Sperm Ultrastructure Research Articles

Spermatozoan morphology of the snails Babylonia lutosa , Babylonia areolata from parental lines of populations in Hainan and Thailand and hybrid lines

Sperm morphology provides an abundant source of potentially informative characters for gastropod taxonomy. In the present study, this character was used to offer additional information concerning the genus Babylonia (Mollusca, Gastropoda), the most promising economic marine gastropods of the present century. Light and transmission electron microscopy was used to study the ultrastructure of spermatozoa in five groups of Babylonia, including B. areolata populations from Hainan, China, from Thailand, and the reciprocal hybrid offspring of these two populations and B. lutosa. Principal component analysis was conducted to examine morphological differences of spermatozoa. The results showed that morphology can contribute to the classification of the two species B. areolata and B. lutosa. The reciprocal hybrid offspring of the two B. areolata varieties exhibited ultrastructure of spermatozoa similar to that of their male parents, and each showed greater similarity to the male parent in the fine structural details. This study aimed to compare the ultrastructure of sperm in five groups of Babylonia and to compare the differences between the parents and the hybrids to provide basic information for genetic studies of Babylonia.

Effect of Cooling and Freezing on Llama (Lama glama) Sperm Ultrastructure.

Semen cryopreservation in South American camelids has a low efficiency. Post-thaw viability of sperm is low, and poor results are obtained when artificial insemination is performed with cryopreserved semen, impeding advances both in accelerated genetic progress and selection. This study aimed to describe the effect of a conventional method of camelid semen cryopreservation on the llama sperm ultrastructure during cooling and freezing, using transmission and scanning electron microscopy (TEM, SEM). Sperm motility, vigor, viability, and DNA integrity during those steps were also examined. Ejaculates from five fertile adult llama males were obtained by electroejaculation. For cooling, semen samples were washed with Hepes-balanced salt solution (HBSS), diluted in Tris-citric acid-fructose egg yolk extender (TCF-EY), and then cooled until 5°C for 24 h. For freezing, sperm samples were washed with HBSS, diluted in TCF-EY and cooled until 5°C for 2.5 h. Samples were equilibrated with TCF-EY, supplemented with 6% glycerol at 5°C for 20 min, and then stored in liquid nitrogen for a month before thawing. TEM and SEM analyses were carried out on sperm samples prior to cryopreservation, after cooling down until 5°C for 2.5 and 24 h, and after the freeze-thaw process. Ultrastructural injury was noticed during cooling, even though sperm motility, vigor, viability, and DNA integrity were not significantly affected. Analysis revealed plasma membrane and acrosome damage, loss of mitochondria, and axoneme and periaxonemal structure disorganization after 2.5 h of cooling. During freezing, a significant decrease in sperm motility and viability was observed after thawing. TEM and SEM revealed prominent signs of post-thawing damage. The plasma membrane was lost or exhibited various degrees of swelling, undulation, and perforations. Besides, the sperm presented vacuoles in the nucleus and broken acrosomes. Mitochondria in the midpiece showed vacuolization and structural disorganization. In conclusion, SEM and TEM revealed that cryopreservation induced ultrastructural damages in llama sperm that initiated during cooling and intensified during freezing. These details provide valuable data for further studies to minimize cryodamage in camelid sperm.

Ultrastructure of sperm and complete mitochondrial genome in Meretrix sp. (Bivalvia: Veneridae) from Taiwan

Spermatozoan ultrastructure and complete mitochondrial genome in the marine bivalve mollusk Meretrix sp. (Taiwan) from Taiwan are described and contrasted with other bivalves, especially within Meretrix. We have examined the features of the mature gonadal spermatozoa of Meretrix sp. (Taiwan) and provided comparisons with the other four Meretrix species (M. petechialis, M. meretrix, M. lyrata, and M. lamarckii). The morphological characteristics of these spermatozoa are diagnostic for each of the species studied here. The most marked interspecific difference was found in the acrosome. Meretrix sp. (Taiwan) is genetically distinct and is a different species from M. petechialis and M. lusoria (Japan) based on complete mitochondrial genome data. Sperm data for Meretrix are limited but show remarkable congruence with the molecular results. We suggest use Meretrix formosa Gwo and Hsu as the scientific name for Taiwanese hard clams, Meretrix sp. (Taiwan). Additional species, particularly the Japanese hard clam (M. lusoria) require examination before this tentative conclusion can be verified.

Bovine sperm-oviduct interactions are characterized by specific sperm behaviour, ultrastructure and tubal reactions which are impacted by sex sorting

To date sperm-oviduct interactions have largely been investigated under in vitro conditions. Therefore we set out to characterize the behaviour of bovine spermatozoa within the sperm reservoir under near in vivo conditions and in real-time using a novel live cell imaging technology and a newly established fluorescent sperm binding assay. Sperm structure and tubal reactions after sperm binding were analysed using scanning and transmission electron microscopy and histochemistry. As a model to specify the impact of stress on sperm-oviduct interactions, frozen-thawed conventional and sex-sorted spermatozoa from the same bulls (n = 7) were co-incubated with oviducts obtained from cows immediately after slaughter. Our studies revealed that within the oviductal sperm reservoir agile (bound at a tangential angle of about 30°, actively beating undulating tail), lagging (bound at a lower angle, reduced tail movement), immotile (absence of tail movement) and hyperactivated (whip-like movement of tail) spermatozoa occur, the prevalence of which changes in a time-dependent pattern. After formation of the sperm reservoir, tubal ciliary beat frequency is significantly increased (p = 0.022) and the epithelial cells show increased activity of endoplasmic reticula. After sex sorting, spermatozoa occasionally display abnormal movement patterns characterized by a 360° rotating head and tail. Sperm binding in the oviduct is significantly reduced (p = 0.008) following sexing. Sex-sorted spermatozoa reveal deformations in the head, sharp bends in the tail and a significantly increased prevalence of damaged mitochondria (p < 0.001). Our results imply that the oviductal cells specifically react to the binding of spermatozoa, maintaining sperm survival within the tubal reservoir. The sex-sorting process, which is associated with mechanical, chemical and time stress, impacts sperm binding to the oviduct and mitochondrial integrity affecting sperm motility and function.

Characterization of seminal parameters, sperm morphometry, micromorphology, and ultrastructure in gray brocket deer (Mazama gouazoubira, Fischer, 1814).

Populations of gray brocket deer (Mazama gouazoubira) are declining; yet, knowledge on the reproductive biology of this species remains limited. Therefore, this study aimed to describe morphology, viability, membrane integrity, mitochondrial activity, morphometry, micromorphology, and ultrastructure of the gray brocket deer sperm. Three adult male gray brocket deer were used in the study. Semen collection was performed using electroejaculation. Semen were analyzed by evaluating pH, motilities, vigor, mass movement, volume, concentration, viability, membrane integrity, mitochondrial activity, morphology, and morphometry. Micromorphology and ultrastructure of sperm were analyzed using scanning and transmission electron microscopy (SEM and TEM), respectively. There was no significant difference among males regarding on pH, motilities, vigor, mass movement, volume, concentration, viability. High values for membrane integrity, mitochondrial activity, and normal sperm were observed. The most frequent defects were simple bent tail and bowed midpiece. The head length, and width, midpiece, and tail length were 8.5, 4.4, 11.5, and 41.3 μm, respectively. SEM sperm showed paddle-shaped heads, with apical ridge and serrated band on the equatorial segment. TEM revealed the nucleus, acrosome, plasma membrane, mitochondria sheath, proximal centrioles, segmented columns, axoneme, outer dense fibers, and fibrous sheath. SEM and TEM showed the presence of some abnormalities. These results are expected to provide baseline values of diverse semen parameters, contributing toward the development of reproductive biotechnologies for gray brocket deer and, other deer species at risk of extinction.

Short Communication: Effect of cryopreservation on ultrastructure and mitochondrial function of albino Pangasius catfish spermatozoa

Abstract. Hasanah U, Binawanto, Alimuddin A, Boediono A, Kursini E. 2020. Short Communication: Effect of cryopreservation on ultrastructure and mitochondrial function of albino Pangasius catfish spermatozoa. Biodiversitas 21: 4524-4528. Cryopreservation techniques have been carried out on many endangered species and animals with unique characteristics. Successful cryopreservation techniques vary between species depending on various factors. The study used cryopreserved spermatozoa of the albino Pangasius catfish as samples. The cryopreserved spermatozoa were analyzed by its ultrastructure, functional mitochondria, and viability. The cryopreservation was performed using a combination of 10% methanol, skim milk, and fish ringer extender. A deep freezer is used for cryopreservation at -80 °C with a storage period of 14 days. Observations were made on fresh spermatozoa, post-equilibration spermatozoa, and frozen-thaw spermatozoa. This study found there were differences in ultrastructure and morphology in the three treatments. Fresh spermatozoa and post-equilibration spermatozoa appeared intact membrane, mitochondrial, and flagellar structures. In contrast, in frozen-thaw spermatozoa, there was damage to the cell membrane. The study showed different percentages of yields on functional mitochondria of fresh spermatozoa (98 ± 2%), spermatozoa post-equilibration (57 ± 7%), frozen-thaw spermatozoa (42 ± 3.21%). Cell viability showed that there were differences in viability of fresh spermatozoa and frozen-thaw (p &lt;0.05), the results of fresh spermatozoa (92 ± 0.57%) spermatozoa post-equilibration (80 ± 3.51%), frozen-thaw spermatozoa (61 ± 2.30%).The study concluded that the spermatozoa cryopreservation affects the ultrastructure, mitochondrial function, and viability in albino Pangasius catfish spermatozoa.

Structure, development, and evolutive patterns of spermatozoa in rhabditid nematodes (Nematoda: Rhabditida).

Spermatogenesis of five rhabditid nematodes was studied using transmission electron microscopy and is described herein. Structure and development of nematode sperm in all available representatives of the extensive order Rhabditida have been analysed and the main characteristics of each infraorder are discussed. The ancestral sperm of the order Rhabditida was reconstructed using maximum likelihood and Bayesian methods based on 44 ultrastructural sperm characters. The hypothetical ancestral spermatogenesis of the order Rhabditida agrees with the previously suggested "rhabditid" pattern and appears to be conserved throughout the order Rhabditida. Despite the enormous variation of rhabditid nematodes, few groups deviate from the ancestral pattern. This conserved pattern can be informative within the phylum Nematoda at order level, but poses limitations when used in taxonomic and phylogenetic analysis within Rhabditida.

Semen Biochemical Components in Varicocele, Leukocytospermia, and Idiopathic Infertility

The evaluation of the seminal plasma plays a relevant role in the definition of male infertility and in assisted reproduction outcomes; for this reason, it would be recommended to find biochemical markers able to characterize sperm pathology. In this study, 53 infertile patients (grouped by the presence leukocytospermia, idiopathic infertility, or varicocele) and 10 fertile men were selected. Spermiogram was performed by light microscopy, and sperm ultrastructure was evaluated by transmission electron microscopy (TEM) mathematically elaborated. Testosterone (TESTO), estradiol (E2), ferritin (FERR), iron (Fe), transferrin (TRSF), triglycerides (TRG), cholesterol (CHOL), and isoprostanes (F2-IsoPs) were detected in seminal plasma. Sperm characteristics and biochemical components were correlated by Spearman’s rank correlation coefficient in the whole population and in each group. The levels of TESTO and E2 were positively correlated with sperm quality in particular, and E2 was correlated with fertility index expressing the number of sperm free of ultrastructural defects evaluated by TEM. On the contrary, the indices of iron metabolism (FERR, Fe, and TRSF) were positively associated with low sperm quality and sperm necrosis, particularly in leukocytospermia and varicocele groups, pathologies in which an inflammatory status and oxidative stress condition are present. The study of the seminal plasma composition deserves attention because the levels of the various components seem to be associated with specific reproductive pathologies.

Reproductive outcomes predicted by phase imaging with computational specificity of spermatozoon ultrastructure

The ability to evaluate sperm at the microscopic level, at high-throughput, would be useful for assisted reproductive technologies (ARTs), as it can allow specific selection of sperm cells for in vitro fertilization (IVF). The tradeoff between intrinsic imaging and external contrast agents is particularly acute in reproductive medicine. The use of fluorescence labels has enabled new cell-sorting strategies and given new insights into developmental biology. Nevertheless, using extrinsic contrast agents is often too invasive for routine clinical operation. Raising questions about cell viability, especially for single-cell selection, clinicians prefer intrinsic contrast in the form of phase-contrast, differential-interference contrast, or Hoffman modulation contrast. While such instruments are nondestructive, the resulting image suffers from a lack of specificity. In this work, we provide a template to circumvent the tradeoff between cell viability and specificity by combining high-sensitivity phase imaging with deep learning. In order to introduce specificity to label-free images, we trained a deep-convolutional neural network to perform semantic segmentation on quantitative phase maps. This approach, a form of phase imaging with computational specificity (PICS), allowed us to efficiently analyze thousands of sperm cells and identify correlations between dry-mass content and artificial-reproduction outcomes. Specifically, we found that the dry-mass content ratios between the head, midpiece, and tail of the cells can predict the percentages of success for zygote cleavage and embryo blastocyst formation.

Ultrastructure of Spermatozoa of Elaphe schrenckii (Reptilia, Squamata)

Elaphe schrenckii (Serpentes, Colubridae), a kind of large nonvenomous snakes and great significance to maintain the stability of ecosystem in China. We provide detailed descriptions of the sperm microstructure and ultrastructure of E. schrenckii, experimented by light microscope and transmission electron microscope. The spermatozoon of E. schrenckii is filiform and consists of head and tail regions. The cross-section of acrosomal vesicle is always rounded and divided into medulla inside and cortex outside. The ultrastructure of acrosome complex observed the unilateral ridge, the single perforatorium, the perforatorium base plate, the epinuclear lucent zone, the subacrosomal space and the nuclear fossa at the end of nucleus connect the neck region. The neck region is short with the stratified laminar structure and observed the distal centriole and the proximal centriole are perpendicular and both consisted of nine triplets. Midpiece is long and observed the extracellular microtubules, the multilaminar membranec, the mitochondria with the dense bodies discontinuity distribting, the fibrous sheath, and the axoneme. The principal piece is after the annulus with no mitochondrias and the end piece with no mitochondrias neither the fibrous sheath. Our study contrasted the spermatozoa ultrastructure of 8 species belong to 5 families and 6 genera and added the sperm measurement compare, summarized that three Colubridae snakes are more similar than others momentarily but some specific characteristics in E. schrenckii and proved that the ultrastructure of sperm related to phylogeny in some ways.

Diminution in sperm quantity and quality in mouse models of Duchenne Muscular Dystrophy induced by a myostatin-based muscle growth-promoting intervention.

Duchenne Muscular Dystrophy is a devastating disease caused by the absence of a functional rod-shaped cytoplasmic protein called dystrophin. Several avenues are being developed aimed to restore dystrophin expression in boys affected by this X-linked disease. However, its complete cure is likely to need combinational approaches which may include regimes aimed at restoring muscle mass. Augmenting muscle growth through the manipulation of the Myostatin/Activin signalling axis has received much attention. However, we have recently shown that while manipulation of this axis in wild type mice using the sActRIIB ligand trap indeed results in muscle growth, it also had a detrimental impact on the testis. Here we examined the impact of administering a powerful Myostatin/Activin antagonist in two mouse models of Duchenne Muscular Dystrophy. We report that whilst the impact on muscle growth was not always positive, both models showed attenuated testis development. Sperm number, motility and ultrastructure were significantly affected by the sActRIIB treatment. Our report suggests that interventions based on Myostatin/Activin should investigate off-target effects on tissues as well as muscle.

Diminution in sperm quantity and quality in mouse models of Duchenne Muscular Dystrophy induced by a myostatin-based muscle growth-promoting intervention

Duchenne Muscular Dystrophy is a devastating disease caused by the absence of a functional rod-shaped cytoplasmic protein called dystrophin. Several avenues are being developed aimed to restore dystrophin expression in boys affected by this X-linked disease. However, its complete cure is likely to need combinational approaches which may include regimes aimed at restoring muscle mass. Augmenting muscle growth through the manipulation of the Myostatin/Activin signalling axis has received much attention. However, we have recently shown that while manipulation of this axis in wild type mice using the sActRIIB ligand trap indeed results in muscle growth, it also had a detrimental impact on the testis. Here we examined the impact of administering a powerful Myostatin/Activin antagonist in two mouse models of Duchenne Muscular Dystrophy. We report that whilst the impact on muscle growth was not always positive, both models showed attenuated testis development. Sperm number, motility and ultrastructure were significantly affected by the sActRIIB treatment. Our report suggests that interventions based on Myostatin/Activin should investigate off-target effects on tissues as well as muscle.

Diminution in sperm quantity and quality in mouse models of Duchenne Muscular Dystrophy induced by a myostatin-based muscle growth-promoting intervention

Duchenne Muscular Dystrophy is a devastating disease caused by the absence of a functional rod-shaped cytoplasmic protein called dystrophin. Several avenues are being developed aimed to restore dystrophin expression in boys affected by this X-linked disease. However, its complete cure is likely to need combinational approaches which may include regimes aimed at restoring muscle mass. Augmenting muscle growth through the manipulation of the Myostatin/Activin signalling axis has received much attention. However, we have recently shown that while manipulation of this axis in wild type mice using the sActRIIB ligand trap indeed results in muscle growth, it also had a detrimental impact on the testis. Here we examined the impact of administering a powerful Myostatin/Activin antagonist in two mouse models of Duchenne Muscular Dystrophy. We report that whilst the impact on muscle growth was not always positive, both models showed attenuated testis development. Sperm number, motility and ultrastructure were significantly affected by the sActRIIB treatment. Our report suggests that interventions based on Myostatin/Activin should investigate off-target effects on tissues as well as muscle.

Ultrastructure of the spermatozoon of Lecithostaphylus parexocoeti (Digenea, Microphalloidea, Zoogonidae) parasite of the flying fish Cheilopogon pinnatibarbatus (Teleostei, Exocoetidae) off Senegal and their implication on the phylogenetic relationships in Microphalloidea

This study shows the ultrastructure of the mature spermatozoon of Lecithostaphylus parexocoeti (Digenea, Microphalloidea, Zoogonidae) parasite of the flying fish Cheilopogon pinnatibarbatus (Exocoetidae) captured in the Atlantic Ocean near Dakar (Senegal). This is the first report of L. parexocoeti from Senegal and from this host species. The mature spermatozoon is filiform and exhibits two axonemes of the 9+“1” pattern of the Trepaxonemata, an external ornamentation associated with spine-like bodies, parallel cortical microtubules, a nucleus, two mitochondria and granules of glycogen. The particularities of the spermatozoon of L. parexocoeti are the location of the first mitochondrion and the morphology of its anterior extremity with only cortical microtubules and external ornamentations of the plasma membrane. To our knowledge, this type of anterior extremity has never been described in the superfamily Microphalloidea. However, the ultrastructure of the posterior extremity of the spermatozoon corresponds to the fasciolidean type that was previously described in the Zoogonidae.

Some Data on Reproductive Biology of Spotted Ghoul Inimicus sinensis (Synanceiidae)

Gonadal structure, cytological condition of oocytes, ultrastructure of egg envelope, and spermatozoa morphology are studied in the individuals of Inimicus sinensis 120–230 mm TL with the gonads mainly at maturity stage IV. The males possess hypertrophied urinary bladders. Based on oocytes’ size distribution in the ovaries, the oogenesis is continuous and the spawning is multiple. Oocytes of the maturation period are 600–700 µm in diameter. The batch fecundity is 15 380 oocytes on average. The zona radiata of oocytes that have completed growth is 10.2 µm in width on average and it consists of two layers. The narrow outermost layer of the egg envelope approximately 0.34 µm width is most likely chorion. Spermatozoon ultrastructure of I. sinensis does not notably differ from that in other representatives of the suborder Scorpaenoidei with external insemination. The centriolar complex is characterized by orthogonal position of centrioles. Based on ovarian structure, the representatives of the genus Inimicus do not differ from the majority of teleost fishes and viviparous species of the genera Sebastes and Sebastiscus. However, many representatives of the suborder Scorpaenoidei have unique ovarian structure with the central ovigerous stroma attached to the anterior part of the ovary. Based on the current phylogenetic schemes, this ovarian type evolved independently in several evolutionary lineages.

Spermatozoa ultrastructure, spermatogenesis and reproductive system of Acanthodasys australis (Gastrotricha, Macrodasyida)

Spermatozoon morphology, spermatogenesis and reproductive system of Acanthodasys australis (Thaumastodermatidae) were studied at ultrastructural level. The spermatozoon is a filiform cell formed by a cork-screw-shaped acrosome, a helical nuclear–mitochondrial complex and a long flagellum. The acrosome contains an axial tubular structure composed of a pile of electron-dense rings. The helical nucleus is thin and wraps a single, giant mitochondrion. A large and thick perinuclear helix surrounds the nuclear–mitochondrial complex extending for its whole length, except the two most basal coils. The flagellum with typical 9 × 2 + 2 axoneme is surrounded by a monolayered obliquely striated cylinder, in the terminal part of which the axoneme disappears. The general architecture of the spermatozoon of Acanthodasys species agrees with the general sperm model described for the family Thaumastodermatidae, and perfectly matches that observed in Acanthodasys aculeatus and Diplodasys ankeli, so confirming the great uniformity of the sperm ultrastructure morphology within the subfamily Diplodasyinae. Spermatogenesis includes the early and simultaneous development of acrosome and flagellum, the elongation of the nucleus and the fusion of mitochondria into a single giant mitochondrion, which later sinks into the nucleus, and the final appearance of a perinuclear helix and a peraxonemal striated cylinder. The process takes place through the same main steps already observed in all Macrodasyida species studied so far. The reproductive system consists of paired testes arrangement laterally to the intestine, a single ovary located dorsally to the posterior part of the intestine, a caudal organ located ventrolaterally to the intestine end, and, a dorsal frontal organ. The latter is in contact with mature oocyte posteriorly and with a compact cellular band connecting to the ‘rosette’ organ anteriorly. The general layout of the reproductive apparatus of A. australis perfectly matches that of the members of Diplodasyinae.

Cryopreservation effects on canine sperm morphometric variables and ultrastructure: Comparison between vitrification and conventional freezing

Semen cryopreservation is an increasingly demanded technique in canids, particularly in order to preserve and spread high genetic value material. Sperm vitrification may represent an interesting alternative to costly and time consuming conventional freezing. The objective of this study was to evaluate the effect of sperm vitrification on sperm morphometry and ultrastructure compared to conventional freezing. Pools of nine beagle dogs were both frozen and vitrified. Computerized morphological parameters (length, wide, area and perimeter) and sperm ultrastructure, using scanning and transmission microscopy, were analysed in both fresh and in thawed/warmed samples. There were no differences (p > 0.05) between post-thaw and fresh morphometric variables of the sperm heads. However, cluster analysis revealed that sperm-heads turned out to be smaller after thawing (p < 0.05) in two of the four subpopulations. Vitrification-warming process led to an overall increase in sperm-head size. Furthermore, the sperm head size increased after warming in two subpopulations (p < 0.05). In conclusion, the variations in the sperm head area depended on the cryopreservation procedure (conventional freezing or vitrification). Conventional freezing tended to decrease the head dimensions, at least in some subpopulations, and vitrification led to an overall increase in the sperm head size. Decondensation of chromatin and plasma membrane blebbing in the head region was observed by transmission electron microscopy in several vitrified sperm, which might explain the increase of head dimensions detected by CASA-Morph system.

Biallelic variants in MAATS1 encoding CFAP91, a calmodulin-associated and spoke-associated complex protein, cause severe astheno-teratozoospermia and male infertility

BackgroundMultiple morphological abnormalities of the flagella (MMAF) consistently lead to male infertility due to a reduced or absent sperm motility defined as asthenozoospermia. Despite numerous genes recently described to be...

Sperm ultrastructure of three species of ladybirds (Coleoptera, Coccinellidae)

The sperm of three coccinellid species belonging to the subfamilies Chilocorinae, Coccinellinae and Epilachninae were studied under light and transmission electron microscopy. The basic sperm structure of these ladybirds is common to that of the other previously studied species, especially the acrosome in front of the basal body and not the nucleus, with this latter running parallel with the flagellar components. In the Chilocorinae Platynaspis luteorubra (Platynaspidini) the sperm are of the type 1, as in Scymnini and Coccinellini, since they exhibited a cylindrical basal body with 9 + 0 and then 9 + 2, microtubules continuing further in an initial flagellar portion with the only axoneme devoid of accessory structures. The sperm exhibits a thin nucleus and mitochondrial derivatives. Such uniformity of sperm ultrastructure could be indicative of the occurrence of a close relationship between Platynaspidini and Scymnini as also proposed in the previous studies. Conversely, they differ markedly from the sperm of type 3 observed in the Chilocorini Exochomus quadripustulatus. In the Coccinellini Propylea quatuordecimpunctata the sperm are also of the type 1, but they can be easily differentiated from those of the other Coccinellinae studied so far, because of their very short acrosome without the posterior extension, the relatively thicker mitochondrial derivatives and the cylindrical nucleus. Epilachna clandestina sperm resemble those of E. quadripustulatus but differ from them, because they exhibit an elliptical nucleus which is anteriorly very thin, and the asymmetrical mitochondrial derivatives in the anterior extremity, with the greater one starting at the same level of the basal body, rather than at the nucleus level, as it occurs in E. quadripustulatus. Because of the differences observed in the sperm ultrastructure we propose a new sperm type (Type 4) for E. clandestina. This study on ladybird spermatozoon ultrastructure clearly indicates that the current classifications of coccinellids do not reflect the natural history of this well-known insect family.

Aluminum, at an environmental concentration, associated with acidic pH and high water temperature, causes impairment of sperm quality in the freshwater teleost Astyanax altiparanae (Teleostei: Characidae)

Given the toxicity of metals, including aluminum (Al), and the effects of water temperature on ectotherms, we investigated the individual or association effect of these variables (Al + acidic pH + temperature changes) on sperm quality of Astyanax altiparanae. Mature males were divided into nine experimental groups based on the combination of each of three water temperatures (20, 25, and 30 °C) with neutral and acidic pH values (7.0 and 5.5, respectively) with or without 0.5 mg L−1 Al. The fish were subjected to subacute, semi-static exposure and at 24 and 96 h were evaluated for seminal parameters: (1) pH; (2) osmolality; (3) sperm concentration; (4) sperm morphology; (5) sperm kinetics; and (6) sperm ultrastructure. At 30 °C, Al caused a reduction in osmolality (24 and 96 h) and sperm concentration (24 h). When analysing sperm kinetics (30 s post-activation), Al caused a reduction in total motility at all temperatures (24 h), and when this exposure time was longer (96 h), both acidic pH and Al addition to the water caused sperm motility reduction. By analysing curvilinear velocity (VCL) 30 s after sperm activation (24 and 96 h), the acidic pH caused a reduction in sperm movement at 20 and 30 °C, but at 25 °C Al triggered this reduction. Finally, Al in the water caused ultrastructural changes in the sperm head, midpiece, and flagella regardless of water temperature. Also, it was found that the combination of Al at 30 °C caused a reduction in sperm head area while at 20 °C, Al triggered a reduction in the midpiece area. Therefore, acidity influenced some A. altiparanae sperm parameters but Al in the water accentuated these effects on seminal quality, especially seminal osmolality and sperm concentration, kinetics, and ultrastructure. This toxicity was also influenced by changes in water temperature.