of the acrosomal enzymes released at the site of sperm-egg (zona) binding, along with the hyperactivated beat pattern of the bound spermatozoon, are important factors in directing the sperm to penetrate the ZP and fertilize the egg. The role of Ca(2+)-signaling in sperm capacitation and induction of the acrosome reaction (acrosomal exocytosis) has been of wide interest. However, the precise mechanism(s) of its action remains elusive. In this article, we intend to highlight data from this and other laboratories on Ca(2+) signaling cascades that regulate sperm functions.
Article
Chronic ethanol consumption coupled with folate deficiency leads to rapid liver fat accumulation and progression to alcoholic steatohepatitis (ASH). However, the specific effects of alcohol on key liver lipid metabolic pathways involved in fat accumulation are unknown. It is unclear whether lipid synthesis, lipid export, or a combination of both is contributing to hepatic steatosis in ASH. In this study we estimated the flux of fatty acids (FA) through the stearoyl-CoA desaturase (SCD), phosphatidylethanolamine-N-methyltransferase (PEMT), and FA elongation pathways in relation to liver triacylglycerol (TG) content in Yucatan micropigs fed a 40% ethanol folate-deficient diet with or without supplementation with S-adenosyl methionine (SAM) compared with controls. Flux through the SCD and PEMT pathways was used to assess the contribution of lipid synthesis and lipid export respectively on the accumulation of fat in the liver. Liver FA composition within TG, cholesterol ester (CE), phosphatidylethanolamine, and phosphatidylcholine classes was quantified by gas chromatography. Alcoholic pigs had increased liver TG content relative to controls, accompanied by increased flux through the SCD pathway as indicated by increases in the ratios of 16:1n7 to 16:0 and 18:1n9 to 18:0. Conversely, flux through the elongation and PEMT pathways was suppressed by alcohol, as indicated by multiple metabolite ratios. SAM supplementation attenuated the TG accumulation associated with alcohol. These data provide an in vivo examination of liver lipid metabolic pathways confirming that both increased de novo lipogenesis (e.g., lipid synthesis) and altered phospholipid metabolism (e.g., lipid export) contribute to the excessive accumulation of lipids in liver affected by ASH.
Article
An increasingly exploited strategy for the isolation of stem cells is based on the increased efflux of Hoechst 33342 lipophilic dye mediated by ABCG2, an ATP-binding cassette transporter which is highly expressed in various stem cells. We found ABCG2 expression to be present at later stages of spermatogenesis. Western blot analysis using an anti-ABCG2 antibody revealed expression of a 72kDa band in mature sperm obtained from mice, rats, bulls or humans. Immunocytochemistry studies revealed acrosomal staining pattern of ABCG2 in spermatozoa. Experiments using the Hoechst 33342 ABCG2 substrate and the ABCG2-specific inhibitor FTC demonstrated efflux activity of ABCG2 in mature sperm. Incubation of sperm in capacitating medium in the presence of the ABCG2-inhibitor FTC resulted in decreased cholesterol depletion compared to sperm incubated in the absence of FTC. Our results demonstrate that ABCG2 is expressed at the acrosome in mature sperm. ABCG2 may thus serve to mediate cholesterol removal.
Article
Mammalian spermatozoa lose plasma membrane cholesterol during their maturation in the epididymis and during their capacitation in the female reproductive tract. While acceptors such as high-density lipoproteins (HDL) and apolipoproteins A-I (apoA-I) and J have been found in male and female reproductive tracts, transporters that mediate cholesterol efflux from plasma membranes of spermatozoa to such acceptors have not yet been defined. Candidate transporters are members of the ATP-binding cassette (ABC) transporter superfamily including ABCA1, ABCA7, ABCG1 and ABCG4, which have all been implicated in the transport of sterols and phospholipids to apolipoproteins and HDL. Here we show that mouse spermatozoa in the seminiferous tubules and epididymis express ABCA1, ABCA7 and ABCG1, but not ABCG4. Moreover, we show that ABCA1, ABCA7, and ABCG1 antibodies decrease cholesterol efflux from spermatozoa to lipid acceptors apoA-I and albumin and inhibit in vitro fertilization.
Article
Plasma membranes were isolated from rat and mouse livers, a transplanted rat hepatoma, two transplanted mouse hepatomas and spontaneous mouse hepatomas. Phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, lysophosphatidylcholine, free fatty acids and triglycerides were separated and their fatty acid profiles determined. The various lipid classes of rat and mouse liver plasma membranes each demonstrated more or less specific fatty acid profiles. The number of double bonds decreased in the order: phosphatidylserine greater than or equal to phosphatidylethanolamine greater than phosphatidylinositol greater than phosphatidylcholine greater than or equal to sphingomyelin and lysophosphatidylcholine. Small species differences were noted in most lipid classes. A marked sex difference was observed in sphingomyelin of mouse liver membranes but in none of the phospholipids of rat liver membranes. The increased cholesterol content of all hepatoma versus liver plasma membranes was accompanied by a decrease of fatty acyl poly-unsaturation in most lipid classes of the rat hepatoma but not of the mouse hepatoma membranes. The fatty acid profiles of the mouse hepatoma membranes deviated much less from those of mouse liver than did the pattern of rat hepatoma versus rat liver. The results were discussed in relation to lipid fluidity of which fatty acyl unsaturation and cholesterol are the main parameters.
Article
The cholesterol content of spermatozoa with highly unsaturated phospholipids, i.e., ram and bull, which are very susceptible to cold-shock was compared with that of rabbit and human spermatozoa which have more saturated phospholipids and a greater resistance to cold-shock. The level of cholesterol in the seminal plasma was also estimated.Cholesterol was present in ram and bull spermatozoa in comparable amounts (280–346 μg) and at approximately half the level (in micrograms per 109 spermatozoa) in rabbit and human spermatozoa (545–556 μg). The molar ratio of cholesterol: phospholipid in rabbit and human spermatozoa was 0.88 and 0.99, respectively, which is similar to the ratio of these lipids in erythrocytes and higher than the ratio of 0.38 and 0.45 for ram and bull spermatozoa, respectively. This ratio is of some importance in determining the nature and degree of packing in the spermatozoan membrane; higher levels of cholesterol result in a more cohesive, rigid, and impermeable structure. A definite relationship is apparent between the ratio of cholesterol: phospholipid, the ratio of polyunsaturated: saturated phospholipid-bound fatty acids, and the susceptibility of the spermatozoa to cold-shock.
Article
Techniques for freezing bull sperm developed over the past 40 years have not yielded protocols for preserving sperm from other species. Recent advances in our understanding of cell membrane structure function and metabolism now permit alternative modes of investigation. These data will allow development of unique studies which should have a higher probability of yielding successful protocols for sperm from other species. In this review the authors will: (1) provide a general overview of cryopreservation; (2) review emerging concepts of membrane structure and the relationship of membrane composition to water and cryoprotectant movement; (3) emphasize how these parameters affect cell volume and surface areas; (4) focus attention on the concept that cryoprotectants will alter membrane structure and function in addition to their well-recognized effects on bulk solvent; and (5) emphasize the effect of the processing protocol on metabolic balance. These concepts are reintroduced in the context of the established and successful protocol for freezing bull sperm to illustrate the molecular responses that may be necessary to survive a freeze-thaw cycle.
Article
Binding assays with [3H] heparin were performed using spermatozoa collected prior to, during, and following summer heat stress to dairy bulls. Ejaculates collected in August 1983 after a period of ambient temperatures exceeding 29.4 degrees C exhibited a high frequency of abnormal sperm, and motility was reduced in some samples. Sperm in samples collected during heat stress possessed dissociation constants for binding [3H] heparin ranging from 134.5 to 163.2 nmol. In contrast, sperm in semen collected prior to and after heat stress had significantly lower dissociation constants (higher affinity) for [3H] heparin, 12.9 to 56.4 nmol. The number of binding sites for [3H] heparin on sperm did not change among collection periods. It was concluded that the binding affinity for [3H] heparin may reflect membrane integrity of bull sperm.
Article
Receiver operating characteristic curves and accuracy parameters were computed for traditional sperm characteristics (concentration, motility, morphology) and the number of peroxidase negative cells, and the concentration of adenosine triphosphate (ATP) in semen from populations of fertile and infertile men, and men who achieved a pregnancy after varicocele treatment. The percentage and concentration per millilitre of spermatozoa with rapid linear progressive motility, and the ATP concentration, provided the best discrimination between fertile and treated fertile from infertile men. The misclassification rate was higher for sperm morphology, total progressive motility and viability, whereas sperm concentration and the total sperm count per ejaculate had the worst discriminating power. The number of peroxidase negative cells per 100 spermatozoa was highly specific in identifying men who achieved pregnancy after varicocele treatment. The lower limit of normality of sperm characteristics was remarkably different between fertile men and men achieving pregnancy after treatment or during infertility work-up.
Article
The composition of phospholipids extracted from bovine testicular, epididymal and ejaculated spermatozoa was examined. Extracts from testicular spermatozoa contained approximately twice the amount of phospholipid extracted from cauda epididymal or ejaculated spermatozoa. The concentration of most of the major phospholipid components of bovine spermatozoa including phosphatidylcholine, phosphatidylethanolamine, cardiolipin, phosphatidylserine and sphingomyelin, decreased markedly during passage through the epididymis. However, the concentration of the principal phospholipid component, choline plasmalogen, appeared to change only slightly. Cauda epididymal and ejaculated spermatozoa had a similar phospholipid composition.The phospholipid-bound fatty acids in bovine spermatozoa also changed during migration through the epididymis. Most of the major fatty acid species, including palmitic, stearic, oleic and arachidonic acids, decreased both in their percentage composition and in their absolute concentration per spermatozoon. The greatest loss was in the palmitic acid fraction, which decreased in both epididymal and ejaculated spermatozoa to about a third of the amount present in testicular extracts. The polyunsaturated fatty acid, docosahexaenoic acid, which is the principal fatty acid component of ejaculated bull sperm phospholipids, changed only slightly in concentration, although, owing to the large palmitic acid loss, the percentage by weight of this substance increased by about 60 % prior to ejaculation. The phospholipidbound aldehyde fraction decreased in concentration during the maturation process but there was little alteration in composition of the various aldehyde components. The principal fatty aldehyde species of the three types of spermatozoa was palmitaldehyde.
Article
Bull, boar, rabbit and human semen was fractionated into seminal plasma, sperm tails and sperm heads. The fatty acid composition of total lipid, phopholipid, phosphatidyl choline, triglycerides and diglycerides was determined in each fraction. Qualitative and quanti- tative differences in the fatty acid contents were found between the species and between the semen fractions. Bull and rabbit seminal plasma contained relatively higher proportions of polyunsaturated acids (PUFA) (\g=w\6and \g=w\3)than that of man and boar. The sperm tails con- tained in all four species more PUFA than did the sperm heads. The higher metabolites of oleic acid were not present in measurable amounts. The 20- and 22-carbon acids were present in semen in much larger quantities than thus far reported, in any other mammalian tissue.