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A follow-up study of sperm preparation for IVF by swim-up in a solution of hyaluronate.

Spermatozoa prepared for in-vitro fertilization (IVF) by swim-up in a balanced salt solution containing hyaluronate gave rates of fertilization, cleavage and pregnancy which were not significantly different from those obtained with sperm prepared by swim-up in standard IVF medium followed by centrifugation. However, the content of prostaglandin F2alpha in the final sperm suspension was higher using hyaluronate but this seemed to be of no consequence for IVF. Thus, preparation of normal sperm samples for IVF may be simplified by performing swim-up in a balanced salt solution containing hyaluronate.

Cell Separation↗

Effect of swim-up, Percoll and Sephadex sperm separation methods on the hypo-osmotic swelling test.

The separation of spermatozoa from the seminal plasma is required to prepare the spermatozoa for intrauterine insemination, in-vitro fertilization and sex selection. This study evaluated the effects of sperm preparation techniques on the functional integrity of the sperm membrane, as measured by the hypo-osmotic swelling test (HOS). Thirty-four semen specimens obtained from the male partner of infertile couples were evaluated. A semen analysis and HOS test were performed on each specimen. The remainder of the specimen was divided into three equal aliquots, the first prepared using Percoll, the second using a swim-up method and the third using a Sephadex column. After the preparation, a semen analysis and HOS test was performed on each aliquot. The mean and standard deviation for the HOS test was 72.9 +/- 8.5% initially, 71.2 +/- 13.1 after Percoll, 75.2 +/- 15.1 after swim-up and 62.4 +/- 14.5 after Sephadex. Analysis of variance showed that the mean HOS score was the same after Percoll and swim-up as it was initially but significantly lower after preparation with Sephadex. There was also a higher proportion of abnormal semen specimens (HOS less than 50%) after preparation with Sephadex than after the other preparation methods. We recommend the use of the HOS test as part of a screening panel for sperm separation.

Cell Membrane↗

Enhanced recruitment of motile spermatozoa by prostasome inclusion in swim-up medium.

Prostasomes, which are prostate-derived organelles, were purified from human seminal plasma for inclusion in Earle's balanced salt solution (EBSS) medium with or without human serum albumin. These media were used for swim-up experiments and the subsequent analyses of sperm motility parameters at different incubation times. The yield of motile spermatozoa after swim-up in EBSS medium enriched with boiled prostasomes was increased by 32% compared with EBSS containing albumin. Native prostasomes were less active. Combinations of albumin and either prostasomes or boiled prostasomes significantly increased the recovery of motile spermatozoa and also increased the percentage of spermatozoa displaying progressive motility after 1 h of incubation. Media lacking albumin showed lower values regarding progressive motility after 22 h of incubation. A beneficial effect of prostasomes was noted on lateral head displacement and percentage of hyperactive spermatozoa during the first 6 h of incubation. These results suggest that inclusion of prostasomes, especially boiled prostasomes, in swim-up media may improve the recovery of hyperactive motile spermatozoa for up to 6 h in cases of established male factor infertility, and consequently increase the opportunities for fertilization.

Hot Temperature↗

The combination of two semen preparation techniques (glass wool filtration and swim-up) and their effect on the morphology of recovered spermatozoa and outcome of IVF-ET.

The aim of this prospective study was to relate sperm quality, especially sperm morphology, to the outcome of IVF results, when glass wool filtration (GWF) and swim-up were used in combination for the preparation of spermatozoa. A total of 60 ejaculates were analysed. GWF was compared with glass wool filtration/swim-up (GWF-SU) by using aliquots from the same semen samples to increase the precision of the comparison and to establish the cumulative effect of these two semen preparation methods on the morphology of the recovered spermatozoa. Sperm parameters were examined in native semen, in semen preparation samples after GWF and GWF-SU. The mean percentages for motility, morphology and velocity were improved significantly over those in fresh semen only when GWF-SU was used. GWF alone resulted only in a significantly higher recovery of motile spermatozoa. In semen preparation, 10% of spermatozoa with normal morphology appeared to be the cut-off point as there was a significantly higher recovery in fresh semen samples containing > 10% morphologically normal spermatozoa after GWF-SU. Outcome of IVF-ET following preparation with GWF-SU showed better results in comparison to the swim-up procedure alone, though this difference was not statistically significant. The cut-off point was also > 10% morphologically normal spermatozoa. Improvement in all aspects of IVF-ET occurred when native semen contained > 10% morphologically normal spermatozoa. It is concluded that GWF alone did not produce significantly better results but, in combination with swim-up, it resulted in significantly better sperm morphology and in an improved outcome of IVF-ET for fresh semen samples with > 10% morphologically normal spermatozoa.

Adult↗

Human sperm swimming in flow.

To study the movement of human sperm, we have developed a microflow cell by miniaturizing our design for a preparative fractionation flow column. The microflow cell enabled us to view the movement of sperm over periods as long as 2 min. Sequential steps of filming, editing, and analysis revealed that the curved swimming patterns of sperm swimming in stagnant fluid become nearly straight tracks when the flow velocity is increased. However, the net swimming speed remained unchanged. Motile sperm accumulated near solid wall surfaces surrounding the fluid and oriented against the direction of the current; the velocity gradient was steepest in these regions. A laminar-flow preparative column separated motile sperm from dead sperm by carrying the nonmotile sperm and debris with the stream while leaving the motile sperm near the surrounding walls.

Adult↗

Hypoosmotic swelling test in normo-, oligo-, astheno- and oligoasthenozoospermic men before and after swim-up separation of spermatozoa.

Semen samples from a total 58 men were examined by routine semen analyses and the hypoosmotic swelling test. Samples were classified as normal, oligo-, astheno- or oligoasthenozoospermic on the basis of spermatogram findings. The latter three groups showed a significant decrease in the percentage of HOS positive forms in comparison to normal spermograms. All these samples were treated with the swim up technique to select motile spermatozoa, using a procedure similar to that routinely employed in clinical settings for homologous intrauterine insemination (IUI). Following swim-up, the ejaculate supernatant and residual precipitate were subjected to the hypoosmotic swelling test (HOS test), and the percentage of normal forms was determined in the three groups. The results showed greater percentages of HOS positive, normal and HOS positive-normal forms in the group of normal individuals than in any of the other three groups. The supernatant used in IUI showed a significant increase in percentage HOS positive spermatozoa, normal forms and spermatozoa which were both normal and HOS positive in comparison with the other two groups in normal and oligozoospermic samples, but not in samples which presented suboptimal motility (astheno- and oligozoospermia). In conclusion, the swim-up technique is effective in separating high-quality spermatozoa in normo- and oligozoospermic samples, although its effectiveness with astheno- and oligoasthenozoospermic samples should be questioned.

Humans↗

Morphology of seminal and swim-up spermatozoa and the outcome of in vitro fertilization and embryo transfer.

Tubal infertility was treated by in vitro fertilization-embryo transfer (IVF-ET) in 112 couples. Twenty-eight pregnancies were obtained in 140 treatment cycles. Couples are accepted for treatment in our IVF-ET programme if previous semen samples fulfil the inclusion criteria: ejaculate volume greater than 1.5 ml, concentration of spermatozoa greater than 15 x 10(6) ml-1, greater than 40% motile spermatozoa, and greater than 25% spermatozoa with normal morphology. In order to determine to which extent IVF-ET treatment results are influenced by sperm morphology, within this selected group of patients, we have retrospectively analysed the data from both original semen samples and swim-up preparations. The sperm morphology was not related to the outcome of treatment in terms of fertilization (ovum cleavage rate), early embryo development, or pregnancy. Nor was any relationship detected between early embryo development or pregnancy and the degree of improvement in morphology resulting from the swim-up procedure. However, if improvement in morphology by swim-up was high, ovum cleavage rate was low. Sperm morphology within the limits set by our inclusion criteria could not predict the outcome of IVF-ET treatment. It is further concluded that the presence of abnormal spermatozoa at the site of fertilization may be without harm if only the number of normal sperms is high enough.

Embryo Transfer↗

Comparisons of sperm quality, morphometry and function among human sperm populations recovered via SpermPrep II filtration, swim-up and Percoll density gradient methods.

The purpose of this study was to compare the morphology/morphometry and fertilizing capacity of human spermatozoa recovered via swim-up method, Percoll density gradient method, and SpermPrep II filtration method. Thirty-three ejaculates were equally divided into 2 aliquots. Aliquot 1 was processed via the direct swim-up method, whereas aliquot 2 was filtered via a SpermPrep II column. The Percoll density gradient method was compared with the SpermPrep II method in a similar protocol using 43 ejaculates. Sperm populations recovered via the SpermPrep II filtration method showed significantly higher hypoosmotic swelling test results, acrosin profiles, and percentage of hyperactivated spermatozoa than sperm fractions recovered by the swim-up method. Furthermore, significant differences were found in most of sperm morphometric parameters between the above sperm populations. However, sperm fractions recovered via the SpermPrep II method did not show significantly different values for these same tests and for most of sperm morphometric parameters compared to the Percoll density gradient method. These results suggest that the SpermPrep II filtration and Percoll density gradient method are equally efficient in isolating sperm subpopulations with better functional parameters than the swimup method.

Cell Separation↗

Transformation of a bop-hop-sop-I-sop-II-Halobacterium halobium mutant to bop+: effects of bacteriorhodopsin photoactivation on cellular proton fluxes and swimming behavior.

We have transformed Pho81, a Halobacterium halobium mutant strain which does not contain any of the four retinylidene proteins known in this species, with the bop gene cluster to create Pho81BR, a BR+HR-SR-I-SR-II-strain. The absorption spectrum, pigment reconstitution process, light-dark adaptation and photochemical reaction cycle of the expressed protein are indistinguishable from those of native bacteriorhodopsin (BR) in purple membrane of wild type strains. Strain Pho81BR permits for the first time characterization of effects of BR photoactivation alone on cell swimming behavior and energetics in the absence of the spectrally similar phototaxis receptor sensory rhodopsin I (SR-I) and electrogenic chloride pump halorhodopsin (HR). A non-adaptive upward shift in spontaneous swimming reversal frequency occurs following 3 s of continuous illumination of Pho81BR cells with green light (550 +/- 20 nm). This effect is abolished by low concentrations of the proton ionophore carbonylcyanide m-chlorophenylhydrazone. Although BR does not mediate phototaxis responses in energized Pho81BR cells under our culture conditions, proton pumping by BR in Pho81BR cells partially deenergized by inhibitors of respiration and adenosine triphosphate synthesis results in a small attractant response. Based on our measurements, we attribute the observed effects of BR photoactivation on swimming behavior to secondary consequences of electrogenic proton pumping on metabolic or signal transduction pathways, rather than to primary sensory signaling such as that mediated by SR-I. Proton extrusion by BR activates gated proton influx ports resulting in net proton uptake in wild-type cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacteriorhodopsins↗

On torque and tumbling in swimming Escherichia coli.

Bacteria swim by rotating long thin helical filaments, each driven at its base by a reversible rotary motor. When the motors of peritrichous cells turn counterclockwise (CCW), their filaments form bundles that drive the cells forward. We imaged fluorescently labeled cells of Escherichia coli with a high-speed charge-coupled-device camera (500 frames/s) and measured swimming speeds, rotation rates of cell bodies, and rotation rates of flagellar bundles. Using cells stuck to glass, we studied individual filaments, stopping their rotation by exposing the cells to high-intensity light. From these measurements we calculated approximate values for bundle torque and thrust and body torque and drag, and we estimated the filament stiffness. For both immobilized and swimming cells, the motor torque, as estimated using resistive force theory, was significantly lower than the motor torque reported previously. Also, a bundle of several flagella produced little more torque than a single flagellum produced. Motors driving individual filaments frequently changed directions of rotation. Usually, but not always, this led to a change in the handedness of the filament, which went through a sequence of polymorphic transformations, from normal to semicoiled to curly 1 and then, when the motor again spun CCW, back to normal. Motor reversals were necessary, although not always sufficient, to cause changes in filament chirality. Polymorphic transformations among helices having the same handedness occurred without changes in the sign of the applied torque.

Escherichia coli↗

Attachment of Vibrio alginolyticus to glass surfaces is dependent on swimming speed.

The attachment of Vibrio alginolyticus to glass surfaces was investigated with special reference to the swimming speed due to the polar flagellum. This bacterium has two types of flagella, i.e., one polar flagellum and numerous lateral flagella. The mutant YM4, which possesses only the polar flagellum, showed much faster attachment than the mutant YM18, which does not possess flagella, indicating that the polar flagellum plays an important role. The attachment of YM4 was dependent on Na+ concentration and was specifically inhibited by amiloride, an inhibitor of polar flagellum rotation. These results are quite similar to those for swimming speed obtained under the same conditions. Observations with other mutants showed that chemotaxis is not critical and that the flagellum does not act as an appendage for attachment. From these results, it is concluded that the attachment of V. alginolyticus to glass surfaces is dependent on swimming speed.

Amiloride↗

The Yersinia enterocolitica motility master regulatory operon, flhDC, is required for flagellin production, swimming motility, and swarming motility.

The ability to move over and colonize surface substrata has been linked to the formation of biofilms and to the virulence of some bacterial pathogens. Results from this study show that the gastrointestinal pathogen Yersinia enterocolitica can migrate over and colonize surfaces by swarming motility, a form of cooperative multicellular behavior. Immunoblot analysis and electron microscopy indicated that swarming motility is dependent on the same flagellum organelle that is required for swimming motility, which occurs in fluid environments. Furthermore, motility genes such as flgEF, flgMN, flhBA, and fliA, known to be required for the production of flagella, are essential for swarming motility. To begin to investigate how environmental signals are processed and integrated by Y. enterocolitica to stimulate the production of flagella and regulate these two forms of cell migration, the motility master regulatory operon, flhDC, was cloned. Mutations within flhDC completely abolished swimming motility, swarming motility, and flagellin production. DNA sequence analysis revealed that this locus is similar to motility master regulatory operons of other gram-negative bacteria. Genetic complementation and functional analysis of flhDC indicated that it is required for the production of flagella. When flhDC was expressed from an inducible ptac promoter, flagellin production was shown to be dependent on levels of flhDC expression. Phenotypically, induction of the ptac-flhDC fusion also corresponded to increased levels of both swimming and swarming motility.

Bacterial Proteins↗

Inactivation of swmA results in the loss of an outer cell layer in a swimming synechococcus strain.

The mechanism of nonflagellar swimming of marine unicellular cyanobacteria remains poorly understood. SwmA is an abundant cell surface-associated 130-kDa glycoprotein that is required for the generation of thrust in Synechococcus sp. strain WH8102. Ultrastructural comparisons of wild-type cells to a mutant strain in which the gene encoding SwmA has been insertionally inactivated reveal that the mutant lacks a layer external to the outer membrane. Cryofixation and freeze-substitution are required for the preservation of this external layer. Freeze fracturing and etching reveal that this additional layer is an S-layer. How the S-layer might function in motility remains elusive; however, this work describes an ultrastructural component required for this unique type of swimming. In addition, the work presented here describes the envelope structure of a model swimming cyanobacterium.

Bacterial Proteins↗

A swimming robot actuated by living muscle tissue.

Biomechatronics is the integration of biological components with artificial devices, in which the biological component confers a significant functional capability to the system, and the artificial component provides specific cellular and tissue interfaces that promote the maintenance and functional adaptation of the biological component. Based upon functional performance, muscle is potentially an excellent mechanical actuator, but the larger challenge of developing muscle-actuated, biomechatronic devices poses many scientific and engineering challenges. As a demonstratory proof of concept, we designed, built, and characterized a swimming robot actuated by two explanted frog semitendinosus muscles and controlled by an embedded microcontroller. Using open loop stimulation protocols, the robot performed basic swimming maneuvers such as starting, stopping, turning (turning radius ~400 mm) and straight-line swimming (max speed >1/3 body lengths/second). A broad spectrum antibiotic/antimycotic ringer solution surrounded the muscle actuators for long term maintenance, ex vivo. The robot swam for a total of 4 hours over a 42 hour lifespan (10% duty cycle) before its velocity degraded below 75% of its maximum. The development of functional biomechatronic prototypes with integrated musculoskeletal tissues is the first critical step toward the long term objective of controllable, adaptive and robust biomechatronic robots and prostheses.

Journal Article↗

Physiology and behaviour of free-swimming Atlantic cod (Gadus morhua) facing fluctuating salinity and oxygenation conditions

1. Atlantic cod (Gadus morhua L.) acclimated to a temperature of 5 &deg;C and 30 salinity were equipped with ultrasonic transmitters which allowed continuous monitoring of their heart rate and their position in the water column. Fish were placed in a 125 m3 tower tank which permitted various environmentally relevant modifications of the salinity and oxygenation conditions. Cod physiological and behavioural responses were followed in parallel to the environmental manipulations. Some of the experimental conditions studied in the tower tank were also reproduced in a swimming respirometer where fish oxygen consumption and heart rate were monitored at various levels of activity. 2. Lowering salinity from 30 to 26 did not change resting oxygen consumption, but increased active oxygen consumption. 3. Lowering salinity from 30 to 26 increased heart rate over the whole range of swimming speeds except at maximum speed. 4. Lowering oxygen tension to 9 kPa decreased oxygen consumption over the whole range of swimming speeds and decreased resting heart rate. 5. Low salinity did not significantly affect the relationship between heart rate and oxygen consumption. 6. Low oxygen levels decreased the oxygen transported per heart beat. 7. In the tower tank, bursts of activity were associated with tachycardias. 8. In uniform conditions, fish swam more deeply during the day than at night. 9. After an exploratory period of approximately 6 h, fish chose to remain in a low-salinity upper layer of the tank. Thereafter, high salinities were avoided. Fish tended to select low salinities if a choice was provided. 10. Fish generally avoided zones of low oxygen (<9 kPa) but continued voluntarily to enter regions with values as low as 3.0 kPa for short excursions or if food was offered.

Journal Article↗

Hydroplaning by ducklings: overcoming limitations to swimming at the water surface

Rapid escape behavior by mallard (Anas platyrhynchos) ducklings is restricted to burst swimming at the water surface. Maximum speed may be limited because of the pattern of waves created as the duckling's body moves through the water (hull speed). Burst speeds for 9-day-old ducklings were compared with predicted hull speeds, based on the waterline length of ducklings either resting in water or actively swimming. Kinematic analysis of video tapes showed a mean maximum burst speed of 1.73 m s-1, which was four times greater than the predicted hull speed. At burst velocities, stroke frequency was 1.9 times higher than the stroke frequency measured during steady low-speed paddling. Transition to burst speeds from steady paddling occurred near predicted hull speed. The paddling motions of the webbed feet were used to generate both thrust and lift. By using lift to raise the body above the water surface, the influence of waves in restricting maximum swimming speed is negated. The duckling's body becomes a planing type of hull and skims on the water surface.

Journal Article↗

Pectoral fin locomotion in the striped surfperch. II. Scaling swimming kinematics and performance at a gait transition

In this study, we report the first allometric equations relating gait parameters and swimming speed to body size for fish employing pectoral fin locomotion. Comparisons of locomotor kinematics and performance among striped surfperch (Teleostei: Embiotocidae) are made at the pectoral&shy;caudal gait transition speed (Up-c). Up-c is considered to elicit physiologically equivalent levels of exercise in animals varying over 100-fold in body mass (Mb) by virtue of dynamically similar pectoral fin movements (constant duty factor, length-specific stride length and fin-beat amplitude) and size-independent propulsive efficiency. At Up-c, pectoral fin-beat frequency scales in proportion to Mb-0.12&plusmn;0.03, a size-dependence consistent with that observed for stride frequency in fishes swimming by axial undulatory propulsion and in many running tetrapods. It is proposed that the similarity in the scaling of frequency in these vertebrate groups reflects an underlying similarity in the allometry of the maximal velocity of muscle shortening. Absolute Up-c (m s-1) generally increases with body size, but the fastest speeds are not exhibited by the largest animals. A pattern of declining performance in fish 23 cm in standard length and longer may be related to their disproportionately small fin areas and aspect ratios. The pronounced negative allometry of Up-c expressed as standard body lengths per second indicates that a given length-specific speed does not induce comparable levels of activity in large and small fish. Thus, normalization of swimming speed to body length may not be a sufficient correction for kinematic comparisons across size.

Journal Article↗

Fish foot prints: morphology and energetics of the wake behind a continuously swimming mullet (Chelon labrosus Risso).

The structure of the wake behind a continuously swimming mullet was analysed qualitatively and quantitatively by applying two-dimensional particle image velocimetry. A detailed analysis of the flow pattern and of the swimming movements of the fish allowed us to derive a kinematic explanation of the flow pattern as well as an estimate of the relative contributions of the body and the tail to thrust production. During active propulsion, the undulatory swimming fish shed a wake consisting in the medio-frontal plane of a rearward, zigzagging jet flow between alternating vortices. The fish shed one vortex per half tailbeat when the tail reached its most lateral position. Part of the circulation shed in the vortices had been generated previously on the body by the transverse body wave travelling down the body. This undulatory pump mechanism accounted for less than half of the energy shed in the wake. The remainder was generated by the tail. The vortex spacing matched the tailbeat amplitude and the stride length.

Journal Article↗