Effects of bovine sperm cryopreservation using different freezing techniques and cryoprotective agents on plasma, acrosomal and mitochondrial membranes

The success of semen cryopreservation is influenced by several factors, such as freezing curves and cryoprotectants. These two factors are of special interest once they may lead to many important physical-chemical changes resulting in different degrees of damage in spermatozoa structure. This experiment was designed to compare the effect of bull semen cryopreservation using two freezing techniques: conventional (CT cooling rate of -0.55 degrees C min-1 and freezing rate of -19.1 degrees C min-1) and automated (AT cooling rate of -0.23 degrees C min-1 and freezing rate of -15 degrees C min-1), performed with different curves, and with three cryoprotectants (glycerol, ethylene glycol and dimethyl formamide) on bovine sperm motility and integrity of plasma, acrosomal and mitochondrial membranes. These variables were simultaneously evaluated using the fluorescence probes propidium iodide, fluorescein-conjugated Pisum sativum agglutinin and MitoTracker Green FM. The effects of freezing techniques, as well as of different cryoprotectants were analysed by the analysis of variance. The means were compared by Fishers test. There were no significant differences between freezing techniques (P > 0.05). Glycerol showed higher percentages of motility, vigour and integrity of plasma, acrosomal and mitochondrial membranes than other two cryoprotectants (P < 0.05). Ethylene glycol preserved higher motility and integrity of plasma and mitochondrial membranes than dimethyl formamide (P < 0.05). Sperm motility with glycerol was 30.67 +/- 1.41% and 30.50 +/- 1.06%, with ethylene glycol was 21.17 +/- 1.66% and 21.67 +/- 1.13% and with dimethyl formamide was 8.33 +/- 0.65% and 9.17 +/- 0.72% to CT and AT curves, respectively. The percentage of spermatozoa with simultaneously intact plasma membrane, intact acrosome and mitochondrial function (IPIAH) was 14.82 +/- 1.49% (CT) and 15.83 +/- 1.26% (AT) to glycerol, 9.20 +/- 1.31% (CT) and 9.92 +/- 1.29% (AT) to ethylene glycol 4.65 +/- 0.93% (CT) and 5.17 +/- 0.87% (AT) to dimethyl formamide. Glycerol provided the best results, although nearly 85% of spermatozoa showed some degree of injury in their membranes, suggesting that further studies are required to improve the results of cryopreservation of bovine semen.

Effect of varicocele on sperm function and semen oxidative stress

OBJECTIVE To assess the effect of varicocele on sperm DNA integrity, mitochondrial activity, lipid peroxidation and acrosome integrity. PATIENTS AND METHODS In all, 30 patients with a clinically diagnosed varicocele of grade II or III and 32 men without a varicocele were evaluated for sperm DNA fragmentation (comet assay), mitochondrial activity (3,3'-diaminobenzidine assay), lipid peroxidation (malondialdehyde) and acrosome integrity (fluorescent probe labelled peanut agglutinin). RESULTS The varicocele group showed fewer spermatozoa with intact DNA (grade II, P = 0.040), more cells with inactive mitochondria (class III, P = 0.001), fewer cells with active mitochondria (class I, P = 0.005) and fewer spermatozoa with intact acrosomes (P < 0.001). Finally, no significant differences were observed in lipid peroxidation levels. CONCLUSION Men with varicocele showed an increase in sperm DNA fragmentation and a reduction in mitochondrial activity and acrosome integrity. However, lipid peroxidation levels remained unchanged.

Evaluation of Sperm DNA Peroxidation in Fertile and Subfertile Dogs

Contents Oxidative stress (OS) has been recognized as one of the most important causes of male infertility. The antioxidant activities of seminal plasma and epididymal fluid are not enough to prevent OS, which can damage sperm membranes and DNA, so antioxidant supplementation has been used as a treatment of male infertility. The aim of this experiment was to evaluate the DNA peroxidation before and after antioxidant supplementation with vitamin C and E in dogs with and without fertility problems. A total of eleven dogs were used and were divided in two groups: fertile group (G1), dogs with normal spermiogram (n=5); subfertile group (G2): dogs with low sperm count (<20x106sptz/ml) and/or more than 30% of total sperm pathology (n=6). Both groups received 500mg/day of vitamin C and 500mg/day of vitamin E for 60days. A semen sample was collected before (M1) and after (M2) oral supplementation. Samples were analysed for DNA peroxidation by measuring the 8-hydroxy-2'-deoxyguanosine concentration. No significant difference was observed between groups at either time. Oral supplementation with 500mg/day of vitamin C and 500mg/day of vitamin E did not change the DNA peroxidation in fertile and subfertile dogs.

Sperm head morphometry in ejaculates of adult marmosets (Callithrix jacchus): a model for studying sperm subpopulations and among-donor variations

In humans and other mammals, sperm morphology has been considered one of the most important predictive parameters of fertility. The objective was to determine the presence and distribution of sperm head morphometric subpopulations in a nonhuman primate model (Callithrix jacchus), using an objective computer analysis system and principal component analysis (PCA) methods to establish the relationship between the subpopulation distribution observed and among-donor variation. The PCA method revealed a stable number of principal components in all donors studied, that represented more than 85% of the cumulative variance in all cases. After cluster analysis, a variable number (from three to seven) sperm morphometric subpopulations were identified with defined sperm dimensions and shapes. There were differences in the distribution of the sperm morphometric subpopulations (P < 0.001) in all ejaculates among the four donors analyzed. In conclusion, in this study, computerized sperm analysis methods combined with PCA cluster analyses were useful to identify, classify, and characterize various head sperm morphometric subpopulations in nonhuman primates, yielding considerable biological information. In addition, because all individuals were kept in the same conditions, differences in the distribution of these subpopulations were not attributed to external or management factors. Finally, the substantial information derived from subpopulation analyses provided new and relevant biological knowledge which may have a practical use for future studies in human and nonhuman primate ejaculates, including identifying individuals more suitable for assisted reproductive technologies. (c) 2012 Elsevier Inc. All rights reserved.

Meiotic segregation and interchromosomal effect in the sperm of a double translocation carrier: a case report

Abstract Background Infertility is a natural mechanism of selection intended to prevent the delivery of a child with malformations or mental retardation. Male infertility is closely related to chromosomal abnormalities. This study was focused on the analysis of meiotic segregation involving a Robertsonian translocation, 45,XY,der(13;13) [56]/45,XY,der(13;14) [44] and the evaluation of possible interchromosomal effects. Results Hybridisation with LSI 13q14 and subtelomere 14q probes and WCP13 SpectrumGreen and WCP14 SpectrumOrange probes showed a high proportion of unbalanced gametes, corresponding to 71.2% of the spermatozoa. The disomic frequencies of the sexual chromosomes and chromosome 18 of the patient were higher (5.28% and 2.55%, respectively) than those of the control (0.6% and 0.59%, respectively). Conclusion Meiotic segregation studies in sperm are an important tool for genetic counselling of chromosomal aberrations, allowing for a prediction of the risks and consequent implications for the reproductive life. The patient with this rare translocation exhibited meiotic segregation fidelity, and a high rate of unbalanced gametes with disomic spermatozoa.

Sexual selection in Pomatoschistus – nests, sperm competition, and paternal care

Sexual selection arises through variation in reproductive success. This thesis investigates different aspects important in sexual selection, namely nest building, sperm competition, paternity and paternal care, and their mutual interrelationships. In the studied species, the sand goby (Pomatoschistus minutus) and the common goby (Pomatoschistus microps), sperm competition did arise when small males, so called sneakers, sneaked into other males nests and released sperm. They seemed to use female behaviour as their prime cue for a sneaking opportunity. However, also nest-holders, both with and without eggs, were found to fertilize eggs in the nests of other males. Clearly, nest-holding males tried to prevent other males from spreading their sperm in their nests, since they showed aggression towards such males. A nest building experiment indicated that the small nest-openings found in the sneaker male treatment were sexually selected through protection against sneaking or by female choice. Yet, no behavioural or genetical support for the hypothesis that the nest functions as a physical or visual defence, or that sneaker males prefer to sneak upon nests with wide nest-openings, were found in the other studies. Still, individual nest-holding males showed a higher mucus preparation effort inside the nest in the presence of a sneaker male than when alone. In close relatives, such mucus contains sperm, suggesting an importance in sperm competition. However, the mucus may also have pheromone and anti-bacterial functions and may constitute a mating effort, as found in other gobies. Both a behavioural and a mate choice experiment suggested that the males were not less eager to spawn in the presence of a sneaker male. Sneak intrusion did not affect nest defence, fanning or filial cannibalism, nor had paternity an effect on filial cannibalism. This and various life history aspects, together with the fact that the parasitic male only fertilized a fraction of the clutches, would predict females to ignore sneaker males. This was also the case, as the presence of sneaker males was found not to affect female spawning decision. Still, several females spawned in two nests, which coincided with parasitic spawnings, suggesting a cost of disturbance for the females and thus a substantial cost to the nest-holding males in terms of lost mating success. However, females paid attention to other traits in their choice of mate since spawning was associated with sand volume of the nest, but not with nest-opening width. Also, female (but not male) courtship was correlated with partial clutch filial cannibalism, indicating that females are able to anticipate future male cannibalism. In a partial correlation of nest opening, sand volume, male courtship display, displacement fanning and male size, a large number of traits were correlated both positively and negatively with regard to how we may expect them to be appreciated by females. For instance, males which fan well also build large nests or display intensely (but not both). Together with all the other results of this thesis, this shows the entangled selection pressures working on breeding animals, as well as the different male and female tactics employed to maximize their reproduction.

Seasonality and diel variation in blue whale D calls recorded in the Southern California Bight

Passive acoustic data have been collected using HARPs (High-frequency Acoustic Recording Packages) and were used to assess (1) the seasonality of blue whale D calls in the Southern California Bight, (2) their interannual abundance during 2007-2012 and (3) their diel variation. This goal has been achieved running the GPL (Generalized Power-Law) automated detector. (1) Blue whale D calls were detected in the Southern California Bight from May through November with a peak in July, even though few detections were from December to April as well. A key predictor for blue whale distribution and movement in the California Current region has been identified with zooplankton aggregations, paying a particular attention to those euphausiid species, such as E. pacifica and T. spinifera, which are blue whale favorite krill. The Southern California Bight experiences seasonal upwelling, resulting in an increase of productivity and prey availability. The summer and early fall have been marked as the most favorable periods. This supports the presence of blue whales in the area at that time, supposing these marine mammals exploit the region as a feeding ground. (2) As to the interannual abundance during 2007-2012, I found a large variability. I observed a great increase of vocalizations in 2007 and 2010, whereas a decrease was shown in the other years, which is well marked in 2009. It is my belief that these fluctuations in abundance of D calls detections through the deployed period are due to the alternation of El Nino and La Nina events, which occurred in those years. (3) The assessment of the daily timing of D calls production shows that D calls are more abundant during the day than during the night with a peak at 12:00 and 13:00. Assuming that D calling is associated with feeding, the daily pattern of D calls may be linked to the prey availability. E. pacifica and T. spinifera are among those species of krill which undertake daily vertical migrations, remaining at depth during the day and slowly coming up towards the surface at night. Because of some anatomical arrangements, these euphausiids are very sensitive to the light. Given that we believe D calls have a social function, I hypothesize that blue whales may recognize the hours at the highest solar incidence as the best moment of the day in terms of prey availability, exploiting this time window to advert their conspecifics.

Sperm analysis of patients after successful treatment of childhood acute lymphoblastic leukemia with chemotherapy

Survivors of childhood acute lymphoblastic leukemia (ALL) treated with radiotherapy are at risk for impaired fertility. Whether chemotherapy alone is also long-term gonadotoxic is unclear. We assessed gonadal function in 11 male ALL-survivors treated with the same chemotherapy regimen and compared sperm analysis to healthy men. While sex hormone levels were normal in all subjects, 5/11 survivors showed pathological sperm concentration and 4/11 a decreased total sperm count compared to WHO criteria. Compared to healthy controls, all quantitative parameters in semen analysis of survivors were decreased. This suggests that treatment with chemotherapeutic agents alone, even in moderate doses, might have a gonadotoxic effect.

Effects of TNF antagonists on sperm characteristics in patients with spondyloarthritis

To study the influence of tumour necrosis factor (TNF) antagonists on spermatogenesis in a cohort of patients with spondyloarthritis (SpA).

A double role of sperm in scorpions: the mating plug of Euscorpius italicus (Scorpiones: Euscorpiidae) consists of sperm

Mating plugs occluding the female gonopore after mating are a widespread phenomenon. In scorpions, two main types of mating plugs are found: sclerotized mating plugs being parts of the spermatophore that break off during mating, and gel-like mating plugs being gelatinous fluids that harden in the female genital tract. In this study, the gel-like mating plug of Euscorpius italicus was investigated with respect to its composition, fine structure, and changes over time. Sperm forms the major component of the mating plug, a phenomenon previously unknown in arachnids. Three parts of the mating plug can be distinguished. The part facing the outside of the female (outer part) contains sperm packages containing inactive spermatozoa. In this state, sperm is transferred. In the median part, the sperm packages get uncoiled to single spermatozoa. In the inner part, free sperm is embedded in a large amount of secretions. Fresh mating plugs are soft gelatinous, later they harden from outside toward inside. This process is completed after 3-5 days. Sperm from artificially triggered spermatophores could be activated by immersion in insect Ringer's solution indicating that the fluid condition in the females' genital tract or females' secretions causes sperm activation. Because of the male origin of the mating plug, it has likely evolved under sperm competition or sexual conflict. As females refused to remate irrespective of the presence or absence of a mating plug, females may have changed their mating behavior in the course of evolution from polyandry to monandry.

Sperm of colourful males are better protected against oxidative stress

Sperm cells are highly vulnerable to free radicals, and sperm quality and male fertility are critically affected by oxidative stress. Recently, sexual ornaments, particularly carotenoid-based colourful traits, have been proposed to depend on a male's capacity to resist oxidative stress, and thus to signal sperm quality. We conducted an experimental test of this hypothesis on great tits Parus major, in which adults are sexually dichromatic in carotenoid-based breast plumage. We report the first evidence that ornaments and sperm quality may be linked through oxidative stress. When experimentally subjected to oxidative stress resulting from increased workload, less colourful males suffered a greater reduction in sperm motility and swimming ability, and increased levels of sperm lipid peroxidation compared to more colourful males. Moreover, the level of sperm lipid peroxidation was negatively correlated with sperm quality. Finally, carotenoid supplementation increased sperm quality of less colourful males, suggesting that pale males are deficient in carotenoid antioxidants.

Sperm morphology, swimming velocity, and longevity in the house sparrow Passer domesticus

Sperm competition exerts strong selection on males to produce spermatozoa with an optimal morphology that maximizes their fertilization success. Long sperm were first suggested to be favored because they should swim faster. However, studies that investigated the relationship between sperm length and sperm competitive ability or sperm swimming velocity yielded contradictory results. More recently, ratios of the different sections of a spermatozoon (the head, midpiece, and flagellum) were suggested to be more crucial in determining swimming velocity. Additionally, sperm ability to remain and survive in the female storage organs may also influence fertilization success, so that optimal sperm morphology may rather maximize sperm longevity than velocity. In this study, we investigated how sperm morphology is related to sperm velocity and sperm longevity in the house sparrow Passer domesticus. Sperm velocity was found to be correlated with head/flagellum ratio. Sperm with small heads relative to their flagellum showed higher swimming velocity. Additionally, shorter sperm were found to live longer. Finally, we found sperm morphological traits to vary substantially within males and the head/flagellum ratio to be unrelated to total sperm length. We discuss the hypothesis that the substantial within-male variation in sperm morphology reflects a male strategy to produce a diversity of sperm from long, fast-swimming to short, long-living sperm to maximize their fertilization success in a context of sperm competition.

Immune Activation Reduces Sperm Quality in the Great Tit

Mounting an immune response against pathogens incurs costs to organisms by its effects on important life-history traits, such as reproductive investment and survival. As shown recently, immune activation produces large amounts of reactive species and is suggested to induce oxidative stress. Sperm are highly susceptible to oxidative stress, which can negatively impact sperm function and ultimately male fertilizing efficiency. Here we address the question as to whether mounting an immune response affects sperm quality through the damaging effects of oxidative stress. It has been demonstrated recently in birds that carotenoid-based ornaments can be reliable signals of a male's ability to protect sperm from oxidative damage. In a full-factorial design, we immune-challenged great tit males while simultaneously increasing their vitamin E availability, and assessed the effect on sperm quality and oxidative damage. We conducted this experiment in a natural population and tested the males' response to the experimental treatment in relation to their carotenoid-based breast coloration, a condition-dependent trait. Immune activation induced a steeper decline in sperm swimming velocity, thus highlighting the potential costs of an induced immune response on sperm competitive ability and fertilizing efficiency. We found sperm oxidative damage to be negatively correlated with sperm swimming velocity. However, blood resistance to a free-radical attack (a measure of somatic antioxidant capacity) as well as plasma and sperm levels of oxidative damage (lipid peroxidation) remained unaffected, thus suggesting that the observed effect did not arise through oxidative stress. Towards the end of their breeding cycle, swimming velocity of sperm of more intensely colored males was higher, which has important implications for the evolution of mate choice and multiple mating in females because females may accrue both direct and indirect benefits by mating with males having better quality sperm.

Repression of common bull sperm flora and in vitro impairment of sperm motility with Pseudomonas aeruginosa introduced by contaminated lubricant

Semen collected from clinically healthy bulls at an artificial insemination centre was examined for bacterial diversity. While bacteria that are normally present in the common flora of bovine semen were absent, such as Mycoplasma sp., Proteus sp. and Corynebacterium sp., all semen samples contained an unusually high number of Pseudomonas aeruginosa strains. Analysis via pulsed field gel electrophoresis demonstrated that one particular P. aeruginosa strain, present in a sealed bottle of lubricant, was widespread in bull semen. This strain was shown to secrete substances that inhibited both the growth of bacteria constituting the normal bull sperm flora and the motility of spermatozoa in vitro. This study demonstrated that commercially available lubricants might contain bacteria that can spread amongst breeding bulls and affect the quality of semen. Bacteriological controls and species' identification are necessary at several production levels, including lubricants and extenders, to ensure high semen quality and avoid the spread of pathogens.