青岛文昌鱼精子运动特征及计算机辅助分析

青岛文昌鱼的精子在精浆中不运动,在与精浆等渗的生理溶液中也不运动,把精液稀释到过滤海水或人工海水后,精子运动被激活。文昌鱼新鲜精液中精子浓度为(9.4±1.6)×1010/ml;在人工海水中,运动精子占总精子数的85.8%±6.9%,精子运动持续的平均时间是22.7±2.6min。文昌鱼精子经人工海水激活0.5min后,可观察到四种运动方式:(1)向前直线运动,其平均速度93.6±23.7μm/s,这类精子所占的百分比为27.8%±3.1%;(2)弧形曲线运动,平均速度55.6±18.9μm/s,所占的百分比为44.3%±2.5%;(3)左右摆动,平均前进速度27.4±13.4μm/s,所占百分比为14.7%±1.8%;(4)运动速度小于5μm/s的精子,视为不运动的精子,所占百分比为13.2%±1.8%。随着激活时间的延长,文昌鱼精子的运动状态发生改变,向前直线运动和弧形曲线运动的精子逐渐减少,而左右摆动和不运动的精子逐渐增多。     

Identification of sperm subpopulations with defined motility characteristics in ejaculates from Florida goats

The aims of this study were to test the presence of discrete sperm subpopulations in Florida goat ejaculates using a computer-assisted sperm analysis (CASA) system and to establish the relationship between the distribution of the subpopulations found and individual buck, total motility, and sperm concentration. Clustering methods and discriminant analysis were applied to identify motile sperm subpopulations within the semen samples. Principal component analysis revealed that three principal components represented more than the 88% of the variance. After the cluster analysis was performed four motile sperm subpopulations were identified. Subpopulation 1 consisted of rapid and linear sperm (39.84%), Subpopulation 2 consisted of slow but linear spermatozoa (33.23%), Subpopulation 3 consisted of rapid, high ALH but non-linear spermatozoa (14.63%), and Subpopulation 4 consisted of slow and non-linear spermatozoa (12.31%). There were significant differences in the distribution of the four subpopulations (P < 0.001) as well as in the percentage of total motility and the overall sperm concentration (P < 0.05) in fresh ejaculates among the four bucks tested. In conclusion, four well-defined motile sperm subpopulations were identified in Florida goat ejaculates. The relationship between the distribution of the sperm subpopulations and individual buck, total motility, and sperm concentration shows that the spermatozoa of each have different motility patterns. Therefore, the study of discrete subpopulations of motile spermatozoa could lead to a substantial increase in information acquired during caprine semen analysis.     

Use of single-layer centrifugation with Androcoll-C to enhance sperm quality in frozen-thawed dog semen

The aim of this study was to investigate whether single-layer centrifugation (SLC) with Androcoll-C could select good quality spermatozoa, including those with specific motility patterns, from doses of frozen dog semen. Semen from five dogs was collected and cryopreserved following a standard protocol. After thawing, the semen samples were divided in two aliquots, one of which was used as a control and the other one processed by SLC. Assessment of sperm motility (assessed by computer-assisted semen analysis), morphology (Diff-Quick staining), viability (dual staining with propidium iodine/acridine orange), and acrosome integrity (dual staining with propidium iodine/isothiocyanate-labeled peanut [Arachis hypogaea] agglutinin) were performed on aliquots of fresh semen, frozen-thawed control samples, and frozen-thawed SLC-treated preparations. A multivariate clustering procedure separated 57,577 motile spermatozoa into three subpopulations (sP): sP1 consisted of poorly active and nonprogressive spermatozoa (48.8%), sP2 consisted of moderately slow but progressive spermatozoa (13.3%), and sP3 consisted of highly active and/or progressive spermatozoa (37.8%). SLC with Androcoll-C yielded sperm suspensions with improved motility, viability, and acrosome integrity (P < 0.01). The frozen-thawed SLC-treated samples were enriched in sP3, representing 38.5% of the sperm population. Likewise, sP2 was more frequently observed after SLC, but not significantly so. From these results, we concluded that for dog semen samples selected by SLC with Androcoll-C after thawing, the sperm quality parameters, including motility patterns, are better than in frozen-thawed control samples.     

Tracking algorithms chase down pathogens

Understanding subcellular dynamic processes governing pathogenic mechanisms is a necessary step towards the development of new drugs and strategies against infectious diseases. Subcellular pathogenic mechanisms, such as viral invasion processes involve highly dynamic nanometric-scale objects and rapid molecular interactions that require the study of individual particle paths. Single-particle tracking methods allow visualizing and characterizing the dynamics of biological objects, and provide a straightforward and accurate means to understand subcellular processes. This review describes a number of particle-tracking methods in time-lapse microscopy sequences and provides examples of using such techniques to investigate mechanisms of host-pathogen interactions.     

Changes in extracellular osmolality initiate sperm motility in freshwater teleost rosy barb Puntius conchonius

The objective was to investigate the effects of extracellular osmolality and membrane osmotic-sensitive channels on the initiation of sperm motility and to explore mechanisms of sperm initiation in rosy barb (Puntius conchonius). We found that (1) sperm were immotile in seminal plasma and remained quiescent in electrolyte or nonelectrolyte solutions isotonic to seminal plasma; (2) sperm movement was initiated when the sperm were exposed to hypo-osmotic electrolyte or hypo-osmotic nonelectrolyte solutions, and that the responsiveness of sperm to changes in the extracellular osmolalities (100, 200, 250, 270, and 300 mOsm/kg) differed among sperm cells (P < 0.05); (3) sperm movement could be initiated and terminated repeatedly by decreasing and increasing the osmolality (in increments of 100 and 300 mOsm/kg) of a nonelectrolyte mannitol solution, respectively (P < 0.05); (4) gadolinium (20, 40, and 80 μM) inhibited the initiation of sperm motility and abolished the sperm activation caused by the hypo-osmotic media treatment in dose- and time-dependent manners (P < 0.05); and (5) sperm activation in a hypo-osmotic medium and inhibition in an isotonic solution were associated with swelling and shrinkage of the sperm sleeves, respectively. Therefore, we concluded that osmolality was a critical physiologic signal in regulating the initiation and termination of sperm motility in freshwater teleost rosy barb. Furthermore, we inferred that rosy barb sperm were hypo-osmotic–dependent conformers, and the osmotic-sensitive channel could be involved in the mechanism of sperm initiation.     

Relationship between sperm motility, morphology and the fertility of stallions

Sperm quality has an important role in determining fertility. Although there have been numerous studies to document the relationship between sperm quality and fertility, the methods of determining this association and conclusions vary. In the present study, computer-assisted sperm analysis (CASA) was used for evaluation of sperm motility, and differential interference contrast (DIC) microscopy was used for evaluating sperm morphologic features of breeding stallions. Fertility was measured using three endpoints: seasonal pregnancy rate (PR), percent pregnant/cycle (PC), and percent pregnant/first cycle (FCP). Increased total sperm motility (P = 0.08) and progressive path velocity (P = 0.06) tended to be associated with higher PR, whereas percent coiled tails (P = 0.02) was associated with a lower PR. Sperm motility variables associated with an increase in PC and FCP included total, progressive, and rapid sperm motility, and increased path and progressive velocity. Percent pregnant/first cycle was the only fertility measure able to discriminate among high, average, and low fertility groups, based on total and progressive sperm motility. Percent normal sperm was the only morphology variable associated with an increased PC and FCP, whereas increased levels of most sperm morphologic abnormalities (including abnormal and detached heads, proximal and distal droplets, general midpiece abnormality, and coiled tails) were associated with a decline in PC and FCP. Sperm quality variables most highly correlated with fertility included percent total sperm motility (PR, r = 0.37, P < 0.05; PC, r = 0.59, P < 0.05; and FCP, r = 0.64, P < 0.05), and percent morphologically normal sperm (PC, r = 0.42, P < 0.05; and FCP, r = 0.39, P < 0.05).     

Thiotepa, a reliable marker for sperm precedence measurement in a polyandrous moth

The determination of paternity of offspring produced by polyandrous females is essential for the understanding of sperm competition mechanisms. The sterile male technique using radiation is one of the most commonly employed methods for this purpose. However, sterilization using radiation is likely to be restricted by the equipment availability and cost. Chemosterilization may thus be a cheaper and easier alternative for sterilizing male insects in sperm competition studies. Here we report a reliable chemomarker, thiotepa (N,N′,N″-triethylenethiophosphoramide), for the study of sperm competition and precedence in a polyandrous moth, Ephestia kuehniella (Lepidoptera: Pyralidae). Dipping heads of male moths in 1% thiotepa aqueous solution for 10 s resulted in complete sterilization, i.e. their sperm still fertilized eggs but those eggs did not hatch. The sterilization treatment did not significantly affect male copulation ability, female fecundity, and sperm transfer, motility and fertilization. Our results indicate that 86% offspring of the twice-copulated females were fathered by the second males and 14% by the first males. Males treated with 5% thiotepa aqueous solution died within 24 h while those treated with 0.5% thiotepa were not fully sterilized.     

Microencapsulation of canine sperm and its preservation at 4 °C

The objective of this study was to develop a preservation method for canine sperm using microencapsulation. Pooled ejaculates from three beagles (Canis familiaris) were extended in egg yolk Tris extender and were encapsulated in gel (alginate only) or polycation (poly-l-lysine membrane bound) microcapsules at 0.75% and 1.0% alginate concentration. In Experiment 1, characteristics of microcapsule and microencapsulated sperm were evaluated during chilling storage for 48 h. Gel microcapsules at 0.75% alginate concentration had a teardrop-like structure with fragility, whereas those at 1.0% alginate had a solid spherical structure. In all groups, diameter of the microcapsules increased with duration of storage (P < 0.05). Alginate concentration did not affect the sperm recovery rate from microcapsules. Total average recovery rate of sperm from polycation microcapsules was lower than that of gel microcapsules (P < 0.05). Progressive motility of polycation microencapsulated sperm and unencapsulated sperm (control) was higher than that of the gel microencapsulated sperm, both at 0.75% and 1.0% alginate concentration (P < 0.05), although viability of sperm was similar among the three groups. In Experiment 2, to evaluate the sperm longevity after chilling storage, sperm were microencapsulated in polycation microcapsules at 1.0% alginate concentration, stored at 4 °C for 0, 1, 4, and 7 d, and then cultured at 38.5 °C for 0, 6, and 24 h. Progressive motility and viability of microencapsulated sperm were higher than those of unencapsulated spermatozoa at 0 to 24 h of culture after 4 and 7 d of chilling storage (P < 0.05). In conclusion, polycation microencapsulation at 1.0% alginate concentration can be successfully applied for chilling storage of canine sperm by maintaining motility and viability for up to 7 d.     

Cryopreservation effects on sperm function and fertility in two threatened crane species

The capacity to cryopreserve semen from captive cranes facilitates production of offspring from behaviorally incompatible or geographically separated pairs, and allows for long-term preservation of valuable genetic materials. The present study sought to develop effective cryopreservation protocols for whooping (Grus americana) and white-naped (Grus vipio) cranes, through examining the influences of two permeating (DMA and Me₂SO) and one non-permeating (sucrose) cryoprotectants, as well as vitamin E on post-thaw sperm survival. In Study 1, ejaculates (whooping: n = 10, white-naped: n = 8) were collected and cryopreserved in one of six cryo-diluents (crane extender with: DMA; DMA+0.1M sucrose; Me₂SO; Me₂SO+0.1M sucrose; 0.1M sucrose; 0.2M sucrose) using a two-step cooling method. Frozen samples were thawed and assessed for overall motility, motion characteristics, membrane integrity, morphology, and ability to bind to the inner perivitelline membrane (IPVM). In Study 2, whooping crane ejaculates (n = 17) were frozen in crane extender containing Me₂SO alone or with vitamin E (5 μg/mL or 10 μg/mL). Frozen samples were thawed and assessed as in Study 1, except the binding assay. White-naped crane sperm were more tolerant to cryopreservation than whooping crane (15% vs 6% post-thawed motility). In both species, sperm cryopreserved in medium containing Me₂SO alone displayed higher post thaw survival and ability to bind to IPVM than the other cryodiluent treatments. Vitamin E supplementation exerted no benefits to post thaw motility or membrane integrity. The findings demonstrated that there was species specificity in the susceptibility to cryopreservation. Nevertheless, Me₂SO was a preferred cryoprotectant for sperm from both whooping and white-naped cranes.     

Evaluation of ram semen quality using polyacrylamide gel instead of cervical mucus in the sperm penetration test

Fertility is a very complex biological function that depends on several properties of the spermatozoa, including sperm motility. Two objectives are analyzed in this study: (1) Replace the cervical mucus by a synthetic medium in a sperm penetration test, and (2) evaluating the results of this test objectively analyzing the sperm number that migrates. In experiment 1, we have tested eight concentrations of acrylamide (1%-2%). Rheological properties of media were analyzed. The plastic straws, loaded with acrylamide, were placed vertically on the semen sample tube for 15 min at 39 °C. After, the acrylamides were placed, by segments of 5 mm, into wells of a 24-well plate, dyed with Hoechst 33342 and the number of spermatozoa were calculated by automated microscopy analysis. The 1.55% and 1.6% acrylamide gel showed a number of spermatozoa emigrating closer to that seen with natural mucus. In experiment 2, we applied the sperm penetration in acrylamide 1.6% and 1.55% using fresh semen and cooled semen at 15 °C and 5 °C. The spermatozoa counts were performed for each segment of 10 mm. Semen chilled at 15 °C presented intermediate values of sperm counts in comparison with fresh semen (higher) and 5 °C chilled semen. The sperm counts do not differ between acrylamides but the rheological properties of acrylamide 1.6% were more similar to those of the natural cervical mucus. In experiment 3, we have observed significant correlations between the number of spermatozoa and several sperm quality parameters (positive: progressive motility and velocity according to the straight path; negative: damaged acrosomes and apoptotic cells) in 1.6% acrylamide media. We conclude that the size of the cell subpopulation, objectively calculated, that migrate beyond 20 mm in 0.5-mL straws filled with acrylamide is a useful parameter in ram sperm quality assessment and further studies are needed to evaluate its relationship with field fertility.     

Toxicity evaluation of parboiled rice effluent using sperm quality of zebrafish as bioindicator

This study aimed to assess the acute toxicity of raw and treated wastewater generated by the rice parboiling industry using zebrafish (Danio rerio) sperm quality as a bioindicator. Toxicity bioassays were conducted comparing physicochemical parameters of sperm quality for zebrafish at sublethal conditions (n = 150 fish, 50 per treatment). Acute toxicity was detected in all sperm quality parameters assessed for both raw and treated wastewater, when contrasted to the control (p < 0.05). For zebrafish exposed to raw effluent, negative correlations with parameters of sperm quality were observed for the concentration of iron, phosphorus and total suspended solids (p < 0.05). Salinity, the biochemical oxygen demand and the concentration of total suspended solids were negatively correlated with parameters of sperm quality for zebrafish exposed to treated effluent (p < 0.05). In comparison with the levels observed for the raw effluent, most physicochemical parameters of the treated effluent were reduced to levels within the limits required by the environmental legislation. Despite the physical and chemical parameters measured in the treated wastewater meeting environmental legislation thresholds, acute toxicity persisted. These results show that the sperm quality can be used as a bioindicator for wastewater toxicity and release of wastewater to surface water could affect the fertility of fishes.     

Cryopreservation of sperm from farmed Pacific abalone,Haliotis discus hannai

This study aimed to improve a sperm cryopreservation protocol for farmed Pacific abalone, Haliotis discus hannai. Dimethyl sulfoxide (Me₂SO), glycerol, ethylene glycol (EG), propylene glycol (PG), and methanol were chosen as cryoprotectants (CPAs). Four different equilibration time (5, 10, 30, and 60 min), and two types of equilibration temperature (4 °C and 20 °C) were selected at the present experiment. Most equilibration temperatures with each CPA showed significant differences among different equilibration time. Post-thaw sperm motility of five CPAs showed no significant difference at two equilibration temperature. Based on these results, 8% Me₂SO, 8% EG, 6% PG, 2% glycerol, and 2% methanol were chosen to determine optimal conditions for sperm cryopreservation of H. discus hannai. The highest post-thaw sperm motility (8% Me₂SO: 50.6%, 8% EG: 45.6%, 2% glycerol: 44.5%, 6% PG: 28.7%, 2% methanol: 25.4%) was achieved after exposing sperm to liquid nitrogen (LN₂) vapor for 10 min at 5 cm above the LN₂ surface and then submerging them in LN₂ for at least 2 h followed by thawing at 60 °C with seawater and recovering them at 20 °C with seawater. In this study, 8% Me₂SO and 2% glycerol were chosen to check post-thaw sperm quality to estimate percentages of plasma membrane integrity (PMI), mitochondrial potential analysis (MP), and acrosome integrity (AI) using fluorescent techniques. No significant difference in PMI, MP, and AI was found between sperm cryopreserved with 8% Me₂SO and those cryopreserved with 2% glycerol. The current study has demonstrated that 8% Me₂SO was optimal for sperm cryopreservation for H. discus hannai with 5 min of equilibration time, 5 cm of rack height and 60 °C of thawing temperature. The present research provides more effective cryopreservation methods for H. discus hannai sperm than previous studies.     

The effect of seminal plasma on alpaca sperm function

In order to advance the development of assisted reproductive technologies in alpacas and other Camelids, the objective of this study was to explore the role of seminal plasma concentration on motility and functional integrity of alpaca sperm. Sixteen male alpacas > 3 y of age were used. In Experiment 1, epididymal sperm were incubated for 0 to 6 h in 0, 10, 25, 50, or 100% seminal plasma and motility was assessed. In Experiment 2, epididymal sperm were incubated in 0, 10, or 100% seminal plasma for 3 h and motility, acrosome integrity and DNA integrity were assessed. In Experiment 3, ejaculated sperm were incubated in 10, 25, 50, or 100% seminal plasma for 0 to 6 h and motility assessed. In Experiment 4, ejaculated sperm were incubated in 10 or 100% seminal plasma for 3 h and motility, acrosome integrity, DNA integrity, and viability were assessed. Epididymal and ejaculated sperm maintained motility longer when incubated in the presence of 10% seminal plasma compared to 0, 25, 50, or 100% seminal plasma (P < 0.001). The mean ± SEM percentage of epididymal sperm with intact acrosomes was less (P < 0.001) in samples incubated in 0% seminal plasma (39.4 ± 3.73) compared to 10% (75.3 ± 1.20) or 100% (77.4 ± 0.90) within 1 h after incubation. However, DNA integrity of ejaculated and epididymal sperm was not significantly affected by seminal plasma concentration. The mean viability of ejaculated sperm was reduced in the presence of 100 (12.7 ± 2.33) compared to 10% (36.2 ± 4.68) seminal plasma (P < 0.001) within 1 h of incubation. We concluded that alpaca semen should be diluted to a final concentration of 10% seminal plasma to prolong motility, preserve acrosome integrity, and maintain viability of sperm.     

Pycnogonid sperm. An example of inter- and intraspecific axonemal variation

Summary The flagella of the motile sperm cells of Nymphon leptocheles and N. rubrum (Pyonogonida, Arthropoda) exhibit a 12+0 and a 9+0 axoneme pattern, respectively. Central tubules, central sheath, spokes and arms are absent. The doublets are connected by a circular nexus. The functional significance of this axonemal composition is discussed. Aberrant axonemes occurring in high frequencies both within the species and within single specimens are probably explained by the loose axonemal connection, due to the absence of a central complex. This absence is further suggested to have facilitated the evolution from the 9+0 type to the 12+0 type.     

Novel and traditional traits of frozen-thawed porcine sperm related to in vitro fertilization success

Cryopreserved semen allows the use of single ejaculates for repeated analyses, potentially improving IVF consistency by eliminating interejaculate variability observed with fresh semen. However, the freezing and thawing processes result in compromised sperm function and IVF success. Semen samples are often screened for motility before use for IVF. Samples that are below a designated motility threshold may be discarded. Our objectives were to determine if post-thaw sperm motility, other traits that may be indicative of sperm function, or a novel assay of oviduct binding were related to IVF success. Semen from 16 boars was cooled to 15 °C for overnight shipment before cryopreservation. Semen was thawed and motility was recorded microscopically and confirmed using computer-automated sperm assessment. Each sample was tested by IVF in two to three independent replicates. Regression and correlation analyses were employed to determine the interrelationships between sperm traits and the relationships between post-thaw motility, sperm-oviduct binding and IVF outcomes. Among the sperm traits examined, sperm acrosome integrity was negatively correlated with post-thaw motility (r² = 0.64) but not with IVF results. The number of sperm bound to oviduct aggregates was correlated with IVF polyspermy rates (r² = 0.62, P < 0.05) but less with overall IVF rates (r² = 0.31, P > 0.10). There was some relationship of post-thaw motility with IVF monospermic fertilization (P = 0.06, r² = 0.08) but not to other IVF outcomes. Our results indicate that post-thaw motility of frozen-thawed boar sperm is strongly related to acrosome integrity but has limited use for predicting IVF success. The number of sperm bound to oviduct cells was related to IVF polyspermy rates and may be more indicative of in vitro sperm function than traditional sperm motility and acrosome status evaluation.     

Altering the speract-induced ion permeability changes that generate flagellar Ca2+ spikes regulates their kinetics and sea urchin sperm motility

Speract, an egg-derived sperm-activating peptide, induces changes in intracellular Ca2+, Na+, pH, cAMP, cGMP, and membrane potential in sperm of the sea urchin Strongylocentrotus purpuratus. Ca2+ is a key regulator of motility in all sperm and, in many marine species, is required for generating turns interspersed with straighter swimming paths that are essential for chemotaxis towards the egg. We show that speract triggers a train of increases in flagellar Ca2+, and that each individual Ca2+ fluctuation induces a transient increase in flagellar asymmetry that leads to a turn. We also find that modifying the amplitude, duration and interval between individual Ca2+ fluctuations by treating sperm with niflumic acid, an inhibitor of Ca2+-activated Cl(-) channels, correspondingly alters the properties of the sperm turns. We conclude that Ca2+ entry through a fast flagellar pathway not only induces sperm turns, but the kinetics of Ca2+ entry may shape the nature of these turns, and that these kinetics are tuned by other channels, possibly including Cl(-) channels. In addition, the speract-induced changes in sperm motility closely resemble those seen during chemotaxis in other marine organisms, yet speract is not a chemoattractant. This implies the Ca2+-induced motility changes are necessary but not sufficient for chemotaxis.     

Seasonal variations in seminal plasma and sperm characteristics of wild-caught and cultivated Atlantic cod, Gadus morhua

The objective was to investigate changes, throughout the spawning season, in body size attributes and quantitative semen characteristics of wild-caught and cultivated Atlantic cod, Gadus morhua L. Sperm velocity increased significantly throughout the spawning season of cod from both origins. Curvilinear velocity (VCL; 30 sec post-activation) increased from 78.9 ± 6.5 to 128.2 ± 6.5 μm/sec (mean ± SEM) between the beginning and end of the spawning season, respectively, for wild-caught cod, whereas for cultivated fish, it increased from 26.6 ± 2.4 to 48.9 ± 3.1 μm/sec between January and March. Spermatocrit did not undergo a significant seasonal change in wild-caught cod but did thicken for cultivated cod (24.6 ± 4.2% in January to 40.5 ± 4.4% in April; P < 0.01). Sperm head area, perimeter, length, and width declined significantly at the end of the spawning season of cod from both origins (all P values < 0.01). Seminal plasma osmolality and Na⁺ ion concentration followed a dome-shaped function through the spawning season for both wild-caught and cultivated cod (P < 0.05). For cultivated cod, seminal plasma pH was significantly lower at the start of the spawning season (P < 0.001), whereas Ca²⁺ increased then decreased (P < 0.05). Body size attributes, spermatocrit, and seminal plasma constituents had significant relationships with sperm activity variables. These relationships varied as a function of time post-activation, month, and fish origin. Our findings may be used to (i) assess spermiation stage without killing males; (ii) optimize semen collection for hatchery production; (iii) characterize the potential impact of farming on sperm quality; and (iv) improve success of sperm cryopreservation and short-term storage.     

Immediate and delayed (after cooling) effects of centrifugation on equine sperm

The objectives of this study were to determine the effects of centrifugation on equine sperm total and progressive motility, viability, and acrosomal integrity. We hypothesized that although high centrifugation forces would be detrimental to equine Equus caballus sperm, recovery rates would increase. Ejaculates from six stallions were collected, extended to a concentration of 25 × 10⁶ cells/mL, and subjected for 10 min to (1) no centrifugation (NC) or (2) centrifugation at 400 × g, (3) 900 × g, or (4) 4500 × g. Before and after centrifugation (Day 0), and after 24 h of cooling (Day 1), sperm motility was assessed by computer-assisted semen analysis, and samples were stained with SYBR-14/propidium iodide (PI) for viability and with PI/fluorescein isothiocyanate (FITC)-Peanut aglutinin (PNA) (Arachis hypogaea) for acrosomal integrity. The effect of treatment and day on motility, viability, and acrosomal integrity was determined using a mixed linear model. Compared with the other treatments, centrifugation at 4500 × g reduced all end points measured (P < 0.05). Both 400 × g and 900 × g yielded lower recovery rates than that of 4500 × g (NC = 100.0 ± 0.0%; 400 × g = 54.4 ± 8.6%; 900 × g = 75.0 ± 7.1%; 4500 × g = 97.9 ± 2.8%; P < 0.05). Centrifugation at 400 × g or 900 × g did not damage equine sperm. Based on these findings, further studies of centrifugal forces between 900 × g and 4500 × g are warranted to determine the optimal force that maximizes recovery rate, minimizes sperm damage, and does not affect fertility.     

Maturation of hamster epididymal sperm motility and influence of the thiol status of hamster and rat spermatozoa on their motility patterns

A method for objective quantification of hamster sperm movement parameters as an indicator of maturation along the epididymis was established using a computerised system. Analysis of spermatozoa released into medium from five epididymal regions showed that the most drastic increases in percentage motility and curvilinear velocity (VCL) occurred from the distal corpus to the beginning of the proximal cauda and in straight-line velocity (VSL) from the beginning to a more distal site within the proximal cauda region. Both high osmolarity (400 mOsm/kg) and the thiol-oxidising agent diamide (10 μM) increased flagellar straightness of distal corpus spermatozoa, but VSL was increased only with the latter. The thiol-reducing agent dithiothreitol (DTT, 1mM) stimulated and maintained percentage motility and velocities of spermatozoa from the caput, stimulated only percentage motility of distal corpus sperm, but decreased velocities of those from the proximal cauda in prolonged incubation. In rats, diamide increased path straightness but not velocities of caput spermatozoa and yet caused immotility within 15 min, whereas DTT prolonged the maintenance of in vitro motility. The slight increases in kinematic parameters in the presence of DTT were enhanced by a 2-min preincubation with diamide. The finding that the effects of DTT and diamide were not compensatory suggests that the influence of the SH/S-S status on sperm movement is multifaceted, with decreasing sensitivity to stimulation upon sperm maturation. © 1994 Wiley-Liss, Inc.     

Seasonal dynamics of sperm morphometric subpopulations and its association with sperm quality parameters in ram ejaculates

Sperm morphologic assessment is considered an irreplaceable part of standard laboratory routine analyses in the diagnosis of male fertility. Thus, in an attempt to quantify the effects of season on sperm morphology and its functional significance in relation to sperm quality parameters, sperm head morphometric traits were analyzed by using an objective computerized analysis combined with principal components analysis (PCA) cluster analysis to establish the relationship between the distribution of the subpopulations found and sperm quality in each season. There were slight variations on sperm motility and sperm membrane integrity indexes (P > 0.05). However, the mean values for sperm concentration substantially changed among seasons in all individuals studied (P < 0.01). There were significant differences in sperm morphometric parameters (P < 0.01) as well as in the distribution of morphometric subpopulations between seasons (P < 0.001). In conclusion, this study confirmed that there was an important seasonal effect on sperm morphometric traits. In addition, the distribution of these subpopulations seems to be related to the season studied and the ejaculate quality which would be a very important indicator of sperm function. The substantial information derived from these morphometric subpopulations has provided new knowledge which can be used in future studies using sperm morphometry as a seasonal indicator in ram ejaculates.