Morphology of the male reproductive system and spermatozoa in Centris Fabricius, 1804 (Hymenoptera: Apidae, Centridini)

The genera Centris and Epicharis constitute the Centridini and are widely distributed in the Neotropical region. Centris is also found in the southern portion of the Neartic region, although both genera are more abundant in the humid tropical regions. To describe the structure of the male reproductive system and spermatozoa, light and transmission electron microscopy were used. The male reproductive system of Centris sp. is formed by a pair of testes, a pair of deferent ducts, a pair of seminal vesicles, a pair of accessory glands and an ejaculatory duct connected to the external genitalia, the aedeagus. In this species, testes and the pre-vesicular deferens ducts as well as the seminal vesicles are encapsulated in a single conjunctive capsule, the scrotal membrane. Each testis consists of four testicular follicles, made up of cysts with up to 64 germinative cells. Histologically, the seminal vesicles are formed by a simple cylindrical epithelium, basal membrane and muscular tunic. The spermatozoa of Centris analis, C. fuscata, C. tarsata and Centris sp. are morphologically similar. They have two easily distinguishable regions: the head and flagellum. The head is formed by the two-layer acrosome, the linear nucleus and the flagellum, the centriole adjunct, the axoneme of pattern 9+9+2 microtubules, two asymmetric mitochondrial derivatives and two accessory bodies. These Centris species share various morphological characteristics of the male reproductive system and spermatozoa with the other bees previously described, indicating that several characteristics are synapomorphic for the family Apidae. Studies on the morphology of the male reproductive system and spermatozoa in Hymenoptera have demonstrated the diversity of the information provided by these reproductive structures, which can be used in taxonomy studies and the phylogeny of this important group of insects.     

Structural and ultrastructural characterization of male reproductive tracts and spermatozoa in fig wasps of the genus Pegoscapus (Hymenoptera, Chalcidoidea)

The three Pegoscapus species present the same internal reproductive tract features comprising testes with a single testicular tubule, seminal vesicles, vasa deferentia, accessory glands and an ejaculatory duct. The seminal vesicle shows two morphologically distinct portions although they do not resemble the separate chambers found in other Chalcidoidea. The anterior portion of the seminal vesicle shows a prominent epithelium and stores the mature spermatozoa, while the posterior region is formed by a thicker muscular sheath that participates on ejaculation. The sexual maturation in Pegoscapus is achieved at emergence, when the testicular degeneration occurs. The spermatozoa of Pegoscapus reveal a basic structure similar to that of other Chalcidoidea. In Pegoscapus sp1. and Pegoscapus sp2. they present the same features, whereas Pegoscapus tonduzi comprises some different characteristics. It measures approximately 160 μm in Pegoscapus sp1. and Pegoscapus sp2., while in P. tonduzi the spermatozoa measure about 360 μm. The extracellular sheath thickness is another difference among the species. While Pegoscapus sp1. and Pegoscapus sp2. show a thick extracellular sheath, in P. tonduzi this sheath is very thin resulting in a large space intervening between the extracellular sheath and the nucleus. Despite these differences, the three species analyzed share some characteristics that allow the establishment of an identity to the spermatozoon of the genus Pegoscapus: the seminal vesicle not divided in chambers; the absence of acrosomal structures in the spermatozoa; the length of the extracellular sheath; the central microtubules being the firsts to terminate in the sequence of microtubular cutoff at the final axonemal portion.     

Structure and ultrastructure of the spermatozoa of Trypoxylon (Trypargilum) albitarse Fabricius 1804 (Hymenoptera: Apoidea: Crabronidae)

The ultrastructure of the spermatozoa of Trypoxylon (Trypargilum) albitarse is described here for the first time within this genus. Testes and seminal vesicles were dissected and processed for transmission electron microscopy. In the testicular follicles, the spermatids are arranged in a maximum number of 32 for each cyst. The spermatozoa are slender and they measure approximately 150 μm in length. The head is about 17 μm long and is formed by the acrosome and the nucleus. The flagellum consists in an axoneme, two mitochondrial derivatives, two accessory bodies and, at the nucleus–flagellum transition, a symmetric centriolar adjunct. The axoneme presents the typical 9 + 9 + 2 microtubule pattern. In the terminal region, the central microtubules and nine doublets finish first, followed by the accessory microtubules. Both mitochondrial derivatives begin together and are inserted in the base of the centriolar adjunct. Along the middle region, the larger derivative has almost twice the area of the smaller one and includes a discrete paracrystalline region. At the tip, the smaller derivative ends before the larger one and both before the axoneme. The characters derived from the ultrastructure of the spermatozoa of T. albitarse show synapomorphies shared with the Apoidea and present characters that are probably apomorphic for the Crabroninae subfamily.     

Morphology of the male reproductive system and spermiogenesis in Hypanthidium foveolatum (Alfken, 1930) (Hymenoptera: Apidae: Megachilinae)

The morphological aspects of male reproductive tract, spermiogenesis and spermatozoa are typical for each species and reflect its evolution, establishing a unique source of characters, which has been used to help solve phylogenetic problems. In Hypanthidium foveolatum the reproductive tract is composed of the testes comprising 28 testicular tubules, deferent ducts, seminal vesicles, accessory glands and an ejaculatory duct. The differentiation of spermatids occurs within cysts of up to 128 germ line cells each one. During the early spermatid phase, the nucleus resembles that of somatic cells. There follows a gradual chromatin condensation with an increase in nuclear electron density. In the spermatozoon, the nucleus contains heterogeneous chromatin with a loose appearance. The acrosome, shaped with the active participation of the Golgi complex, shows an electron-dense perforatorium involved by four electron-lucent acrosomal vesicle projections. The sperm tail presents an axoneme with a 9 + 9 + 2 microtubule pattern and two mitochondrial derivatives, which appear with different sizes. A dense crystalloid is formed initially in the mitochondrial matrix of the large derivative. The mitochondrial derivatives’ differentiation occurs concomitantly with an axoneme outgrowth. The centriolar adjunct is observed near the axoneme, anterior to the smaller mithocondrial derivative and exhibits an approximately triangular shape in cross-sections. Microtubules were observed around the head region and flagellar components during spermiogenesis.     

Ultrastructure of the seminal vesicle and sperm storage in Panorpidae (Insecta: Mecoptera)

The histology and ultrastructure of the seminal vesicle, male accessory gland and the epididymis in Panorpa and Sinopanorpa were observed using light microscopy and scanning and transmission electron microscopy. The seminal vesicle consists of a mono-layered elongated columnar epithelium, which contains abundant electron-dense granules and secretory vesicles, and a small central lumen. In the apical region of the epithelium of the seminal vesicle, the intense secretory activity seems to be effected by means of merocrine mechanisms. The epithelium of the accessory gland is rich in rough endoplasmic reticulum and Golgi complex, and secretes seminal fluid into the lumen via both apocrine and merocrine mechanisms. The seminal vesicle is similar to the accessory gland in the epithelium structure and their secretory activity, mainly serving a secretory function rather than storing sperms. Instead, the sperms are stored in the epididymis, whose epithelium secretes nutrients into the large lumen by merocrine mechanisms. The secretory activity and function of the seminal vesicle are briefly discussed.     

Structure and ultrastructure of spermatozoa of Chrysomya megacephala (Diptera: Calliphoridae)

The spermatozoa of Chrysomya megacephala are similar to those described for other Brachycera. In this species, the spermatozoa are long and thin, measuring about 590 μm in length, of which the head region measures approximately 60 μm. The head includes a monolayered acrosome with electron-lucid material, and the shape of the nucleus, in cross-sections, varies from circular to oval with completely condensed chromatin. The centriole was observed in the zone of flagellar implantation, below the “peg” region. In the region of overlap, the followings structures are observed: nucleus, centriolar adjunct, mitochondrial derivatives and axoneme. The two mitochondrial derivatives are of different lengths but similar diameter. The axoneme is of a conventional insectan type with a 9 + 9 + 2 microtubular arrangement, with accessory tubules flanked by the electron-dense intertubular material. The male internal reproductive tract consists of testis, vas deferens, seminal vesicle, accessory glands and ejaculatory duct.     

High-pressure freezing and freeze-substitution fixation reveal the ultrastructure of immature and mature spermatozoa of the plant-parasitic nematode Trichodorus similis (Nematoda; Triplonchida; Trichodoridae)

The spermatozoa from testis and spermatheca of the plant-parasitic nematode Trichodorus similis Seinhorst, 1963 (Nematoda; Triplonchida; Trichodoridae) were studied with transmission electron microscopy (TEM), being the first study on spermatogenesis of a representative of the order Triplonchida and important to unravel nematode sperm evolution. Comprehensive results could only be obtained using high-pressure freezing (HPF) and freeze-substitution instead of chemical fixation, demonstrating the importance of cryo-fixation for nematode ultrastructural research. The spermatozoa from the testis (immature spermatozoa) are unpolarized cells covered by numerous filopodia. They contain a centrally-located nucleus without a nuclear envelope, surrounded by mitochondria. Specific fibrous bodies (FB) as long parallel bundles of filaments occupy the peripheral cytoplasm. No structures resembling membranous organelles (MO), as found in the sperm of many other nematodes, were observed in immature spermatozoa of T. similis. The spermatozoa from the uterus (mature or activated spermatozoa) are bipolar cells with an anterior pseudopod and posterior main cell body (MCB), which include a nucleus, mitochondria and MO appearing as large vesicles with finger-like invaginations of the outer cell membrane, or as large vesicles connected to the inner cell membrane. The peripheral MO open to the exterior via pores. In the mature sperm, neither FBs nor filopodia were observed. An important feature of T. similis spermatozoa is the late formation of MO; they first appear in mature spermatozoa. This pattern of MO formation is known for several other orders of the nematode class Enoplea: Enoplida, Mermithida, Dioctophymatida, Trichinellida but has never been observed in the class Chromadorea.     

Actin localisation and the effect of cytochalasin D on the osmotic tolerance of cauda epididymidal kangaroo spermatozoa

This study examined the hypothesis that filamentous actin associated with the complex cytoskeleton of the kangaroo sperm head and tail may be contributing to lack of plasma membrane plasticity and a consequent loss of membrane integrity during cryopreservation. In the first study, the distribution of G and F actin within Eastern Grey Kangaroo (EGK, Macropus giganteus) cauda epididymidal spermatozoa was successfully detected using DNAse-FITC and a monoclonal F-actin antibody (ab205, Abcam), respectively. G-actin staining was most intense in the acrosome but was also observed with less intensity over the nucleus and mid-piece. F-actin was located in the sperm nucleus but was not discernable in the acrosome or sperm tail. To investigate whether cytochalasin D (a known F-actin depolymerising agent) was capable of improving the osmotic tolerance of EGK cauda epididymal spermatozoa, sperm were incubated in hypo-osmotic media (61 and 104 mOsm) containing a range of cytochalasin D concentrations (0-200 microM). Cytochalasin D had no beneficial effect on plasma membrane integrity of sperm incubated in hypo-osmotic media. However, when EGK cauda epididymidal sperm were incubated in isosmotic media, there was a progressive loss of sperm motility with increasing cytochalasin D concentration. The results of this study indicated that the F-actin distribution in cauda epididymidal spermatozoa of the EGK was surprisingly different from that of the Tammar Wallaby (M. eugenii) and that cytochalasin-D does not appear to improve the tolerance of EGK cauda epididymidal sperm to osmotically induced injury.     

The effects of different cryoprotectants and the temperature of addition on the survival of red deer epididymal spermatozoa

With the aim of finding an ideal cryoprotectant in a suitable concentration for red deer epididymal spermatozoa conservation, we evaluated the effects of four most commonly used cryoprotectants (CPAs), Glycerol (G), Ethylene glycol (EG), Propylene glycol (PG), and Dimethyl sulfoxide (DMSO), on the sperm survival. Besides, the effects of two temperatures of CPA addition--22 degrees C (ambient temperature) and 5 degrees C--on sperm quality were also tested. For each temperature tested, sperm samples were evaluated after 0, 15, 30 and 60 min of spermatozoa exposition to CPAs. Thus, sperm quality was in vitro judged by microscopic assessments of individual sperm motility (SMI), and of plasma membrane (Viability) and acrosome (NAR) integrities. Overall, DMSO showed the highest toxicity for red deer epididymal spermatozoa, and glycerol the lowest. Thus, at 60 min of incubation SMI results showed that the toxicity to red deer epididymal spermatozoa of the four CPAs are in the following sequence: G approximately = EG approximately = PG < DMSO ('less than' symbol means P < 0.05, and approximate symbol means P = 0.08). Furthermore, our results also showed a differential response of acrosome membrane to temperature of CPAs addition. Regardless of the CPA used, statistically significant variations (P < 0.05) were found between the two temperatures of addition of CPAs for acrosome integrity, the best being 22 degrees C (NAR = 83.8% vs. 69.8%). These data indicate that sperm quality of red deer epididymal spermatozoa, in addition to be affected by the cryoprotectant, can also be influenced by the temperature at which CPAs are added prior to freezing.     

Head dimensions of cryopreserved red deer spermatozoa are affected by thawing procedure

The objective of this study was to evaluate the effects of the thawing procedure on red deer spermatozoa distribution in morphologically distinct subpopulations after freezing and thawing. For this purpose, epididymal spermatozoa were thawed using two different thawing protocols (I = 37 degree celsius for 20 s vs. II = 70 degree celsius for 5 s). The spermatozoa, from 10 Iberian deer stags, were diluted at room temperature in a Triladyl-20 percent egg yolk medium and frozen in nitrogen vapor. Standard sperm freezability was judged by microscopic assessments of sperm motility. The thawing procedure had an effect on sperm motility percentage (P = 0.05), with the best overall recovery rates found with the use of protocol I (76.8 + or - 1.8 vs. 70.6 + or - 1.8). Moreover, the morphometric dimensions for a minimum of 200 sperm heads were analyzed from each sample by means of the Sperm-Class Analysez (SCA), and the mean measurements recorded. Deer sperm heads were significantly (P = 0.01) smaller when spermatozoa were thawed using protocol II than when using procedure I (area = 30.02 square micrometers vs. 30.32 square micrometers; width = 4.47 micrometers vs. 4.51 micrometers; length = 8.05 micrometers vs. 8.11 micrometers), but not for all stags. All sperm head measurements were placed in a statistical database and a multivariate cluster analysis performed. Mean measurements for all parameters of the major clusters for the two different thawing procedures were compared by ANOVA. The mean values for length, width, area, perimeter, shape factor and width/length in the major cluster of sperm head dimensions for thawing protocol I were significantly different from those for protocol II (P = 0.001). In addition, differences were found within all stags for whole morphometric parameters (P = 0.001), with the smallest overall sperm head dimensions found with the use of protocol II. Additionally, the rapid thawing protocol produced a dramatic loss of heterogeneity. Finally, our results showed that the greater the loss of heterogeneity, the greater the degree of sperm cryoinjury.     

Usefulness of MR-seminography using Gd-DTPA in intermediate magnetic field MRI equipment

T(2)-weighted fast advanced spin-echo imaging with gadolinium (hereinafter referred to as "MR-seminography") of the genital tract was evaluated for its usefulness in non-invasive investigation into the morphology of these organs. Twenty healthy male volunteers who are confirmed as free of genital tract obstruction were entered into the study. The genital tract was imaged using the 0.5 T MRI system by fast advanced spin echo (FASE) method. Based on quantitative evaluation, visibility evaluation, and the visualization of the genital tract in each subject, the contrast-enhanced images were assessed in comparison with those at pre-injection of the contrast agent by four specialists. Also examined were the morphology of the seminal vesicle according to age group using "Ishigami-Mori's classification," and the age angle of the main ducts. MR-seminography produced high contrast images and revealed a significantly decreased signal in the pelvic organs under investigation. In the analysis of individual subjects, the area from the proximal portion of the vas deferens to the ampulla and the seminal vesicles was on the whole well visualized except for the distal portion of the vas deferens and the ejaculatory duct. With regard to seminal vesicle morphology, type II was predominant. The age angle showed a trend toward widening with age. MR-seminography is non-invasive, useful, and therefore a highly recommended procedure for observation of the morphology in the area from the proximal portion of the vas deferens to the seminal vesicle.     

Acrosome reaction in Octopus tankahkeei induced by calcium ionophore A23187 and a possible role of the acrosomal screw

The octopod sperm is unique especially in two aspects: the screw-shaped acrosome and its inner layered substructure (striation). The present study aims to investigate morphological changes of Octopus tankahkeei spermatozoa during the acrosome reaction (AR) and to pursue functions of the internal substructures revealed by inducing AR with the calcium ionophore A23187. Gradual changes of the spermatozoa were traced using fluorescence and electron microscopy. The AR process included the bulging, vesiculation, and dehiscence of the plasma membrane around the acrosome and the nucleus, as well as the vesiculation of the mitochondrial sheath. Membrane vesiculation outside the nucleus has never been reported in the order Octopoda. The rigid screw and the inner striation of the acrosome remained intact surmounting the nucleus, suggesting that these two structures have potential functions during fertilization. In addition, the detachment of the sperm head and the tail was commonly observed in this study, both in intact and acrosome-reacted sperm. Fluorescence microscopy revealed that the detached mitochondrial sheath usually gave weaker and more dispersive signals than the joint ones. This phenomenon implied that the intense energy release might promote the detachment of the mitochondrial sheath.     

Fine structure of the spermatozoon of Perinereis macropus (Claparède, 1870) (polychaeta,Nereidae)

The mature spermatozoa of Perinereis macropus were investigated by transmission electron microscopy. The spermatozoon is composed with a large anterior part (head), a short middle piece and a long flagellum. The head contains a large acrosomal complex with a convex acrosomal vesicle, a subacrosomal space, a fibrillar crown and an acrosomal rod which penetrates into the nucleus invagination. The later is U shaped (in longitudinal section). The short middle piece contains about nine to eleven mitochondria and a centriole associated with the flagellum. This centriole, slightly eccentric to the sperm axis, anchors to the plasma membrane by nine satellite rays of the pericentriolar complex. The axoneme has a “9 + 2″ arrangement of microtubules. In cross section, the flagellar membrane extends in two lateral protuberances, aligned with the axis formed by the two central microtubules of the axoneme. The spermatozoon of P. macropus conforms to the primitive type with an acrosomal extension. Nevertheless, the acrosome complex ultrastructure shows noticeable modifications from the basic form. This finding agrees with the previously observed reproductive pattern (broadcast spawning – free-swimming larvae), and may be helpful to classify the sperm type of P. macropus.     

Comparative ultrastructural study of spermatozoa in some oyster species from the Asian-Pacific Coast

Sperm organization in the oysters Crassostrea gigas, Crassostrea nippona, Crassostrea cf. rivularis and Saccostrea cf. mordax inhabiting Asian Pacific coast was studied. The spermatozoa of all studied species had a number of common morphological characters such as a cup-like acrosome with heterogeneous matrix on its top, an axial rod in the subacrosomal space, a barrel-shaped nucleus, four mitochondria in the midpiece, pericentriolar complexes, and a 9+2-organized flagellum. The spermatozoa of C. cf. rivularis differed from the other species by having cytoplasm processes in the midpiece region. Such structures have never been described in the Ostreidae. Additionally, each species could be identified by the shape and size of sperm compartments (acrosome, nucleus, anterior nuclear fossa). The most significant interspecific difference was found in the size of an anterior nuclear fossa. The smallest anterior nuclear fossa was found in C. cf. rivularis (about 0.24 μm in length reaching about 22% of the nuclear length) while the biggest in C. gigas from the Sea of Japan (about 0.53 μm in length reaching about 46% of the nuclear length). The spermatozoa of C. gigas collected from the Sea of Japan and Taiwan Strait differed significantly in almost all the studied parameters. Since sperm morphology has been successfully used for species differentiation, this suggests the existence of two species rather than two populations. The data obtained indicate the species-specific difference in the sperm ultrastructure within the Ostreidae, which may be identified both ultrastructurally and morphometrically.     

Comparative sperm ultrastructure of three species in Siniperca (Teleostei: Perciformes: Sinipercidae)

Siniperca chuatsi, Siniperca kneri, and Siniperca scherzeri are three of the most economically important sinipercid species. The ultrastructure and morphology of the mature spermatozoa of them are examined using transmission electron microscopy (TEM) and scanning electron microscopy (SEM). The sperm consists of an acrosome-less head, a short midpiece and a long flagellum. Ultrastructurally, it has a homogeneously electron-dense nucleus in a granular pattern with nuclear lucent and a nuclear fossa excluding the centriolar complex. One to four mitochondria with lamellar cristae encircle the basal body of the flagellum in the midpiece. The cytoplasm surrounding the centrioles and the cylindric cytoplasmic channel contains glycogen granules and vesicles. Comprising the conventional 9 + 2 axoneme, vesicles and lateral fins, the sperm flagellum is inserted laterally on the nucleus, therefore the spermatozoon is asymmetrical. All of the spermatozoa of the three species are of the primitive or ect-aquasperm form and conform to the teleostean type II spermatozoa instead of the previously supposed type I. Variations in the shape of the heads, angles between the two centrioles, location of the cytoplasmic vesicles, mitochondrial number and structural characteristics of the lateral fins are notable among the three species. S. chuatsi is a sister-group of the other two species and is the most differentiated. The present study provides fresh insights to the comparative spermatology of Siniperca fishes and will be useful to the existing knowledge of the sinipercid fishes in systematic characters, biodiversity conservation and reproduction.     

Cryopreservation of human sperm using rapid cooling rates

The effect of cryopreservation on human spermatozoa has been investigated during the past few decades. The majority of current cryopreservation protocols are carried-out using low cooling rates. However, theoretical calculations have shown that for human spermatozoa an optimal cooling rate is about 7,000 C/min. In our work we have studied the effect of cryopreservation with high cooling rates, variation of osmolarity of the cryoprotectant medium and the glycerol content. The results of experiments have demonstrated that within the range of high cooling rates, after thawing the dependency of sperm survival on the cooling rate has a maximum recovery at 2,500-3,300 C/min in moderately hyperosmolar medium, containing 4-5% glycerol. When decreasing the cooling rate down to 1,750-2,500 C/min there was a statistically significant reduction in sperm motility. Using the cooling rate of 8,000-11,000 C /min only a small percentage of spermatozoa retained their motility.     

Top-down mass spectrometry reveals new sequence variants of the major bovine seminal plasma protein PDC-109

The major protein of bovine seminal plasma, PDC-109, is a 109-residue polypeptide that exists as a polydisperse aggregate under native conditions. The oligomeric state of this aggregate varies with ionic strength and the presence of lipids. Binding of PDC-109 to choline phospholipids on the sperm plasma membrane results in an efflux of cholesterol and choline phospholipids, which is an important step in sperm capacitation. In this study, Fourier transform ion cyclotron resonance mass spectrometry was used to analyze PDC-109 purified from bovine seminal plasma. In addition to the previously known PDC-109 variants, four new sequence variants were identified by top-down mass spectrometry. For example, a protein variant containing point mutations P10L and G14R was identified along with another form having a 14-residue truncation in the N-terminal region. Two other minor variants could also be identified from the affinity-purified PDC-109. These results demonstrate that PDC-109 is naturally produced as a mixture of several protein forms, most of which have not been detected in previous studies. Native mass spectrometry revealed that PDC-109 is exclusively monomeric at low protein concentrations, suggesting that the protein oligomers are weakly bound and can easily be disrupted. Ligand binding to PDC-109 was also investigated, and it was observed that two molecules of O-phosphorylcholine bind to each PDC-109 monomer, consistent with previous reports.     

Evaluation of damage in Pacific oyster (Crassostrea gigas) spermatozoa before and after cryopreservation using comet assay

We examined the applicability of the comet assay (single cell gel electrophoresis assay) to estimate the quality of frozen-thawed Pacific oyster (Crassostrea gigas) spermatozoa. Comet assay was performed on semen before and after cryopreservation followed by fluorescent staining with propidium iodide to assess DNA integrity. After cryopreservation, the percentage of spermatozoa with damaged DNA significantly increased, while only about half of the cells displayed intact DNA, even when protected with 10 percent DMSO. All the considered parameters (head length, head area, head intensity, total length, total area, total intensity, tail length percent, tail area percent, and tail intensity percent) were higher than the oyster sperm protected with 10 percent DMSO-artificial sea water after freezing and thawing. Only tail length percent, tail area percent, and tail intensity percent were increased significantly after cryopreservation. The tail length percent was found to be the most sensitive indicator of the cryopreservation-induced DNA damage. Our freeze-thawing procedure significantly affected oyster sperm DNA, as indicated by the reduced fertilization rate when frozen-thawed oyster sperm are used. Irreversible alteration of the genome may prevent fertilization or alter normal embryonic development. This study is the first to demonstrate that the comet assay is an inexpensive, rapid and sensitive method for determining DNA damage in Pacific oyster sperm quality assessments.     

Size determination of Acipenser ruthenus spermatozoa in different types of electron microscopy

In this study three types of scanning electron microscopes were used for the size determination of spermatozoa of sterlet Acipenser ruthenus – high vacuum scanning electron microscope (SEM, JEOL 6300), environmental scanning electron microscope (ESEM, Quanta 200 FEG), field emission scanning electron microscope (FESEM, JEOL 7401F) with cryoattachment Alto 2500 (Gatan) and transmission electron microscope (TEM, JEOL 1010). The use of particular microscopes was tied with different specimen preparation techniques. The aim of this study was to evaluate to what degree the type of used electron microscope can influence the size of different parts of spermatozoa. For high vacuum SEM the specimen was prepared using two slightly different procedures. After chemical fixation with 2.5% glutaraldehyde in 0.1 M phosphate buffer and post-fixation by 1% osmium tetroxide, the specimen was dehydrated by acetone series and dried either by critical point method or by means of t-butylalcohol. For ESEM fresh, unfixed material was used, which was dropped on microscopic copper grids. In FESEM working in cryo-mode the specimen was observed in a frozen state. Ultrathin sections from chemically fixed and Epon embedded specimens were prepared for TEM observation. Distinct parts of sterlet spermatozoa were measured in each microscope and the data obtained was statistically processed. Results confirmed that the classical chemical procedure of specimen preparation for SEM including critical point drying method led to a significant contraction of all measured values, which could deviate up to 30% in comparison with values measured on the fresh chemically untreated specimen in ESEM. Surprisingly sperm dimensions determinated on ultrathin sections by TEM are comparable with values obtained in ESEM or FESEM.