Effects of lipid peroxide formation in fowl semen on sperm motility, ATP content and fertilizing ability

The ability of samples of semen from individual male fowl to form the products of lipid peroxidation during 5 h aerobic incubation at 40 degrees C varied between 0 and 8 nmol malonaldehyde/10(9) spermatozoa. Formation of higher concentrations of malonaldehyde was associated with a partial or complete loss of fertilizing ability whilst the fertilizing ability of samples producing low or negligible concentrations of malonaldehyde remained unimpaired. The semen of birds which showed a tendency to form high concentrations of malonaldehyde was not readily identifiable as abnormal by assessment of sperm motility, morphology or ATP content. Nor was the loss of fertilizing ability during aerobic incubation associated with an obvious change in these characteristics.     

Effect of cooling rates on post-thaw sperm motility, membrane integrity, capacitation status and fertility of dairy bull semen used for artificial insemination in Sweden

We studied the effects of 2 different cooling rates during equilibration of semen from room temperature to 4 degrees C, at 4.2 degrees C/min (control split sample) or at 0.1 degree C/min (treatment split sample) on in vitro sperm viability post thawing and fertility after AI. Forty batches of split-frozen semen from 14 dairy bulls (Swedish Red and White breed) aged 14 to 16 m.o. or 66 to 79 m.o. were evaluated post-thawing for sperm motility (visual and computer-assisted sperm analysis [CASA], membrane integrity (fluorescent microscopy and flow cytometry post-loading with the combined fluorophores Calcein AM/EthD-1 and SYBR-14/PI); acrosomal status (with Pisum sativum agglutinin [PSA] staining); and capacitation status (CTC-assay). Fertility values (56-d nonreturn rate) of the slow cooling batches (treatment) were 0.4% units higher than for faster cooled (control) batches, but the difference was not statistically significant. Fertility values for the older bulls were 1.6% units higher than for the group of younger sires. No statistically significant correlations were found between semen viability parameters assessed in vitro and 56-d nonreturn rate. Visually assessed sperm motility, membrane integrity, capacitation and acrosomal status post-thawing did not differ significantly between cooling procedures, however the percentage of motile spermatozoa and the kinetic characteristics of spermatozoa--average path velocity (VAP), straight path velocity (VSL) and curvilinear velocity (VCL)--assessed by CASA differed significantly between cooling procedures. The results indicate that most of the in vitro sperm viability parameters post-thawing and the fertility results for bulls after AI did not differ significantly between the 2 semen cooling procedures tested.     

Implications of the pH and temperature of diluted, cooled boar semen on fresh and frozen-thawed sperm motility characteristics

Boar semen is typically collected, diluted and cooled for AI use over numerous days, or frozen immediately after shipping to capable laboratories. The storage temperature and pH of the diluted, cooled boar semen could influence the fertility of boar sperm. Therefore, the purpose of this study was to determine the effects of pH and storage temperature on fresh and frozen-thawed boar sperm motility end points. Semen samples (n = 199) were collected, diluted, cooled and shipped overnight to the National Animal Germplasm Program laboratory for freezing and analysis from four boar stud facilities. The temperature, pH and motility characteristics, determined using computer automated semen analysis, were measured at arrival. Samples were then cryopreserved and post-thaw motility determined. The commercial stud was a significant source of variation for mean semen temperature and pH, as well as total and progressive motility, and numerous other sperm motility characteristics. Based on multiple regression analysis, pH was not a significant source of variation for fresh or frozen-thawed boar sperm motility end points. However, significant models were derived which demonstrated that storage temperature, boar, and the commercial stud influenced sperm motility end points and the potential success for surviving cryopreservation. We inferred that maintaining cooled boar semen at approximately 16 °C during storage will result in higher fresh and frozen-thawed boar sperm quality, which should result in greater fertility.     

The comparison of assessment of pigeon semen motility and sperm concentration by conventional methods and the CASA system (HTM IVOS)

The aim of these experiments was to compare conventional, microscopic methods of evaluating pigeon sperm motility and concentration to those measured by computer-assisted sperm analysis (CASA system). Semen was collected twice a week from two groups of pigeons, each of 40 males (group I: meat-type breed; group II: fancy pigeon) using the lumbo-sacral and cloacal region massage method. Ejaculates collected in each group were diluted 1:100 in BPSE solution and divided into two equal samples. One sample was examined subjectively by microscope and the second one was analysed using CASA system. The sperm concentration was measured by CASA using the anti-collision (AC) system and fluorescent staining (IDENT). There were not any significant differences between the methods of evaluation of sperm concentration. High positive correlations in both groups were observed between the sperm concentration estimated by Thom counting chamber and AC (r=0.87 and r=0.91, respectively), and between the sperm concentration evaluated by Thom counting chamber and IDENT (r=0.85 and r=0.90, respectively). The mean values for CASA measurement of proportion of motile spermatozoa (MOT) and progressive movement (PMOT) were significantly lower than the values estimated subjectively in both groups of pigeons (p< or =0.05 and p< or =0.01, respectively). Positive correlations in MOT and PMOT were noted between both methods of evaluation. The CASA system is very rapid, objective and sensitive method in detecting subtle motility characteristics as well as sperm concentration and is recommended for future research into pigeon semen.     

Sperm motility in the horseshoe crab. III. Isolation and characterization of a sperm motility initiating peptide

Sperm motility in Limulus is initiated by a sperm motility initiating factor (SMI) that emanates from Limulus eggs. This report describes the partial purification of SMI (greater than 230-fold purification with respect to protein content) with 40% recovery. SMI appears to be a hydrophobic peptide of 500–2,000 MW. Although probably not purified to homogeneity, SMI is estimated to be active at a concentration of less than 0.2 μM.     

Initiation of sperm motility depending on a change in external osmotic pressure in the noncopulatory marine cottid fish Gymnocanthus herzensteini

 The initiation of sperm motility in a noncopulatory marine cottid fish, Gymnocanthus herzensteini, was examined. The spermatozoa, which were immotile in seminal plasma, initiated motility at osmolalities of more than 500 mOsm kg⁻¹ in NaCl solution and 400 mOsm kg⁻¹ in KCl and mannitol solutions, indicating that the initiation of sperm motility depends on changes in external osmolality, in contrast with that of the sperm of other marine cottid fish, which are motile in seminal plasma. This study revealed that there are plural manner of initiation of sperm motility in marine cottid fish, which are oviparous but include both copulatory and noncopulatory modes. Received: May 24, 2001 / Revised: December 19, 2001 / Accepted: January 8, 2002     

Reduced prostaglandin levels in the semen of men with very high sperm concentrations.

The prostaglandin levels have been measured in a group of men with sperm concentrations greater than 300 X 10(6)/ml and compared with the levels in men with sperm concentrations of 50 to 150 X 10(6)/ml. The distribution of the PG levels in all groups was highly skewed but the data could be transformed to a normal distribution by taking logarithms. Comparison of the PG levels showed a highly significant lowering of the PG levels in the polyzoospermic group when compared wieth either of the groups with normal sperm concentrations.     

The role of hormones in the acquisition of sperm motility in salmonid fish.

In salmonid fish, spermatozoa taken from the testes are immotile, but acquire motility during their passage through the sperm duct. Using male masu salmon (Oncorhynchus masou), we found that gonadotropin-induced testicular production of 17 alpha, 20 beta-dihydroxy-4-pregnen-3-one (17 alpha, 20 beta-DP), the oocyte maturation-inducing hormone of salmonid fish, is responsible for the acquisition of sperm motility. However, neither testosterone (T) nor 11-ketotestosterone (11-KT), the two major androgens in teleost fish, were effective. We also present evidence that 17 alpha, 20 beta-DP action is mediated through an increase in sperm duct pH, which in turn increases the cAMP content of sperm allowing the acquisition of motility.     

Manual semen collection from a Grevy's zebra stallion (Equus grevyi), onset of sperm production, semen characteristics, and cryopreservation of semen, with a comparison to the sperm production from a Grant's Zebra stallion (Equus burchelli boehmi)

A manual technique was used to collect representative ejaculates from an unrestrained Grevy's zebra stallion beginning at 13 mo of age to determine the onset of sperm production, to calculate the number of spermatozoa produced per ejaculate, and to determine any seasonality associated with sperm production. Spermatozoa first appeared in the ejaculate at 31 mo of age. By 48 mo of age the zebra was producing up to 40 billion spermatozoa per ejaculate. Progressive sperm motility ranged from 75 to 95%. Gel-free semen volume averaged 75 to 120 ml/ejaculate. Gel volume ranged from 0 to 1100 ml/ejaculate. Semen was frozen in 2 different extenders in 0.5-ml PVC straws. The post-thaw motility of cryopreserved spermatozoa ranged from 30 to 70%. A domestic horse mare became pregnant on the first cycle after insemination with frozen-thawed spermatozoa from this zebra. Sperm production data obtained from semen collections made on a Grant's Zebra stallion from 3 to 8 yr of age is presented for comparison of the 2 species.     

Initiation of sperm motility in the newt, Cynops pyrrhogaster, is induced by a heat-stable component of egg-jelly

Sperm motility in amphibians is thought to be initiated by a decrease in environmental osmolarity. However, fertilisation in the newt, Cynops pyrrhogaster, is achieved in an environment without osmotic change. We show here that sperm motility initiating activity is present in jelly layer extract (JE). JE was gel-filtrated and a single peak with sperm motility initiating activity was detected in the fraction corresponding to about 50 kDa. The activity was strengthened by heat treatment of JE at 100 degrees C for 30 min. This suggests that JE includes the inactive form of sperm motility inducing substance (SMIS) in addition to active substance. Thus JE was fractionated before and after the heat treatment. When JE was fractionated first and then each fraction was heated, the activity was detected in the fraction both above 500 kDa and below 500 kDa. When heat-treated JE was fractionated, the activity was detected only in the fraction below 500 kDa. These results suggest that JE includes the inactive form of SMIS of more than 500 kDa in molecular weight. A regulatory mechanism for the initiation of sperm motility in C. pyrrhogaster is proposed according to the results of the present study.     

Studying sperm motility in marine fish: an overview on the state of the art

This contribution reviews existing literature and some new own findings on teleost sperm motility and factors controlling it, emphasizing selected marine species. In marine teleosts with external fertilization (halibut, turbot, sea bass, hake, cod and tuna serving as examples), mainly the osmolality controls sperm motility: movement is activated by transfer from the seminal fluid into sea water, representing a large upward step in osmolality. The exception are flatfishes (such as halibut or turbot) where CO₂ is responsible for flagellar immotility in seminal fluid. In all cases, the duration of motility is short and limited to minutes ranges due to partial exhaustion of the ATP energy and to increase of internal ionic concentration as suggested by studies with de‐membranated/ATP reactivated flagellae. In this overview, we compare motility characteristics (percentage of active spermatozoa, velocity, linearity), flagellar waves parameters (wave length and amplitude, number of waves) and energy content (respiration and ATP concentration) within species where these data have been established. All parameters show a rapid decrease after activation; therefore progressive forward movement needed by the sperm to effectively reach the egg surface, is limited to a short initial period following activation. In two species (turbot and sea bass) the rapid decrease of sperm motility is reflected by a corresponding decrease of the fertilizing ability. Exposure to external environments (sea water) at activation also leads to local defects of the sperm flagella posing additional limitations on motility duration. However, minor flagellar damages as well as energetic exhaustion are reversible: after a resting period in a non‐swimming solution at the end of the motility period, spermatozoa can be re‐activated for a second motility period. From these results and from additional data obtained from de‐membranated/ATP re‐activated spermatozoa, a paradigm has been developed which establishes a link between external osmolality (sea water), internal ionic concentration and control of axonemal activity.     

The indiction by complement of a change in KSCN-dissociable red cell membrane lipids.

During complement lysis of antibody-sensitized sheep erythrocytes (EA) there was a larger loss of membrane phospholipids than during lysis elicited by hypotonic buffer. In addition, membranes prepared from complement-lysed EA had a marked reduction in KSCN (2.4 M)-dissociable membrane cholesterol and phospholipids, as compared to membranes from EA lysed hypotonically. Complement lysis caused a mild reduction in the amount of KSCN-dissociable membrane hexose but no change in the amount of dissociable protein. The impairment in dissociation of membrane lipids was related to the action of C8; it did not occur with membranes from EA that were treated with heat-inactivated (56 degrees C for 30 min) human serum, C4-deficient guinea pig serum, C6-deficient rabbit serum, or the first seven human complement components. EA lysed with limited amounts of complement exhibited a partial impairment in KSCN-dissociable lipids. Membranes from erythrocytes lysed with melittin showed a large increase in dissociation by KSCN of lipids, proteins,and hexoses. Membranes from erythocytes lysed with lysolecithin or phospholipase C showed, in addition to a reduction in dissociable lipid, a much larger reduction in dissociable hexose than a membranes from complement-lysed cells. These profiles of reactivity with 2.4 M KSCN inidcate that the membrane pertubations caused caused by complement may be specific. We conclude that complement-lysis is accompanied by a major rearrangement of membrane cholesterol and phospholipid which could be demonstrated in membranes from cells lysed by only one or very few complement lesions. Therefore, it appears that the lesions induce a propragated change in the lipid organization which extends throughout large areas of the membrane. This change might be responsible for the impairment of membrane permeability that follows the action of complement and results in cell destruction.     

A study of the change in conductivity of the neuronal membrane caused by a generator potential using a method of computer modeling

Injection of cAMP induces in snail neurons generator potential, which is related to an increase of sodium and decrease of potassium permeability of the neuron outer membrane. A model is proposed which takes into account cAMP diffusion inside the neuron from the injection place and interaction of these molecules with the intercellular system controlling permeability of the outer membrane. Resulting impulse generation induces calcium ions current through the outer membrane. The model also considers calcium diffusion toward cAMP and its effect on the rate of the enzyme work destroying cAMP. Agreement between the calculations of ionic current I(t) and the experiment permits determination of the model parameters and calculation of the observed change of time distribution of nerve impulses when calcium input is significant.     

Osmotic tolerance limits and effects of cryoprotectants on the motility, plasma membrane integrity and acrosomal integrity of rat sperm

Osmotic stress is an important factor that can result in cell damage during cryopreservation. The objectives of this study were to determine: (1) isosmotic sperm cell volume; (2) osmotically inactive volume; (3) osmotic tolerance limits of rat sperm; and (4) the effects of addition and removal of glycerol (Gly), ethylene glycol (EG), propylene glycol (PG) or dimethyl sulfoxide (Me(2)SO) on rat sperm function. Sperm from Fischer 344 and Sprague-Dawley rats were used in this study. An electronic particle counter was used to measure the cell volume of rat sperm. Computer-assisted sperm motility analysis and flow-cytometric analysis were used to assess sperm motility, plasma membrane and acrosomal integrity. The isosmotic sperm cell volumes of the two strains were 37.0+/-0.1 and 36.2+/-0.2 microm(3), respectively. Rat sperm behaved as linear osmometers from 260 to 450 mOsm, and the osmotically inactive sperm volumes of the two strains were 79.8+/-1.5% and 81.4+/-2.2%, respectively. Rat sperm have very limited osmotic tolerances. The sperm motility and the sperm plasma membranes of both strains were sensitive to anisosmotic treatments, but the acrosomes of both strains were more sensitive to hyposmotic than hyperosmotic conditions. The one-step addition and removal of Me(2)SO showed the most deleterious effect on rat sperm motility, plasma membrane integrity, and acrosomal integrity among the four cryoprotectants. These data characterizing rat sperm osmotic behavior, osmotic and cryoprotectant tolerance will be used to design cryopreservation protocols for rat sperm.     

The effect of age on the frequency of sperm chromosomal abnormalities in normal men.

It has been suggested that advanced paternal age (independent of maternal age) is associated with an increased incidence of trisomy. However, studies of human liveborn offspring and of data from prenatal diagnosis have yielded conflicting results. To investigate this possible paternal age effect, we have studied sperm chromosome complements from 30 normal men of proven fertility stratified by age, with five males in each of six age categories (20-24, 25-29, 30-34, 35-39, 40-44, and 45+ years). Sperm chromosome complements were visualized after penetration of golden-hamster oocytes. A minimum of 30 complements were analyzed for each male. The analysis was performed blindly, without knowledge of the donor's age. The mean frequency of sperm chromosomal abnormalities in the individual men was 10.4% with means of 4.7% for numerical abnormalities and 6.2% for structural abnormalities. There was no relationship between age and the frequency of numerical abnormalities in sperm. Since there was a significant difference between the frequency of hyperhaploid and hypohaploid complements, these two types of numerical abnormalities were analyzed separately. There was no correlation between the frequency of hypohaploid complements and age. There was a significant negative correlation between age and the frequency of hyperhaploid complements. For structural abnormalities, there was a highly significant positive correlation with age. Thus, our results do not support the hypothesis of an increased risk of trisomy with paternal age.