Intertubular Material Research Articles

Hoof Matters: Developing an Athletic Thoroughbred Hoof.

Conformation of the hooves and distal limbs of foals and factors influencing their morphological development have not been reported in detail for the Thoroughbred breed. In this paper we explore morphogenesis of the equine distal limb in Thoroughbred foals with emphasis on adaptations in response to weight bearing early in life that prepare the foal for an athletic career. Novel data from four studies are presented chronologically during key time periods to illustrate specific aspects of distal limb growth and adaptation. Dorsal epidermal thickness increased from 2.84 ± 0.41 mm in utero to 4.04 ± 1.10 mm by 4 months of age. The increase in thickness was accompanied by decreased tubular density, increased inter-tubular material, and an increase in number and size of tubules at the quarters, which provided a malleable hoof capsule to allow for skeletal growth. Between 4-6 months of age, the hoof widens, and higher loading on the medial side (>60%) vs. the lateral side (<40%) may be factors that influence mature asymmetric hoof shape. Shortly after 12 months-of-age, the dorsal hoof wall angle and dorsal parietal angle of the distal phalanx become parallel, thus optimizing the functional capacity of the hoof capsule in the weanling Thoroughbred.

Hierarchical modeling of elastic moduli of equine hoof wall

This study predicts analytically effective elastic moduli of substructures within an equine hoof wall. The hoof wall is represented as a composite material with a hierarchical structure comprised of a sequence of length scales. A bottom-up approach is employed. Thus, the outputs from a lower spatial scale serve as the inputs for the following scale. The models include the Halpin-Tsai model, composite cylinders model, a sutured interface model, and classical laminate theory. The length scales span macroscale, mesoscale, sub-mesoscale, microscale, sub-microscale, and nanoscale. The macroscale represents the hoof wall, consisting of tubules within a matrix at the mesoscale. At the sub-mesoscale, a single hollow tubule is reinforced by a tubule wall made of lamellae; the surrounding intertubular material also has a lamellar structure. The lamellae contain sutured and layered cells at the microscale. A single cell is made of crystalline macrofibrils arranged in an amorphous matrix at the sub-microscale. A macrofibril contains aligned crystalline rod-like intermediate filaments at the nanoscale. Experimentally obtained parameters are used in the modeling as inputs for geometry and nanoscale properties. The predicted properties of the hoof wall material agree with experimental measurements at the mesoscale and macroscale. We observe that the hierarchical structure of the hoof wall leads to a decrease in the elastic modulus with increasing scale, from the nanoscale to the macroscale. Such behavior is an intrinsic characteristic of hierarchical biological materials. This study can serve as a framework for designing impact-resistant hoof-inspired materials and structures.

The sperm structure and spermiogenesis of the heteropteran Coptosoma scutellatum (Geoffroy) with emphasis on the development of the centriole adjunct

The sperm structure and spermiogenesis of the morphologically peculiar heteropteran Coptosoma scutellatum has been examined with electron microscopy. The sperm has a short monolayer acrosome, a cylindrical nucleus, a 9+9+2 axoneme and two mitochondrial derivatives with crystallized matrix. It shows the synapomorphies typical of the group, consisting of (a) two crystallized areas in the mitochondrial derivatives; (b) the presence of two bridges connecting the intertubular material of the flagellar axoneme to the flattened cisterns adhering to the inner sides of the mitochondrial derivatives; (c) the absence of accessory bodies. The most interesting feature is the presence, in the early spermatid, of an extended centriole adjunct material with the shape of a large ribbon-like structure. This material surrounds the posterior nuclear region and extends anteriorly along the nucleus. It is a microtubule-organizing-center (MTOC) producing the many microtubules surrounding the sperm components At the end of spermiogenesis, the centriole adjunct material modifies into two kidney-like structures accompanying the nuclear posterior end and surrounding the modified centriole.

The sperm structure of Cryptocercus punctulatus Scudder (Blattodea) and sperm evolution in Dictyoptera.

Sperm of the dictyopteran key taxon Cryptocercus punctulatus was examined. It has largely maintained a blattodean groundplan condition, with a three-layered acrosome, an elongate nucleus, a single centriole, a conspicuous centriole adjunct material, two connecting bands (=accessory bodies), and a long functional flagellum with a 9+9+2 axoneme provided with accessory tubules with 16 protofilaments and intertubular material. These sperm characters are shared with several other polyneopterans. The sperm of C. punctulatus is very similar to what is found in Periplaneta americana and species of other groups of roaches, including the sperm of Loboptera decipiens described here for the first time. The general sperm organization here described can be assumed for the groundplan of Insecta and Pterygota. The following evolutionary path can be suggested: after the split between Cryptocercidae and the common ancestor of Isoptera, the typical pattern of sperm formation was altered very distinctly, resulting in a duplication or multiplication (Mastotermitidae) of the centrioles. Mastotermes has maintained a certain sperm motility, but with a very unusual apparatus of multiple flagella with a 9+0 axoneme pattern. After the split into Mastotermitidae and the remaining Isoptera, sperm motility was completely abandoned, and different modifications of sperm components occurred, and even the loss of the sperm flagellum.

The spermatozoon of Mengenilla moldrzyki (Strepsiptera, Mengenillidae): Ultrastructure and phylogenetic considerations

Even though the spermatozoa of several strepsipteran species were described earlier, no data were available for the basal family Mengenillidae. Well-fixed material of the recently described Tunisian species Mengenilla moldrzyki was used for a detailed examination of the sperm ultrastructure. The total length is c. 30 μm. The head region contains a conical acrosome vesicle (0.3-0.35 μm) and an elongated nucleus (7.3 μm) with dense chromatin. Some granular material along with a uniformely dense centriole adjunct and two mitochondrial derivatives are visible at the posterior end of the nucleus. The material of the centriole adjunct does not extend along the flagellum and accessory bodies are absent. The mitochondrial derivatives are elongated structures crossed by a longitudinal crista but lacking parallel transverse cristae and paracrystalline material in the dense matrix. The mitochondrial derivatives gradually reduce their size and end at the most posterior tail region. The flagellar axoneme has a 9 + 9 + 2 pattern and originates beneath the nucleus. In the terminal tail region the axoneme gradually disintegrates. Despite the extreme specialization of the endoparasitc group, strepsipteran spermatozoa are mostly characterized by plesiomorphies. The pattern within the order is largely uniform, but Mengenilla displays several apomorphic features compared to the presumptive strepsipteran groundplan (e.g., absence of crystallizations and cristae in the mitochondrial derivatives). The subdivision of the intertubular material into two compartments with a dense beak-like structure adhering to the tubular wall supports a clade Coleopterida ( = Strepsiptera + Coleoptera) + Neuropterida.

Sperm structure of some Neuroptera and phylogenetic considerations

Spermatozoa from members of Hemerobiidae, Chrysopidae and Mantispidae (Arthropoda; Hexapoda: Neuroptera) have been examined by electron microscopy. In all species examined, the nucleus is surrounded by a nuclear envelope that in its anterior domain, fans out laterally into one (Chrysopidae) or two wings (Hemerobiidae and Mantispidae). Furthermore, the anterior sperm region is surrounded by external dense material. In Mantispidae, sperm dimorphism with two types of spermatozoa is also confirmed: paraspermatozoa (not fertilizing), provided with giant axoneme and mitochondrial derivatives, and euspermatozoa (fertilizing). Spermatozoa of Chrysopidae and Mantispidae are characterized by the lack of an acrosome while sperm cells of Hemerobiidae are provided with a bilayered acrosome. Spermatozoa from all the investigated species have axonemes of the conventional insect type, with a 9+9+2 microtubular pattern and with accessory tubules provided with 16 protofilaments. In all the examined taxa the intertubular material has the same localization also observed in all other previously analysed Neuroptera. The mitochondrial derivatives and the accessory bodies in the three families are also described. Hemerobiidae are characterized by the presence of a large groove of the plasma membrane along the right side of the anterior sperm region, which results in an eccentric position of the axoneme. Chrysopidae have large mitochondrial derivatives, which encircle the axoneme. The peculiar feature regarding the nuclear envelope was not seen in other members of neuropteroid insects. These data are discussed in the light of the phylogenetic relationships of the taxa examined.

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.

Ultrastructural studies on euspermatozoa and paraspermatozoa in Mantispidae (Insecta, Neuroptera)

Previous studies have demonstrated the presence of sperm dimorphism in the Mantispidae Perlamantispa perla. We extended the study on several other mantidflies. In all the examined species the occurrence of euspermatozoa (typical) and paraspermatozoa (atypical) was established. The euspermatozoa are characterized by the presence of a cylindrical nucleus surrounded by an envelope that fans out laterally into two thin wings of different length. The acrosome seems to be missing. The nucleus is surrounded by extracellular material. The flagellum is provided with a 9 + 9 + 2 axonemal pattern; the accessory tubules contain 16 protofilaments and the intertubular material has the distribution typical of the taxon. Two elongated accessory bodies flank partially the axoneme and connect this structure with the mitochondrial derivatives. The flagellar axoneme of paraspermatozoa consists of an axoneme and two giant mitochondrial derivatives filled with large globular units. The axoneme exhibits a 9 + 9 + 2 pattern, in which the central 9 + 2 units have a normal structure, in that the microtubular doublets are provided with both dynein arms and radial links. On the contrary, the nine accessory microtubules have a large diameter and their tubular wall consists of 40 protofilaments. This comparative study provided evidences about the uniformity of sperm ultrastructure in Mantispidae. The function of non-fertilizing giant sperm in mantidflies is discussed.

A novel membrane specialization in the sperm tail of bug insects (heteroptera)

The sperm tail of bug insects has 9 + 9 + 2 flagellar axonemes and two mitochondrial derivatives showing two to three crystalline inclusions in their matrix. During spermiogenesis, the axoneme is surrounded by a membrane cistern which, at sperm maturity, reduces to two short cisterns on the opposite sides of the axoneme adhering to the mitochondrial derivatives. Filamentous bridges connect the intertubular material of the axoneme to these cisterns. Such bridges, which represent a peculiar feature of bug insects, are resistant to detergent treatment, whereas part of the intertubular material and the inner content of microtubular doublets are affected by the treatment. After freeze-fracture replicas, at the insertion of the bridges to the cisternal membrane, the P-face of this membrane shows a characteristic ribbon consisting of four rows of 11 +/- 1 nm staggered intramembrane particles, 13 +/- 2 nm apart along each row. The bridges could be able to maintain the axoneme in the proper position during flagellar beating avoiding distortion affecting sperm motility.

Sperm ultrastructure and spermiogenesis of Coniopterygidae (Neuroptera, Insecta)

The spermiogenesis and the sperm ultrastructure of several species of Coniopterygidae have been examined. The spermatozoa consist of a three-layered acrosome, an elongated elliptical nucleus, a long flagellum provided with a 9+9+3 axoneme and two mitochondrial derivatives. No accessory bodies were observed. The axoneme exhibits accessory microtubules provided with 13, rather than 16, protofilaments in their tubular wall; the intertubular material is reduced and distributed differently from that observed in other Neuropterida. Sperm axoneme organization supports the isolated position of the family previously proposed on the basis of morphological data.

Sperm structure of Limoniidae and their phylogenetic relationship with Tipulidae ( Diptera, Nematocera)

The sperm ultrastructure of a few species of Limoniidae ( Limonia nigropunctata; L. nubeculosa; Chionea n. sp.; C. alpina; C. lutescens) was studied. The two species of Limonia have a monolayered acrosome with crystallized material, a three-lobed nucleus in cross section, a ring of centriole adjunct material and a flagellum which consists of a 9 + 9 + 1 axoneme and a single mitochondrial derivative. The central axonemal tubule is provided with 15 protofilaments in its tubular wall, while the accessory tubules have 13 protofilaments and are flanked by the electron-dense intertubular material. The three species of Chionea share a monolayered acrosome, a nucleus with two longitudinal grooves, a centriole adjunct material which surrounds the centriole and the initial part of the axoneme. The axoneme is of conventional type, with 9 + 9 + 2 microtubular pattern, with accessory tubules provided with 13 protofilaments and intertubular material. However, in C. lutescens the accessory tubules start with 15 protofilaments and transform into a tubule with 13 protofilaments. These data are discussed in the light of the phylogenetic relationship between Limoniidae and Tipulidae. For this purpose, the sperm ultrastructure of Nephrotoma appendiculata was also considered comparatively.

The sperm structure of Galloisiana yuasai (Insecta, Grylloblattodea) and implications for the phylogenetic position of Grylloblattodea

The sperm ultrastructure of the Grylloblattodea Galloisiana yuasai was described and the sperm characters were comparatively examined in several orthopteroid insect orders for inferring the phylogenetic placement of the Grylloblattodea. The spermatozoa of G. yuasai are joined in bundles (spermatodesms) containing 200 units. Major features of these spermatozoa include a monolayered acrosome, a 9+9+2 axoneme with 16-pfs accessory microtubules and expanded intertubular material, and an evident “centriole adjunct”. The diffused material observed between the axoneme and the mitochondrial derivatives is considered to be an extension of the three connecting bands observed in other orthopteroid taxa, similar to what happens in some orthopteran lineages. The presence of the connecting bands, even though modified in G. yuasai, suggests that the Grylloblattodea are to be placed in a clade with Mantophasmatodea, Mantodea and Orthoptera.

Sperm structure and spermiogenesis in Coletinia sp. (Nicoletiidae, Zygentoma, Insecta) with a comparative analysis of sperm structure in Zygentoma

The spermatozoon of Coletinia sp. has a bilayered acrosome, a short nucleus (4μm) and a relatively short sperm tail with two mitochondrial derivatives. The chromatin is uniformly dense except for several electron-lucid channels or strands which permeate the nucleus and which originate in the spermatid as invaginations of the nuclear envelope. The invaginations occur mostly or exclusively along two meridians of the spermatid that are also characterized by the presence of a longitudinal rod of medium electron density. The two rods (designated as ‘mid-spermatid rods’) evidently are instrumental in the formation of the electron-lucid channels. The significance of this elaborate system of intranuclear channels is not understood. The sperm tail has a 9+9+2 axoneme with each of the nine microtubular doublets accompanied by an accessory microtubule; scant intertubular material can also be distinguished. Hence, the tail axoneme resembles that of many pterygote insects. Each of the two mitochondrial derivatives contains a crystalline inclusion that has periodically spaced layers going in different directions on either side of the midline. Two synapomorphic traits appear to be shared by Ateluridae and Nicoletiidae, namely the invaginations of the nuclear membrane along two meridians of the nucleus and the shape of the crystalline inclusions of the mitochondrial derivatives. Four species from the family Lepismatidae were also examined as to their sperm ultrastructure. Three of them, Allacrotelsa kraepelini, Ctenolepisma longicaudata and Ctenolepisma sp., were found to be very similar to the two previously examined lepismatids, Thermobia domestica and Lepisma saccharina. On the other hand, spermatozoa of Tricholepisma aurea were aggregated in small groups rather than pairwise joined as seen in the other lepismatids. Sperm characters are also used to reconstruct a phylogenetic hypothesis which suggests a close relationship between Ateluridae and Nicoletiidae.

Sperm structure and spermiogenesis in Coletinia sp. (Nicoletiidae, Zygentoma, Insecta) with a comparative analysis of sperm structure in Zygentoma

The spermatozoon of Coletinia sp. has a bilayered acrosome, a short nucleus (4 μm) and a relatively short sperm tail with two mitochondrial derivatives. The chromatin is uniformly dense except for several electron-lucid channels or strands which permeate the nucleus and which originate in the spermatid as invaginations of the nuclear envelope. The invaginations occur mostly or exclusively along two meridians of the spermatid that are also characterized by the presence of a longitudinal rod of medium electron density. The two rods (designated as ‘mid-spermatid rods’) evidently are instrumental in the formation of the electron-lucid channels. The significance of this elaborate system of intranuclear channels is not understood. The sperm tail has a 9+9+2 axoneme with each of the nine microtubular doublets accompanied by an accessory microtubule; scant intertubular material can also be distinguished. Hence, the tail axoneme resembles that of many pterygote insects. Each of the two mitochondrial derivatives contains a crystalline inclusion that has periodically spaced layers going in different directions on either side of the midline. Two synapomorphic traits appear to be shared by Ateluridae and Nicoletiidae, namely the invaginations of the nuclear membrane along two meridians of the nucleus and the shape of the crystalline inclusions of the mitochondrial derivatives. Four species from the family Lepismatidae were also examined as to their sperm ultrastructure. Three of them, Allacrotelsa kraepelini , Ctenolepisma longicaudata and Ctenolepisma sp., were found to be very similar to the two previously examined lepismatids, Thermobia domestica and Lepisma saccharina . On the other hand, spermatozoa of Tricholepisma aurea were aggregated in small groups rather than pairwise joined as seen in the other lepismatids. Sperm characters are also used to reconstruct a phylogenetic hypothesis which suggests a close relationship between Ateluridae and Nicoletiidae.

New findings on sperm ultrastructure of Xenos vesparum (Rossi) (Strepsiptera, Insecta)

The systematic position of insect order Strepsiptera is still under debate. It was, therefore, thought of interest to examine the ultrastructure of a strepsipteran in a search for synapomorphies shared with Coleoptera, Diptera, or any other insect order. The fine structure of spermatozoa and the spermatid from Xenos vesparum (Rossi) was re-examined using scanning and transmission electron microscopy and a fixation technique that permits the visualization of the macromolecular organization of the organelles. The spermatozoon was shown to possess several traits that are characteristics of insects in general, such as a 9+9+2 axoneme, two mitochondrial derivatives containing a crystalline material and two ‘zipper lines’ present along the sperm tail. Seventeen protofilaments occurred along most of the accessory tubules, which reduced to 16 posteriorly. An acrosome is absent. The neck region contains a prominent centriolar adjunct, which gives rise to two accessory bodies which adhere to the mitochondrial derivatives, and to slender strands of the so-called intertubular material found between the accessory tubules. Of interest is the finding that the glycocalyx consists of prominent filamentous strands, similar to those found in siphonapterans, mecopterans and basal dipterans.

Sperm ultrastructure of Mantophasma zephyra (Insecta, Mantophasmatodea)

The ultrastructure of Mantophasma zephyra spermatozoa is described. Sperm cells have a trilayered acrosome with conspicuous extra-acrosomal material which expands along the nucleus. The nucleus is crossed anteriorly by a canal and its posterior end is embedded in the centriole adjunct material. A centriole with microtubular triplets is present. The flagellum has a 9+9+2 axonemal pattern, two partially crystallised mitochondrial derivatives, two membranous sacs and three connecting bands. The accessory microtubules are filled with dense material and have 16 protofilaments in their tubular wall. The intertubular material is not very expanded. In the seminal vesicles spermatozoa are stuck together to form spermatodesms, and their heads are also joined by adherens junctions. A cladistic analysis based on sperm features indicates a close relationship of Mantophasmatodea with Mantodea.

Sperm structure and spermiogenesis in Atelura formicaria Heyden (Zygentoma, Insecta)

AbstractThe spermatozoon of Atelura formicaria (Zygentoma) shows several features that are typical of insects: an apical acrosome, an elongated dense nucleus, a centriole with expanded centriolar adjunct material, two large mitochondrial derivatives, and two thin accessory bodies located beneath the nucleus. The axoneme exhibits a 9 + 9 + 2 pattern with accessory tubules formed by 16 protofilaments and intertubular material. However, spermatozoa of A. formicaria show some remarkable features. The sperm cell is short for an insect, being only 50 µm in length. The nucleus is characterized by the presence of two lateral grooves which are filled with numerous infoldings of the nuclear envelope. In a cross‐section the chromatin has the configuration of the Leonardo da Vinci’s ‘Vitruvian man’. Each mitochondrial derivative has a peculiar structure with peripheral cristae and four crystalline bodies in its matrix; two of these crystalline bodies are large and have differently orientated cristal planes. At the end of spermiogenesis, sperm bundles are stored in the proximal part of the testes. Secretions from the epithelial wall of this region give rise to large globular structures. These include sperm bundles intermingled with dense granules, forming the so called ‘spermatolophids’. These formations descend along the deferent duct and are stored in the expanded seminal vesicle. Atelura spermatozoa do not pair as in some Lepismatidae, nor do they fuse as in Tricholepidion (Lepidotrichidae). Thus, sperm aggregation in Zygentoma is realized according to different modalities and can hardly be considered as a synapomorphic trait of its subtaxa.

Micromechanics of the equine hoof wall: optimizing crack control and material stiffness through modulation of the properties of keratin.

Small-scale components of the equine hoof wall were tested to determine their mechanical roles in the morphological hierarchy. Macroscale tensile tests conducted on samples of the inner wall tubules and intertubular material showed a sixfold difference in mean initial stiffnesses (0.47 and 0.08 GPa, respectively), indicating that the inner wall tubules stiffen the wall along its longitudinal axis. The similarity in material properties of tubule and intertubular samples from the mid-wall suggests that tubules in this region offer only minor reinforcement along the longitudinal axis. Microscale tests conducted on rows of keratin strands from the inner wall tubules and intertubular material, and on intertubular keratin strands of the mid-wall, produced estimates of the stiffnesses of the hydrated matrix (0.03 GPa) and intermediate filament (IF; 3-4 GPa) components of the nanoscale ( &agr; -keratin) composite. The results from these tests also suggest that the properties of the keratin composite vary through the wall thickness. Birefringence measurements on inner wall and mid-wall regions agree with these observations and suggest that, although the keratin IF volume fraction is locally constant, the volume fraction changes through the thickness of the wall. These findings imply that modulation of the hoof wall properties has been achieved by varying the IF volume fraction, countering the effects of specific IF alignments which serve another function and would otherwise adversely affect the modulus of a particular region.

Stiffness as a function of moisture content in natural materials:Characterisation of hoof horn samples

Hoof horn, which forms the capsule at the lower part of the legs of many grazing animals including equids (horses, donkeys and mules), is a composite natural material based on α-keratin. Its function is influenced by the tubular and intertubular material and is modulated by the moisture content. There is a requirement to adopt a standard approach to drying regimes and sampling protocols in order to make progress in understanding how the biomechanical properties of hoof horn are related to its structure. In this work the stiffness of donkey hoof has been examined using a three point bending technique and the effect of hydration has been investigated. Also the tubule density properties of this hoof horn material are reported.

Design complexity and fracture control in the equine hoof wall.

Morphological and mechanical studies were conducted on samples of equine hoof wall to help elucidate the relationship between form and function of this complex, hierarchically organized structure. Morphological findings indicated a dependence of tubule size, shape and helical alignment of intermediate filaments (IFs) within the lamellae on the position through the wall thickness. The plane of the intertubular IFs changed from perpendicular to the tubule axis in the inner wall to almost parallel to the tubule axis in the outer wall. Morphological data predicted the existence of three crack diversion mechanisms which might prevent cracks from reaching the sensitive, living tissues of the hoof: a mid-wall diversion mechanism of intertubular material to inhibit inward and upward crack propagation, and inner- and outer-wall diversion mechanisms that prevent inward crack propagation. Tensile and compact tension fracture tests were conducted on samples of fully hydrated equine hoof wall. Longitudinal stiffness decreased from 0.56 to 0.30 GPa proceeding inwardly, whereas ultimate (maximum) properties were constant. Fracture toughness parameters indicated that no compromise results from the declining stiffness, with J-integral values ranging from 5.5 to 7.8 kJ m-2 through the wall thickness; however, highest toughness was found in specimens with cracks initiated tangential to the wall surface (10.7 kJ m-2). Fracture paths agreed with morphological predictions and further suggested that the wall has evolved into a structure capable of both resisting and redirecting cracks initiated in numerous orientations.