Descriptions of the mature spermatozoa of the lizards Crotaphytus bicinctores, Gambelia wislizenii (Crotaphytidae), and Anolis carolinensis (Polychrotidae) (Reptilia, Squamata, Iguania)

The spermatozoa of Crotaphytus bicinctores and Gambelia wislizenii (Crotaphytidae), and Anolis carolinensis (Polychrotidae) exhibit the squamate autapomorphies of a single perforatorium extending anteriorly from the apical tip of the paracrystalline subacrosomal cone, the presence of an epinuclear electron-lucent region, and extension of the fibrous sheath into the midpiece. Crotaphytid sperm differ from those of polychrotids in several respects, including: the structure of the perforatorium, the size of the epinuclear electron-lucent region, aspects of the acrosome complex, the arrangement and structure of intermitochondrial dense bodies, and in the distance the fibrous sheath extends into the midpiece. The sperm of C. bicinctores, G. wislizenii, and A. carolinensis are most similar to those of the agamids and phrynosomatids examined to date. No spermatozoal autapomorphies for Crotaphytidae or Polychrotidae were found. The condition of having the intermitochondrial dense bodies arranged in regular incomplete rings is tentatively defined as a synapomorphy of Iguania (although modified in Chamaeleonidae). Spermatozoal ultrastructure offers no characters that justify the separation of Iguanidae (sensu late) into several separate families. (C) 2001 Wiley-Liss, Inc.

A comparative ultrastructural study of spermatozoa of the teiid lizards Cnemidophorus gularis gularis, Cnemidophorus ocellifer, and Kentropyx altamazonica (Reptilia, Squamata, Teiidae)

The ultrastructure of the spermatozoa of Cnemidophorus gularis gularis, Cnemidophorus ocellifer, and Kentropyx altamazonica is described for the first time. Mature spermatozoa of Cnemidophorus spp. and K. altamazonica differ in the occurrence of a perforatorial base plate, the enlargement of axonemal fibers 3 and 8, and shape of mitochondria. The comparisons of the ultrastructure sperm of Cnemidophorus spp. and K. altamazonica with Ameiva ameiva [J. Morphol. (2002) in press] suggest that Ameiva and Cnemidophorus are more similar to each other than either is to Kentropyx. Statistical analyses reveal that sperm of all three species studied are significantly different in the following dimensions: head, acrosome, distal centriole length, and nuclear shoulders width. There was no variable statistically different between the Cnemidophorus spp. only. The length of the tail, midpiece, entire sperm, and nuclear rostrum are significantly different between K. altamazonica and Cnemidophorus spp. Our results indicate that sperm ultrastructure presents intra and intergeneric variability. (C) 2002 Elsevier Science Ltd. All rights reserved.

Morphology of the spermatozoa of the Microhylidae (Anura, Amphibia)

Microhylid spermatozoa show the autapomorphic condition of possessing a thin post-mitochondrial cytoplasmic collar. Their spermatozoa are apomorphic in several respects. They have lost the distinct nuclear shoulder, endonuclear canal and axial perforatorium observed in urodeles, caecilians and primitive frogs, possess a conical perforatorium and apomorphically lack any fibres associated with the axoneme. The spermatozoa of Cophixalus , however, differ in several respects from those of the other microhylids examined. Cophixalus spermatozoa are longer in almost all measurements, the acrosome vesicle is cylindrical and does not completely cover the putative perforatorium, the perforatorium is asymmetrical and composed of fine fibres, the nucleus is strongly attenuated and narrower, and the mitochondria are elongate. The absence of fibres associated with the axoneme is an apomorphic condition shared with the Ranidae, Rhacophoridae and Pipidae.

Cauda epididymal spermatozoa of the rufous hare wallaby, Lagorchestes hirsutus (Metatheria, Mammalia) imaged by electron and confocal microscopy

The ultrastructure of mature Lagorchestes hirsutus spermatozoa is described for the first time, revealing unusual aspects of sperm structure in macropodid species. The sperm head is ovoid rather than cuneiform, lacks a ventral nuclear groove and has an acrosomal distribution over approximately 85-90% of its dorsal surface. Immediately adjacent to the nuclear membrane the peripheral nucleoplasm in most spermatozoa form an irregular series of distinctive evaginations previously not described in the spermatozoa of any other marsupial. The midpiece is extremely thickened and short, containing no helical network or peripheral plasma membrane specializations. Axonemal structure is unspecialized with no connecting lamellae; dense outer fibres are closely adherent to axonemal doublets. The sperm morphology of this species is highly aberrant in comparison to other macropod taxa and supports the retention of Lagorchestes as a distinctive genus. In light of this new information, skeletal and serological data should be re-evaluated to determine the true taxonomic and phylogenetic position of this species.

Ultrastructure of the mature spermatozoa of caecilians (Amphibia : Gymnophiona)

The spermatozoa of Gymnophiona show the following autapomorphies: 1) penetration of the distal centriole by the axial fiber; 2) presence of an acrosomal baseplate; 3) presence of an acrosome seat (flattened apical end of nucleus); and 4) absence of juxta-axonemal fibers. The wide separation of the plasma membrane bounding the undulating membrane is here also considered to be apomorphic. Three plesiomorphic spermatozoal characters are recognized that are not seen in other Amphibia but occur in basal amniotes: 1) presence of mitochondria with a delicate array of concentric cristae (concentric cristae of salamander spermatozoa differ in lacking the delicate array); 2) presence of peripheral dense fibers associated with the triplets of the distal centriole; and 3) presence of a simple annulus (a highly modified, elongate annulus is present in salamander sperm). The presence of an endonuclear canal containing a perforatorium is a plesiomorphic feature of caecilian spermatozoa that is shared with urodeles, some basal anurans, sarcopterygian fish, and some amniotes. Spermatozoal synapornorphies are identified for 1) the Uraeotyphlidae and Ichthyophiidae, an 2) the Caeciliidae and Typhlonectidae, suggesting that the members of each pair of families are more closely related to each other than to other caecilians. Although caecilian spermatozoa exhibit the clear amphibian synapomorphy of the unilateral location of the undulating membrane and its axial fiber, they have no apomorphic characters that suggest a closer relationship to either the Urodela or Axiura. J. Morphol. 258:179-192, 2003. (C) 2003 Wiley-Liss, Inc.

Studies on the cryopreservation of common wombat (Vombatus ursinus) spermatozoa

In an attempt to develop a gamete-recovery protocol for the northern hairy nosed wombat (Lasiorhinus krefftii), spermatozoa were removed from the cauda epididymides of four common wombats (Vombatus ursinus) and cryopreserved following a variety of prefreeze storage conditions. Spermatozoa stored for 72 h at 4 degrees C within the testicle before cryopreservation tolerated the freeze-thaw procedure remarkably well, resulting in a higher post-thaw viability (% motile P< 0.01; rate of movement P< 0.01; % live P< 0.01) than sperm recovered on the day of post-mortem, stored in a test tube for 72 h at 4 degrees C and then frozen. The effect of post-thaw dilution with Tris citrate fructose (TCF) diluent on the survival of epididymal common wombat spermatozoa was also investigated. Motility (P< 0.05), rate of sperm movement (P< 0.01) and the percentage of live spermatozoa (P< 0.05) were all significantly greater when spermatozoa were thawed and diluted immediately in TCF than when thawed without dilution. The present study also reports, for the first time, a successful pellet method of freezing wombat spermatozoa on dry ice; volumes of 0.25 and 0.5mL resulted in higher post- thaw survival compared with 0.1-mL pellets.

Collection, seminal characteristics and chilled storage of spermatozoa from three species of free-range flying fox (Pteropus spp.)

This study reports observations on the collection and characteristics of semen from free-range populations of flying fox in Brisbane, Australia. Semen was successfully recovered by electroejaculation from 107 of 115 wild flying foxes (Pteropus alecto, Pteropus poliocephalus and Pteropus scapulatus). A proportion of ejaculates collected from all three species contained seminal vesicle secretions, the incidence of which appeared related to breeding season. Ejaculate volume was small (5-160 mu L), requiring a specialised collection vessel and immediate extension to avoid desiccation. Sperm morphological abnormalities and characteristics are described for the first time. In two species (P. scapulatus and P. alecto), sperm quality varied with breeding season. Dilution in Tris-citratefructose buffer and subsequent incubation (37 degrees C) of Pteropus semen for 2-3 h appeared to have a negative impact on sperm motility and the percentage of sperm with intact plasma membranes and acrosomes and represents a concern for the potential development and use of assisted breeding technology in these species. Preliminary attempts to develop a short-term chilled preservation protocol for flying fox semen revealed that spenn viability (percentage motility and percentage live sperm with intact acrosomes) was significantly reduced after 102 h chilled storage at 5 degrees C; nevertheless, approximately 40% of the spermatozoa were still motile and contained intact acrosomes. Glycerol was neither protective nor detrimental to sperm survival during chilled storage. Microbial flora of the prepuce, urethra and semen of all species were isolated and their antibiotic susceptibility tested. Tetracycline, penicillin, ciprofloxacin, and ceftazidime were the most effective antibiotics in preventing growth of all identified bacteria; however, their effects on sperm survival were not investigated. (c) 2005 Elsevier Inc. All rights reserved.

Actin localisation and the effect of cytochalasin D on the osmotic tolerance of cauda epedidymidal 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 mu M). 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.

An investigation into the similarities and differences governing the cryopreservation success of koala (Phascolarctos cinereus: goldfuss) and common wombat (Vombatus ursinus: shaw) spermatozoa

The aim of this study was to determine the relative cryopreservation success of koala and wombat spermatozoa and to investigate reasons for their respective post-thaw survival by examining the sperm's response to a range of osmotic media and determining the presence and distribution of F-actin. An hypothesis was proposed that F-actin may be imparting a degree of structural inflexibility to the koala sperm plasma membrane; hence, exposure of spermatozoa to cytochalasin D (5 mu M), a F-actin depolymerisation agent, should result in increased plasticisation of the membrane and greater tolerance of cell volume changes that typically occur during cryopreservation. In experiment 1, koala (n = 4) and wombat (n = 4) spermatozoa packaged in 0.25 mL straws were cryopreserved using two freezing rates (fast-3 cm above liquid N2 interface; slow-6 degrees C/min in a freezing chamber) and two glycerol concentrations (8 and 14% v/v) in a tris-citrate glucose buffer with 15% (v/v) egg yolk. Wombat spermatozoa showed better (P < 0.01) post-thaw survival (% motile, % intact plasma membranes, % decondensed sperm heads) than koala spermatozoa. When exposed to media of varying osmolality, koala spermatozoa were less tolerant (% intact plasma membrane) of hyper-osmotic conditions (920 and 1410mOsmol/kg) than wombat spermatozoa. F-actin was localised using a monoclonal antibody but only found in the wombat sperm head. When koala and wombat spermatozoa were exposed to media of varying osmolality, cytochalasin D had no beneficial effect on sperm survival (% intact plasma membranes). This study has demonstrated that wombat spermatozoa are highly tolerant of cryopreservation when compared to koala sperm but that spermatozoa from both species show greatest post-thaw survival when frozen slowly in 14% glycerol. Koala sperm are also particularly susceptible to hyper-osmotic environments but lack of detectable F-actin in the koala spermatozoan suggests that poor cryopreservation success in this species is unlikely to be associated with F-actin induced plasma membrane inflexibility. (c) 2006 Elsevier Inc. All rights reserved.

Cryopreservation of spermatozoa of black marlin, Makaira indica (Teleostei : Istiophoredae)

As a first step towards the development of a method for the cryopreservation of black marlin spermatozoa, this study investigated the effect of dimethylsulfoxide (DMSO) concentration and pellet size on post-thaw spermatozoal motility. Spermatozoa were recovered from the spermatic duct of testes retrieved post-mortem from four adult black marlin caught in the Coral Sea spawning grounds of Australia. Undiluted spermatozoa. were stored on ice for 4 to 10 hours during transport to shore, then evaluated for motility after activation in seawater (1:10 v:v). Spermatozoa were prepared for cryopreservation in pellets by extension (1:3 v:v) in a defined fish Ringer's solution to give two final DMSO concentrations of 2.5% or 5.0%. Diluted spermatozoa were frozen directly on a dry ice block in pellet sizes of either 0.25 ml or 0.50 ml. Frozen pellets were thawed in a water bath at 40 degrees C for 60 seconds and assessed for post-thaw motility following activation in seawater. Spermatozoa recovered within 50 minutes of death and chilled on ice for 4 to 10 hours showed a mean (+/- SEM) motility immediately following activation of 91.6 +/- 7.9%. 50% of the spermatozoa remained motile for approximately 4 to 5 minutes. Following cryopreservation; mean motility declined significantly across all cryoprotectant and pellet size combinations (P < 0.001) but spermatozoa frozen in 2.5% DMSO showed higher motility than those frozen in 5.0% DMSO (P = 0.014). Pellet size had no effect on post-thaw motility (P = 0.179).

Ultrastructure, osmotic tolerance, glycerol toxicity and cryopreservation of caput and cauda epididymidal kangaroo spermatozoa

The aim of the present study was to compare cryopreservation, osmotic tolerance and glycerol toxicity between mature and immature epididymal kangaroo spermatozoa to investigate whether the lack of cryopreservation success of cauda epididymidal spermatozoa may be related to the increased complexity of the sperm ultrastructure acquired during epididymal transit. Caput and cauda epididymidal spermatozoa were recovered from red-necked wallabies (RNW; Macropus rufogriseus) and eastern grey kangaroos (EGK; M. giganteus). In Experiment 1, caput and cauda epididymidal spermatozoa were frozen and thawed using a standard cryopreservation procedure in Triscitrate buffer with or without 20% glycerol. Although cryopreservation of caput epididymidal spermatozoa resulted in a significant increase in sperm plasma membrane damage, they were more tolerant of the procedure than spermatozoa recovered from the cauda epididymidis (P< 0.05). In Experiment 2, caput and cauda epididymidal EGK spermatozoa were diluted into phosphate-buffered saline media of varying osmolarity and their osmotic tolerance determined. Plasma membranes of caput epididymidal spermatozoa were more tolerant of hypo-osmotic media than were cauda epididymidal spermatozoa ( P< 0.05). In Experiment 3, caput and cauda epididymidal RNW spermatozoa were incubated in Tris-citrate buffer with and without 20% glycerol at 35 and 4 degrees C to examine the cytotoxic effects of glycerol. At both temperatures, caput epididymidal spermatozoa showed less plasma membrane damage compared with cauda epididymidal spermatozoa when exposed to 20% glycerol ( P< 0.05). These experiments clearly indicate that epididymal maturation of kangaroo spermatozoa results in a decreased ability to withstand the physiological stresses associated with cryopreservation.