As negative control, one high volume culture was set up with a me

As negative control, one high volume culture was set up with a medium without being supplemented with any substrate. Cultures were incubated at 28 °C under shaking by using baffled Erlenmeyer flasks until mid-exponential phase (OD 0.6–0.9) was reached (incubator: INE 800, Memmert, Schwabach, Germany; shaker:

KS501, IKA Labortechnik, Staufen, Germany). Starting from two pre-cultures (50 mL) which had been transferred twice after having been grown to mid exponential phase on glucose, three cultures (50 mL) per substrate of interest (chondroitin sulfate, λ-carrageenan, fucoidan or glucose as reference, 1.8 g/L) were prepared with a 10% (v/v) inoculum (5 mL). The initial OD600 nm was determined and monitored over one week. As negative control, three cultures had no substrate. As positive control, three cultures were grown on medium M13a supplemented with casamino acids (Schlesner, 1994). Growth curves PD0332991 purchase allowed the calculation of growth rates and doubling times. Cell material for downstream processing was harvested by centrifugation and was kept at − 20 °C (− 80 °C for long term storage) until it was processed. Stored cell pellets were thoroughly resuspended in 1–3 mL of TRI reagent (Applied Biosystems, Darmstadt, Germany). The suspension was incubated for 5 min at room temperature. Cells were lysed by beadbeating (lysing matrix B, material: 0.1 mm silica spheres;

MPBiomedicals, Berlin, Germany) applying a FastPrep 24 automated homogenizer (MPBiomedicals). Three steps of 30 s (speed:

6 m/s) were performed, while cooling PR 171 the tubes on ice between beadbeating steps. After the third step, the beadbeater tubes were incubated on ice for additional 10 min. Next, beadbeater tubes were centrifugated at 4 °C for 10 min (5415 C, Eppendorf, Hamburg, Germany; 16,000 × g). Supernatants were transferred into RNase-free, sterile 1.5 mL Eppendorf cups. 200 μL of ice-cold chloroform was added per sample. Suspensions were thoroughly mixed by vortexing for 20 s, followed by a 10 min incubation step at RT. A further centrifugation step was carried out (4 °C, 15 min, 16,000 × g). The aqueous, upper phase was transferred into new, RNase-free and sterile Eppendorf cups. 1 mL of 100% isopropanol was added, followed Vasopressin Receptor by incubation at − 20 °C for 1 h. After the incubation, a 30 min centrifugation step was performed (4 °C, 16,000 × g). The supernatants were discarded and pellets were washed twice in 75% ethanol. Dried pellets were dissolved in 50–100 μL RNase-free water. Extracted RNA was cleaned by using the RNeasy MinElute clean-up kit (Qiagen, Hilden, Germany) following the manufacturer’s instructions. The concentration and quality of eluted RNA were determined by using a NanoDrop® spectrophotometer (Thermo Scientific, Wilmington, USA). The amount and quality of extracted and cleaned up RNA were also documented by RNA agarose gelelectrophoresis.

On the other hand, the development of mouse embryo banks in which

On the other hand, the development of mouse embryo banks in which the strains are cryopreserved at the embryo level have shown great promise. These embryo banks have prevented the discontinuation of strains due to genetic mutation or natural BMS-907351 disasters

and provide a significant cost-savings, including avoiding the need for breeding space [11]. As the cost to maintain rat strains are even higher than that for mice, it is important to preserve rat strains by cryopreserving early-stage embryos. We planned to build a rat embryo bank by cryopreserving early rat embryos. Whittingham [24] modified the slow freezing method used for mouse early-stage embryos and cryopreserved two-, four-, and eight-cell stage rat embryos. In addition, Kono [12], Isachenko [6], Tada [19], Jiang [8], Anzai [2], and Seita [17] cryopreserved rat embryos using the vitrification method. Thus, methods used successfully for other animal species have been applied to rats, usually with some modification. In the present study, we determined the optimal pre-treatment for vitrification and the components of the vitrification solution using rat two-cell embryos. To facilitate manipulation of the collection and embryo transfer, two-cell stage embryos are used for cryopreservation in many mouse embryo banks, and we therefore

examined the cryopreservation of rat embryos using the two-cell stage embryos. Han et al. Han et al. [5] reported that embryo survival and in vivo Alectinib solubility dmso development are improved when two-cell stage rat embryos are exposed to a pretreatment solution containing a low concentration of cell-permeable cryoprotectant, and vitrification of these embryos is then conducted. Based on these findings, we investigated the vitrification method after pretreatment of two-cell stage rat embryos. For pretreatment, as it is necessary to select a cryoprotectant with low cytotoxicity and with a low risk of damaging the embryos due to osmotic expansion, we investigated the permeation rate of cell-permeable

cryoprotectants and fetal development. To prevent damage to the embryos by osmotic expansion after warming without the occurrence of freeze fractures when the vitrification solution vitrifies after cooling, we investigated different types and concentrations of cell-permeable cryoprotectants, sugars, and high molecular weight Selleck Docetaxel molecules added to the vitrification solution. Using the pretreatment and vitrification solutions developed in this study, vitrification of rat two-cell stage embryos was conducted and the survival and in vivo development after warming were investigated. Rats of the BrlHan:[email protected](GALAS) strain (CLEA Japan, Inc., Tokyo Japan) were used for the experiments. The breeding conditions were as follows: room temperature, 22 ± 0.5 °C; humidity, 55 ± 5%; and lighting from 08:00 to 20:00. Rat chow (CA-1; CLEA Japan Inc.) and tap water were available ad libitum.

Assuming a two state model, the observed mean-residue ellipticity

Assuming a two state model, the observed mean-residue ellipticity at 222 nm ([Θ]obs222) was converted into α-helix fraction (fH) using the method proposed

by Rohl and Baldwin (1998) and previously described ( Konno et al., 2001). The lipid bilayers were obtained from giant unilamellar vesicles (GUVs), which were positioned onto the chip check details aperture by application of negative pressure. The GUVs burst as soon as they touch the glass surface of the chip and form a bilayer that spans the aperture (Sondermann et al., 2006). Asolectin (Sigma), a negatively charged mixture of lipids, was used to form artificial membranes. GUVs were formed by electroswelling, using the Nanion Technologies (Munich, Germany) device Vesicle Prep Pro©. 20 μL of 10 mg/mL lipid solution (in chloroform) were deposited onto an indium tin oxide (ITO) coated glass plate and evaporated for 45–60 min. A nitrile ring was placed around the dried lipid film and filled with 350 μL of 250 mM D-Sorbitol dissolved in Milli-Q water. A second ITO coated glass plate was placed on top of the ring. An AC voltage of 3 V peak-to-peak Romidepsin chemical structure amplitude at 5 Hz frequency was supplied to the ITO slides over a

period of 2 h at 36 °C (modified from Sondermann et al., 2006). The formed vesicles were kept in plastic vials under refrigeration (4 °C) until use or used immediately. GUVs suspensions were always observed under light microscope prior to use. The experiments were performed with the automated Patch-Clamp device Port-a-Patch (Nanion Technologies – Munich, Germany), using borosilicate glass chips NPC-1 with aperture

diameter of approximately 1 μm. The resistance of the apertures was approximately 1–3 MΩ in 150 mM HCl solution. Current signals were amplified and recorded by an amplifier EPC-10 (Heka Elektronik, Lambrecht, GPX6 Germany) and an analogical/digital interface ITC-1600. The system was computer controlled by the PatchControl™ software (Nanion) (Fertig et al., 2002 and Sondermann et al., 2006). During the experiments symmetrical solution of 150 mM HCl with 5 mM Tris was used. After a seal was formed (Rm > 500 mΩ), the peptides diluted with Milli-Q water at a 5 μM concentration were added to the cis side of the chip (top) to observe the single channel activity. The volume of peptide solution was never superior to 10% of the solution at the cis side. Voltage pulses were applied at the trans side of the chip (bottom). Usually, single channel activity started approximately 10 min after adding the peptides, as monitored by a constant Vhold of −100 mV. Single channel conductance of incorporated channels was determined under positive and negative voltage pulses. The experiments were performed at room temperature (∼22 °C). The data was analyzed by PatchMaster and Matlab softwares.

When solely cognitive and behavioural responses are encouraged, w

When solely cognitive and behavioural responses are encouraged, without reconceptualising pain, these responses may be counterintuitive for chronic pain Omipalisib molecular weight patients, because pain is still a sign of harm to them (Moseley, 2003b). Therefore education of the central sensitization model relies on deep learning, aimed at reconceptualising pain, based on the assumption that appropriate cognitive and behavioural responses will follow when pain is appraised as less dangerous (Moseley, 2003a). For example, remember the patient with chronic whiplash convinced that the initial neck trauma caused severe cervical

damage that remains invisible to modern imaging methods. Simply providing education about the fear avoidance model to encourage a graded activity approach is unlikely to be beneficial. Detailed pain physiology education is required to reconceptualise pain, and to convince the patient that

hypersensitivity of the central nervous system rather than local tissue damage is the cause of their presenting symptoms. PD-166866 in vivo Educating patients with chronic musculoskeletal pain about central sensitization can be accomplished in one to two face-to-face educational sessions (approximately 30 min per session; depending on the change in cognitions). The aid of a booklet containing detailed written explanation and illustrations about pain physiology and central sensitization processes is recommended. The content of the education sessions can be based on the book 4��8C “Explain Pain” (Butler and Moseley, 2003), covering the physiology of the nervous system in general and of the pain system in particular. Topics that should be addressed during the education sessions include the characteristics of acute versus chronic pain, the purpose of acute pain, how acute pain originates in the nervous

system (nociceptors, ion gates, neurons, action potential, nociception, peripheral sensitization, synapses, synaptic gap, inhibitory/excitatory chemicals, spinal cord, descending/ascending pain pathways, role of the brain, pain memory and pain perception), how pain becomes chronic (plasticity of the nervous system, modulation, modification, central sensitization, the pain neuromatrix theory) and potential sustaining factors of central sensitization like emotions, stress, illness perceptions, pain cognitions and pain behaviour. Acute nociceptive mechanisms are typically explained first and are then contrasted with central sensitization processes i.e. in the case of chronic pain. Illustrations (e.g. Fig. 2 and Fig. 3), examples, and metaphors are frequently used (van Wilgen and Keizer, in press). The education is presented verbally (explanation by the therapist) and visually (summaries, pictures and diagrams on computer and paper). During the sessions patients are encouraged to ask questions and their input should be used to individualise the information.

Loops were optimized using MODLOOP ( Fiser and Sali, 2003b) based

Loops were optimized using MODLOOP ( Fiser and Sali, 2003b) based on the satisfaction of spatial restraints, without relying Epacadostat purchase on a database of known protein structures. The DOPE potential was evaluated for all models, and the model with the lowest global score was selected for explicit solvent molecular dynamics simulation using the GROMACS package (

Lindahl et al., 2001) and the GROMOS-96 (43a1) force field to check its stability and consistency. The overall and local quality of the final model was assessed by VERIFY3D ( Eisenberg et al., 1997), PROSA ( Wiederstein and Sippl, 2007) and VADAR ( Willard et al., 2003). Three-dimensional structures were analyzed and compared using the program PyMoL ( The results obtained were expressed as the mean ± standard deviation (SD) and statistically analyzed by applying a one-way ANOVA, followed by the Tukey method. Differences with p < 0.05 were considered

statistically significant. A new proteinase isolated from the venom of Bothrops Vorinostat mw atrox, which is a snake native to the state of Pará in Brazil, was obtained by two chromatographic procedures. The first step consisted of gel filtration on a Sephadex G-75 column under alkaline conditions (pH 8.0). The chromatogram shown in Fig. 1A illustrates the five major fractions obtained (Ba I to Ba V). Fraction Ba III presented hemorrhagic activity. The SDS-PAGE analysis of the fraction content under reduced conditions ( Fig. 1A insert) shows that Ba III contained two proteins, with one main band presenting a molecular mass of approximately 27 kDa and the second band presenting a molecular mass of approximately 17 kDa. Ba III was submitted to a second purification procedure using anion exchange chromatography ( Fig. 1B). Unbound material was eluted in 50 mM ambic pH 7.4, whereas the bound proteins were

eluted with a linear gradient of increasing concentrations of ambic pH 7.4, up to 500 mM. The resulting fractions (ES I and ES II) were assayed for hemorrhagic activity, Thymidine kinase and fraction ES I was able to induce dorsal skin hemorrhage in mice. SDS-PAGE ( Fig. 1B insert) shows that ES I produced a single protein band of approximately 27 kDa under reducing conditions. To confirm the purity of the fraction, ES I was submitted to reverse phase chromatography on HPLC, which revealed a single homogenous peak ( Fig. 1C). In addition, isoelectric focusing produced a single protein band with a pI of 7.5 ( Fig. 1D). The MALDI-TOF mass spectrometry analysis, based on a single charged molecule, identified a protein with a molecular mass of 22.9 kDa (data not shown). Taken together, these results confirm the isolation of Batroxase, a new protein from Bothrops atrox snake venom. Batroxase was able to induce hemorrhaging after intradermal injection in the dorsal skin of mice, with a DMH of 10 μg (Fig. 2A).

This perceptual attraction can be considered as a subjective cons

This perceptual attraction can be considered as a subjective consequence of the ‘constant conjunction’ of action and effect that underlies our experience of both agency and causation ( Hume, 1763). A convenient measure of this associative aspect of sense of agency is the “intentional binding effect”. When people make a voluntary action to cause a sensory effect a short time

later, they estimate the interval between action and effect as shorter relative to a control condition where the same interval is used ( Engbert et al., 2007; Buehner and Humphreys, 2009; also Haggard et al., 2002). While explicit judgements of agency have been extensively investigated using functional magnetic resonance imaging (MRI) (see above), the implicit sense of agency has been much less investigated. Using positron emission tomography (PET), Elsner et al. (2002) asked participants to make voluntary actions, and followed these by an auditory effect. When participants subsequently listened to mixtures of these previously-caused tones and other, neutral tones, a caudal region of the SMA was increasingly

active as the proportion of previously-caused tones grew. Re-presentation of previously-caused tones was assumed to reactivate associations between action and effect housed in the SMA. This result is consistent with a frontal contribution to sense of agency. However, no measures related to agency STI571 solubility dmso were obtained in the critical trials in their experiment. Miele et al. (2011) asked participants at the end of a video game task how much control they had

experienced during that task. They found a positive correlation between pre-SMA activation and explicit judgements of “sense of control”. However, it is unclear how such synthetic judgements relate to the underlying low-level experience of action events and consequences remain unclear. To our knowledge, the neural correlates of temporal association triclocarban between individual instrumental actions and their effects have not yet been studied using neuroimaging. One transcranial magnetic stimulation (TMS) study ( Moore et al., 2010) used independent estimates of time of action and effect to measure intentional binding, rather than the interval estimation approach used here. Moore et al., found that disrupting the pre-supplementary motor area (pre-SMA) using theta-burst TMS produced a decrease in intentional binding. This result suggests that brain areas that underlie intentional action, such as SMA and pre-SMA, are also involved in the implicit sense of agency. To summarise, previous brain imaging studies suggest that parietal regions may contribute to the explicit judgement of agency. However, existing data do not reveal whether the parietal regions, and the angular gyrus in particular, also play a role in the subjective experience of agency.

First, just

in the river, the Arun, and later, having joi

First, just

in the river, the Arun, and later, having joined the local sea angling club, occasionally out into the Channel when I could persuade my old Mum to part with the hire fee or as a freebie when boat owners were persuaded to keep us kids off the street and out fishing during summer holidays. On one of the latter trips, there was the skipper, his pimply callow mate and four of us boys all around twelve. Getting the rods organised on the way out Torin 1 supplier of harbour, we noticed that the mate was gearing up with a steel trace and huge hook. We asked what he was fishing for and, smirkingly, he said ‘congers’ that were known to live in an old sewerage pipe between our home port of Littlehampton and Worthing. On arrival, baited hooks were lowered, the mate trying to tempt a conger out of its pipe with a whole mackerel. After about an hour’s fishing, the mate’s rod suddenly bent so ferociously that I thought it was going to snap. But, he persevered and another half an hour later he had a gaffed conger at the surface, which he and the skipper now, possibly unwisely in hindsight, eventually managed to get into the boat. It was not so big as the Torquay

individual but, to my young eyes, it PD-0332991 mw was big enough. I would say well over a metre and a half in length, as thick as my waist and, most importantly, not yet dead. Suddenly, it awoke from its torpor and begin thrashing around the boat’s well, snapping at anything and everything. Us boys, me in my brother’s cut-down trousers and plimsolls, were up on the boat’s gunnel before you could say ‘knife’, hanging onto the bits of safety rigging any way we could and cautiously making our way onto the cabin’s roof where the spectacle in the well beneath us could be better enjoyed. The mate was trying to beat the conger

to death with the boat’s club but, not to be outdone, the conger bit him on the toe of his Wellington boot and refused to let go. We now had the spectacle of the wildly thrashing conger shaking the leg of the mate who, in turn, while hopping around on the other one, was still trying to club it senseless but was acutely aware of his own predicament. No more smirking Non-specific serine/threonine protein kinase either, I noted with quiet satisfaction. Eventually, the skipper grabbed hold of the club and was trying to also achieve the conger’s demise, but with a wide-eyed mate now even more frightened of the nightmarish consequences of his catch. It seems like an age later but, ultimately, by dint of discarding his boot to the conger, the beast was overcome and we boys descended to inspect a dead fish in a well of blood and gore and two badly shaken fishermen. It was thereupon decided that that was enough fun for one day’s angling and the boat turned for home. On the return trip, us boys returned to the cabin’s roof to gigglingly mull over the spectacle we had just experienced while the mate tried to clean up the blood-soaked deck.

, 1995) After incubation, cells were washed twice with FACS buff

, 1995). After incubation, cells were washed twice with FACS buffer and were either used for intracellular staining or fixed with a solution of 2% paraformaldehyde in PBS. Incubation with primary antibodies to MHC I (Salomonsen et al., 1987) and MHC II (Kaufman et al., 1990) was followed by Alexa-647 conjugated goat anti-mouse antibody (Life Technologies). Secondary antibody alone or unconjugated goat anti-mouse antibody (Life Technologies) was used as an unstained control for surface MHC staining. Intracellular staining high throughput screening was carried out as described previously (Ariaans et al., 2008). Briefly, splenocytes from challenged birds or non-infected controls were seeded in a 96-well round-bottom plates (Nunc)

at 106 cells/well in a final volume of 200 μl of culture media or culture media supplemented with the different stimuli at the concentration described in the ELISpot technique (except PMA which was used at 50 ng/ml). Cells

were cultured using the conditions described above for ELISpot assays (24 h culture). Volasertib price For intracellular staining, during the last 2 h of culture, cells were treated with Brefeldin A according to the manufacturer’s instructions (Cytofix/Cytoperm™ Plus Fixation/Permeabilization kit, BD Biosciences). To avoid non-specific binding signal, we preincubated cells with Cytofix/Cytoperm™ buffer containing 2% normal mouse serum and further staining steps involved Cytofix/Cytoperm™ washing buffer containing 1% normal mouse serum (Biosource). To confirm the specificity of the anti-IFNγ antibody EH9 we also employed a validated anti-IFNγ [mAb80 (Ariaans et al., 2008)]. Purified fractions of both antibodies were conjugated using Alexa Fluor® 647 monoclonal antibody labeling kit (Molecular Probes) according to the manufacturer’s 4��8C instructions. A mouse isotype matched control antibody IgG1 Alexa Fluor® 647 (Life Technologies) was employed at the same concentration as EH9 and Mab80. For analysis, a gate on the FSC/SSC region of lymphocytes was selected and a minimum of 10,000 events were acquired on a FACSCalibur instrument using Cell Quest software (BD Becton Dickinson). Flow cytometry

analysis indicates that non-adherent CKC were not present at significant levels (data not shown). FlowJo software (TreeStar) was used to analyze flow cytometry data. A paired or unpaired t-student test or one-way ANOVA was performed using GraphPad Prism (version 6.0 for Windows, GraphPad Software, San Diego, California, USA). Screening identified two anti-chicken IFNγ antibodies (clones EH9 and AF10) which were shown by ELISA to bind recombinant chicken IFNγ and to work effectively as an antibody pair in capture ELISA (Supplementary Fig. 2A–C). We subsequently compared this antibody pair with commercially available antibodies [from Life Technologies (Ariaans et al., 2008 and Reemers et al., 2012)] in ELISpot assays.

The MI value of AWW showed a decline from 44 7 to 37 8 to the ext

The MI value of AWW showed a decline from 44.7 to 37.8 to the extent of 15.4% whereas the presence of mannitol in the treatment tubes brought it upto 41.2 exhibiting a recovery of 8.25%. Chromosomal aberration based on its index showed its value for AWW treated A.cepa to be 11.2% compared to 2.6%

for the aquaguard water. The aberration index showed 52% reduction in the presence of mannitol. Interestingly, lead nitrate also exhibited the MI value, the chromosomal aberrations and aberration index very close to those of AWW. Water pollution has attracted a lot of interest in recent years due to its multidimensional hazardous effects. Disposal of the treated as well as untreated disposal of industrial waste material leads to serious problems for human health and survival. Oil refineries as well BMN 673 purchase as other industries generate huge amount of sludge containing both organic and inorganic toxicants which pollute the nearby sites. Many of the constituents in the wastes are carcinogenic and potent immunotoxicants [15]. To monitor the harmful effects of pollutants, a number of toxicity bioassays have been developed. Allium cepa test introduced by Levan [16] has been used frequently and validated by several workers for testing chemical pollutants posing hazardous

environmental effects ( [13], CHIR-99021 order [17], [18] and [19]). Root growth inhibition of Allium cepa was used as an indicator of toxicity of the refinery waste water and Aligarh waste water. The IC50 value in A. cepa system was recorded to be 0.14 X for RWW and 0.10 X for Aligarh waste water in the year 2008. Since water bodies represent a highly dynamic system, the degree of toxicity induced by industrial effluents can surely change over a period of time. Recent study in mafosfamide our lab [20] on the phytotoxic potential

of RWW suggested the IC50 value to be 0.75X. Thus, it can be concluded that from the year 2008 to 2011, there was a definite hike in the IC50 value from 0.14 X to 0.75X. This increase in the IC50 value from 2008 to 2012 signifies a reduced toxic potential of RWW, which might be the outcome of the installation of treatment plants in the refinery. These treatment plants must have detoxified or blocked the release of certain toxicants into water bodies. Toxicity of several other industrial waste samples have been determined in terms of IC50 values employing A. cepa system [21]. In addition to significant phytotoxicity of the RWW, present study also establishes its genotoxic potential in terms of significantly decreased survival of the DNA repair defective mutants of the E. coli K12 ( Figure 2, 3). The efficacy of the E. coli 12 repair defective mutants of E.coli K12 in assaying the genotoxicity of waste water has been well established (IGGE 1990; [22] and [23]).

In the early spermatids ( Fig 7A) the cytoplasm symmetrically en

In the early spermatids ( Fig. 7A) the cytoplasm symmetrically encircles the nucleus, which displays diffuse homogenous chromatin and has a circular outline. The centriolar complex lies laterally to the nucleus and is anchored to the plasma membrane. The proximal centriole is anterior and perpendicular to the distal centriole. The distal centriole differentiates into the basal body and forms the single flagellum. The nucleus rotates toward the centriolar complex ( Fig. 7B) with nuclear rotation of 90° considered complete. A depression is newly formed in the nuclear outline at the level of the centriolar complex that penetrates it ( Fig. 7C). Simultaneous to nuclear rotation, the cytoplasm projects in the direction

of the initial segment of the flagellum forming the cytoplasmic canal and midpiece

( Fig. 7A–C). The midpiece contains the mitochondria, forming vesicles and cytoplasmic canal housing the initial segment of the flagellum ( Fig. Afatinib 7B and C). In the spermatozoon of O. kneri the spherical nucleus (about 1.5 μm in diameter) contains highly condensed homogeneous chromatin interspersed by electron-lucent areas, and is surrounded by a narrow strip of cytoplasm with no organelles ( Fig. 7D buy KU-57788 and E). In the nuclear outline that faces the midpiece there is a medial and moderately deep depression, the nuclear fossa ( Fig. 7D–F). The proximal centriole, initially anterior and perpendicular to distal one, attains an oblique acute angle to the distal centriole. The centrioles are covered by electron dense material and are fastened to one another, to the

nuclear envelope at the nuclear fossa, Cediranib (AZD2171) and to the plasma membrane by stabilization fibrils. The proximal centriole and most of the distal centriole are inside the nuclear fossa ( Fig. 7F and G). The midpiece contains the mitochondria, abundant vesicles and the cytoplasmic canal in which lies the initial segment of the flagellum. The midpiece is slightly asymmetric due to the unequal distribution of mitochondria and vesicles. The mitochondria are elongated and mainly accumulated in the larger portion of the midpiece. Vesicles are elongated and mainly concentrated at the periphery and at the terminal regions of the midpiece ( Fig. 7H–K). The single flagellum contains a classic axoneme (9 + 2) ( Fig. 7L). Information on spermiogenesis in A. cataphractus is not available. In P. granulosus and R. dorbignyi, as in O. kneri, spermatogenesis is cystic and spermiogenesis is Type I. In the spermatozoa of A. cataphractus, P. granulosus and R. dorbignyi the nucleus contains highly condensed homogeneous chromatin and is surrounded by a narrow strip of cytoplasm with no organelles ( Fig. 8A, E, I). The nucleus is flattened at the tip and assumes an ovoid shape in P. granulosus (about 1.2 μm in height by 1.8 μm in width) vs. almost spherical in A. cataphractus (about 1.2 μm in height by 1.3 μm in width) and in R. dorbignyi (about 1.4 μm in height by 1.3 μm in width).