Effects of an anti-gonadoliberin releasing hormone vaccine on testicular, epididymal and spermatogenic development in the horse.

The effects of the GnRH vaccine Improvac® on testicular and epididymal morphometrics, histology and spermatogenesis were measured in 19 young (15-20 months) colts randomly assigned to one control (saline, castration at 57 days, n = 6) or either of two GnRH vaccine-treatment groups, T-57 (castration at 57 days, n = 7) or T-100 (castration at 100 days, n = 6), respectively. All were immunized on Day 0 with a single booster on Day 28. Excised testes and epididymides were weighed and processed for histology to measure tubule, epithelial and muscle dimensions, the ratio of interstitial tissue to seminiferous tubules and determine the stage of spermatogenesis. Testis volume, unchanged within controls, decreased in T-57 and T-100 groups by 50% and 70%, respectively. Treated colts' testes were significantly lighter than controls (64% relative difference); however, epididymal mass showed no significant differences between groups. Proportionally less seminiferous tubule relative to interstitial tissue was observed in both treatment groups (5%) versus controls (22%) with a mean tubule size 28% smaller than controls. Controls exhibited a high proportion of seminiferous tubules with advanced stages of spermatogenesis, whereas treated colts showed a high proportion of tubules in the early stages of spermatogenesis. In conclusion, immunization against GnRH in prepubertal colts was effective at reducing the development of their intra-scrotal reproductive organs and preventing normal spermatogenesis. GnRH vaccination of young colts effectively and consistently reduced testis mass, tubule size and relative proportion of seminiferous tubule tissue while retarding spermatogenesis. The epididymis showed changes with a smaller tubule diameter, lower epithelial height and thicker muscle layer recorded in treated compared to control colts.

Sperm structure of the cattle egret (Bubulcus ibis).

It remains a major concern that sperm structure has continued to be poorly investigated and reported in avian species. To our knowledge, sperm structure in the order Pelecaniforme has not been reported. Although McFarlane (1963; Proceedings of the XIII International Ornithological Congress; Ithaca, NY; American Ornithologists' Union) reported the study of spermatozoa in two genera and two species of the family Ardeidae, he did not provide an account, or the names of the species examined. The present report on the sperm structure of the cattle egret, Bubulcus ibis, is, thus, the first in the order Pelecaniformes (this bird has been placed variably under the order Ciconiiformes, or the order Pelecaniformes). Five sexually mature and reproductively active male cattle egrets were obtained from the wild, humanely euthanized, the reproductive organs dissected out, and tissues from the ducti deferentia were prepared for transmission electron microscopy. The sperm structure of this bird is generally similar to that described for most non-passerine birds. However, the acrosome is a short, conical or bullet-shaped, blunt-ending organelle that lacks a perforatorium. The base of the acrosome is flat and makes contact with the nucleus along, a correspondingly flat plane. The nucleus, thus, ends anteriorly in a flat plane devoid of a concavity or a rostrum, and an endonuclear canal. The acrosomal and nuclear features of this bird are, therefore, main deviations from the situation in the non-passerine clade of birds.

Ultra-imaging in applied animal andrology: The power and the beauty.

Ultrastructural studies of the male gamete provide relevant complementary data of value for the clinical assessment of semen quality and assist in determining phylogenetic and structural/functional relationships. This is illustrated using semen samples and testicular material from vulnerable wild animals (cheetah and rhinoceros), commercially exploited exotic birds (ratites and tinamou) and poultry (chicken and duck). Transmission electron microscopy (TEM) was employed to record sperm and spermatid ultrastructural detail on a comparative basis. The power of the technique was demonstrated using normal and abnormal (the knobbed acrosome defect) formation of the acrosome in the cheetah and rhinoceros. The structural similarities of the defect across species was apparent. The determination of phylogenetic associations was illustrated by comparing structural characteristics between ratites (ostrich, emu and rhea), the tinamou and poultry (chicken and duck), highlighting the morphological peculiarities evident in the midpiece and proximal principal piece of the sperm tail. A clear distinction was obvious between the ratites and tinamou on the one hand and the Galliform and Anseriform birds on the other. The potential power of using molecular techniques in conjunction with ultrastructural studies to explain structural/functional relationships was demonstrated by describing a transient elaboration of the perinuclear theca that occurs during a specific stage of spermiogenesis in ratites, and which can only be imaged using TEM. The inherent aesthetic appeal of the structurally complex normal and defective male gamete was also emphasised.

Adenoviral hepatitis in two Nile crocodile (Crocodylus niloticus) hatchlings from South Africa.

Adenoviral infections may cause mild to severe morbidity or fatality in a large array of animal species. In crocodilians, hatchlings under 5 months of age are usually affected. However, there is a paucity of information on actual incidences in hatchlings originating from South Africa. Two cases of adenoviral hepatitis in crocodile hatchlings about 2 weeks old, bred on a commercial farm in South Africa, are described. Both hatchlings showed typical clinical signs of hepatitis. The identification of intranuclear inclusion bodies in the liver was used to differentiate between adenoviral hepatitis and chlamydial hepatitis. Although vertical transmission has never been proven in crocodiles, the young age of the affected hatchlings raises the possibility of vertical transmission. The lack of epidemiological information on adenoviral hepatitis in crocodiles highlights the need for further characterisation of the virus and targeted surveillance.

Spermatid differentiation, with particular reference to acrosomogenesis, in the passeridan bird, Carib grackle (Quiscalus lugubris).

Only a few studies on the development of the passerine spermatozoon are available, yet species variations in the conformation as well as structure of the generally helical acrosome have been reported. This study of spermiogenesis in the Carib grackle (Quiscalus lugubris) intended to provide a deeper understanding of the development of the sperm, and in particular to investigate the bi-partite nature and development of the acrosome as well as its relationship with the nucleus, in the absence of a perforatorium that is found in most non-passerine birds. The acrosomal vesicle already displays a bi-partite nature in the acrosomal granule within the Golgi complex, and the attachment of the dense granule (future acrosomal core) within the crest part (future acrosomal crest) establishes polarity as it approaches and attaches to the nucleus. Thereafter, they develop variably. The acrosomal crest leads the elongation and spiraling of the acrosome, and the core portion contributes significantly to the formation of the keel of the crest part. The rounded, core-bearing part of the base of the acrosome progressively indents and fits into the concavity, thus formed, at the anterior part of the nucleus. The possible homology of the acrosomal complex (including the perforatorium) and the nucleus between non-passerine and passerine birds was discussed. The centriolar complex comprises both the proximal and distal centrioles in all spermatids and spermatozoa. The mitochondria undergo a number of morphological changes, including size and electron-density, from the round spermatid through to the mature spermatid; changes that are probably influenced by their functional states in the different evolving phases of the spermatids.

The importance of insect sperm: Sperm ultrastructure of Hermetia illucens (black soldier fly).

Sperm structure and ultrastructure of Hermetia illucens was determined by light microscopy and transmission electron microscopy. The main sperm components were similar as for other Dipteran subspecies, while the ultrastructure revealed distinguishing features in the zone of overlap and anterior flagellar region. Sperm varied in size indicating sperm polymorphism. The head region is lacking an acrosome. The zone of overlap consisted of uniquely organized centriolar adjunct material, partly forming electron dense areas to finally form an outer ring separating the mitochondrial derivatives from the 9 + 9 + 2 axoneme. Accessory bodies arising from the zone of overlap are flanked by smaller to large mitochondrial derivatives into the anterior flagellum. This study confirms sperm structure diversity between brachyceran subspecies and support its relationship with nematoceran subspecies.

Effect of using frozen-thawed bovine semen contaminated with lumpy skin disease virus on in vitro embryo production.

Lumpy skin disease (LSD) is an important transboundary animal disease of cattle with significant economic impact because of the implications for international trade in live animals and animal products. LSD is caused by a Capripoxvirus, LSD virus (LSDV), and results in extensive hide and udder damage, fever and pneumonia. LSDV can be shed in semen of infected bulls for prolonged periods and transmitted venereally to cows at high doses. This study examined the effects of LSDV in frozen-thawed semen on in vitro embryo production parameters, including viral status of media and resulting embryos. Bovine oocytes were harvested from abattoir-collected ovaries and split into three experimental groups. After maturation, the oocytes were fertilized in vitro with frozen-thawed semen spiked with a high (HD) or a lower (LD) dose of LSDV, or with LSDV-free semen (control). Following day 7 and day 8 blastocyst evaluation, PCR and virus isolation were performed on all embryonic structures. After completing sufficient replicates to reach 1,000 inseminated oocytes, further in vitro fertilization (IVF) runs were performed to provide material for electron microscopy (EM) and embryo washing procedures. Overall, in vitro embryo yield was significantly reduced by the presence of LSDV in frozen-thawed semen, irrespective of viral dose. When semen with a lower viral dose was used, significantly lower oocyte cleavage rates were observed. LSDV could be detected in fertilization media and all embryo structures, when higher doses of LSDV were present in the frozen-thawed semen used for IVF. Electron microscopy demonstrated LSDV virions inside blastocysts. Following the International Embryo Transfer Society washing procedure resulted in embryos free of viral DNA; however, this may be attributable to a sampling dilution effect and should be interpreted with caution. Further research is required to better quantify the risk of LSDV transmission via assisted reproductive procedures.

Effect of semen processing methods on lumpy skin disease virus status in cryopreserved bull semen.

Lumpy skin disease is an economically important disease of cattle, caused by the lumpy skin disease virus (LSDV; Capripoxvirus). It has a variable clinical appearance but, in severely affected animals, is associated with extensive skin damage, pneumonia and death. The LSDV can be found in the semen of infected bulls for prolonged periods of time, from where it can be transmitted by mating or artificial insemination and cause clinical disease in heifers and cows. In this study, an ejaculate was collected from a LSDV seronegative bull and confirmed free from LSDV DNA by PCR. The ejaculate was split into a control sample (C), a sample spiked with a 4 log TCID50 dose of an LSDV isolate (HD) and a 103 dilution of the virus suspension (ND) and frozen routinely. Two straws from each of the different semen treatment groups (HD, ND and C) were subsequently thawed and subjected to swim-up, single layer centrifugation, Percoll® density gradient and a Percoll® density gradient with added trypsin. For one set of straws, semen quality variables were recorded, and viral DNA status determined using PCR; the other set was used for positive staining electron microscopy. Samples determined to be positive for LSDV DNA by PCR were then subjected to virus isolation (VI). Complete elimination of LSDV from semen did not occur with use of any of the processing methods. Trypsin did reduce the viral load, and eliminated LSDV from the ND sample, but severely negatively influenced semen quality. The LSDV virions, as assessed by electron microscopy, were associated with the sperm plasma membrane. Further investigation is needed to establish the efficacy of immuno-extenders for rendering semen free from LSDV.

Structural features of the spermatozoon of a passeridan bird, the Carib grackle, Quiscalus lugubris.

The spermatozoon of the Carib grackle, Quiscalus lugubris, a member of the family Icteridae, is generally similar in organization to the passerine-type of spermatozoon, in being highly elongated and displaying a helical structure of the acrosome, nucleus and principal piece of the tail. There are subtle variations in acrosomal structural features between this organelle in the grackle and that in some of the very few passerine species of birds in which the spermatozoon has been studied. The proximal centriole is present, and, thus, the Carib grackle is the third passeridan bird in which this organelle, hitherto regarded as absent in passerine birds, has been described in the spermatozoon. The spermatozoon of this bird also possesses a granular helix, which feature has been found variably even in the scanty available reports on passerine spermatozoa. It is advocated that the spermatozoon be studied in many more species of this large clade of birds. This report provides a basis for the study of spermiogenesis in the Carib grackle, with the aim of exposing, inter alia, a number of developmental features and processes of certain organelles that have received attention, recently, in the spermatozoa of passerine birds.

Sperm head shaping in ratites: New insights, yet more questions.

Head shaping in mammalian sperm is regulated by a number of factors including acrosome formation, nuclear condensation and the action of the microtubular manchette. A role has also been suggested for the attendant Sertoli cells and the perinuclear theca (PT). In comparison, relatively little information is available on this topic in birds and the presence of a PT per se has not been described in this vertebrate order. This study revealed that a similar combination of factors contributed to head shaping in the ostrich, emu and rhea, although the Sertoli cells seem to play a limited role in ratites. A fibro-granular structure analogous to the mammalian PT was identified, consisting of sub- and post-acrosomal components. The latter was characterized by stage-specific finger-like projections that appeared to emanate from the cytoplasmic face of the nuclear envelope. They were particularly obvious beneath the base of the acrosome, and closely aligned, but not connected to, the manchette microtubules. During the final stages of chromatin condensation and elongation of the sperm head the projections abruptly disappeared. They appear to play a role in stabilizing the shape of the sperm head during the caudal translocation of the spermatid cytoplasm.

Morphological features of Herbst corpuscles in the oropharynx of the ostrich (Struthio camelus) and emu (Dromaius novaehollandiae).

The distribution of Herbst corpuscles in the oropharynx of the ostrich and emu has recently been documented. However, although the morphology of these mechanoreceptors is well known in neognathous birds, little structural information is available on the Herbst corpuscles of ratites. Tissue sections from those regions of the oropharynx known to possess a high concentration of Herbst corpuscles were sampled from ostrich and emu heads collected after slaughter and prepared for light and transmission electron microscopy. Intra-oral Herbst corpuscles in the ostrich and emu displayed the same basic components (capsule, outer zone, inner core and axon) described in neognathous birds. However, some important differences were observed, notably, the presence of myofibroblasts in the capsule, sensory cilia in cells of the outer layers, a relatively larger, less organized outer zone and narrower inner core, and variations in the shape of the axon. The previously unreported presence of myofibroblasts in the capsule possibly indicates its ability to contract, thus altering the tension of the capsule, which in turn has implications for the conduction of vibrational stimuli. The sensory cilia in the myofibroblasts of the capsule bordering the outer zone, and in the fibroblasts of the outer zone itself, may play a regulatory role in controlling the contraction of the capsule. Such a function has not previously been reported for Herbst corpuscles in any species of bird.

A morphological classification of sperm defects in the ostrich (Struthio camelus).

Commercial ostrich farming is constrained by the absence of a formal animal recording and evaluation scheme as well as by current farming practices. Artificial insemination may have an important role in overcoming these limitations, but requires a thorough knowledge of sperm morphology. Although the morphological characteristics of normal ostrich sperm have been documented, little information is available on the incidence and structural peculiarities of defective sperm in this species. Semen smears were prepared from the ejaculates of five ostriches (Struthio camelus), stained and evaluated. Defects were observed in 17% of sperm studied. Tail defects constituted the most common anomaly. Various forms of bending were the main tail defect, ranging from gentle to acute bends of the principal piece, Dag-like coiling at the head base, sharp reflexes of the midpiece as well as coiling of the endpiece. In contrast, head defects were comparatively low in frequency, with macrocephalic sperm being the defect most frequently observed in this region. Bent, microcephalic, acephalic and round sperm heads were also noted but were few in number. Cytoplasmic droplets occurred frequently in the fixed smears, either associated with the sperm or as free-lying droplets. A small percentage of sperm with multiple defects was recorded. The incidence of morphologically normal sperm in ostrich semen compared favorably with that reported in emu semen, another commercially farmed ratite. However, the range of defects differed appreciably between the two species. Sperm tail anomalies were the most frequent category in the ostrich, whereas head defects comprised the main grouping in the emu.

A re-evaluation of sperm ultrastructure in the emu, Dromaius novaehollandiae.

Existing reports on sperm structure in the emu do not adequately illustrate or describe all the salient ultrastructural features necessary for a meaningful comparison of normal and abnormal sperm in this species. As sperm morphology forms an important parameter in determining semen quality, and in view of the proposed role of artificial insemination in the farming of ratites, this article re-evaluates and complements the existing data on the topic, provides a fully illustrated description of emu sperm ultrastructure, and documents some unreported morphologic features. Conventional transmission and scanning electron microscopy and high resolution scanning electron microscopy were used to describe the ultrastructure of sperm harvested from the distal deferent duct of sexually mature birds slaughtered during the breeding season. In addition to broadly confirming the basic ultrastructural characteristics previously described for emu sperm, this study revealed a number of unreported morphologic features. These included distinct differences in surface properties between the acrosome and nucleus, the presence of a thread-like appendage near the base of the nucleus, variable positioning of the annulus relative to structures located at the midpiece-principal piece junction and regional differentiation of the principal piece. Although the emu displayed similar basic morphologic features to sperm of other ratites and the tinamou, marked structural peculiarities were obvious, notably the lack of an endonuclear canal and a perforatorium and the presence of significantly more mitochondria in the midpiece coupled with an absence of intermitochondrial cement. Although the broad morphologic features of emu sperm would appear to add credence to the general view that the ratites, together with the tinamous, form a monophyletic group at the base of the avian phylogenetic tree, it is also clear that emu sperm are distinctly different from those of the ostrich, rhea, and tinamou which together share morphologic affinities. This observation may lend some support to the alternate view that the Australasian ratites represent a separate clade that developed independently from flightless ancestors.

Light microscopic features and morphometry of sperm in the emu (Dromaius novaehollandiae).

A comprehensive morphologic description of emu sperm at the light microscopy level, an essential prerequisite for the routine evaluation of semen quality in this species, is not currently available. In this study, sperm morphology and morphometry were evaluated using conventionally prepared Romanowsky-stained semen smears of samples collected from the distal ductus deferens from 15 adult birds and fixed in 2.5% glutaraldehyde. Examination of the smears using phase contrast under 100× magnification readily resolved the various components of the cell, namely, the acrosome, nucleus, midpiece, principal piece, and endpiece. This technique was simple to use and produced consistently reproducible results. Normal emu sperm were typically filiform in appearance and closely resembled sperm of the ostrich and other non-passerine species, particularly poultry. A previously undescribed cytoplasmic appendage, associated with the base of the head, was a novel morphologic feature. The acrosome was short (1.84 ± 0.31 µm; mean ± standard deviation), whereas the nucleus measured 11.77 ± 0.93 µm in length. The length of the segments of the flagellum were 2.91 ± 0.4 µm for the midpiece, 47.45 ± 2.8 µm for the principal piece, and 3.69 ± 0.82 µm for the endpiece. The total sperm length was 67.64 ± 3.13 µm (range, 60.14-79.49) and the head:tail ratio was 1:4. Sperm dimensions in the emu were similar to those of other ratites.

A novel transient structure with phylogenetic implications found in ratite spermatids.

BACKGROUND: A novel transient structure was observed in the spermatids of three ratite species using transmission electron microscopy. RESULTS: The structure first appeared at the circular manchette stage of sperm development, was most prominent during the longitudinal manchette phase and disappeared abruptly prior to spermiation. It was composed of regularly-spaced finger-like projections which were closely associated with the outer nuclear membrane, giving the nucleus a cogwheel-like appearance. The projections were approximately 30 nm long and 14 nm wide. Although a similar structure has been described in certain lizard and crocodile species, this is the first report of a similar structure in the developing spermatids of birds. CONCLUSIONS: The potential value of non-traditional characters, such as spermiogenesis and sperm ultrastructure, as phylogenetic markers has recently been advocated. The morphologically unique structure found in ratite spermatids provides additional evidence of a possible phylogenetic link between the reptiles and birds. It also endorses the basal positioning of the ratites as a monophyletic group within the avian phylogenetic tree.

Abaxial tail implantation in the emu, Dromaius novaehollandiae: morphological characteristics and origin of a rare avian sperm defect.

Abaxial tail implantation is a defect occurring in the neck region of spermatozoa and is characterized by misalignment of the centriolar complex relative to the head base. This defect has been described in a number of mammalian species, but is rarely reported in birds. In this study, a detailed description of the defect in emu sperm is presented as well as morphological evidence of its origin in the testis. Despite their low incidence defective sperm could readily be identified using light (LM) and transmission electron microscopy (TEM). Affected sperm displayed obvious misalignment of the head and flagellum with many cells additionally showing unilateral swelling and caudal extension of the nuclear base. This material overlapped the anterior aspect of the centriolar complex. More subtle forms of the defect which were not resolved by LM were revealed by TEM. Abaxial sperm development could be identified in the testis during the early elongated spermatid stage of spermiogenesis. At this stage the centriolar complex was clearly misaligned with respect to the longitudinal axis of the condensing and elongating nucleus. The rare occurrence and low incidence of this defect in the emu would suggest that it has little effect on fertility.

Incidence, structure and morphological classification of abnormal sperm in the emu (Dromaius novaehollandiae).

Little detailed information is currently available on the incidence and morphological characteristics of abnormal sperm in the emu (Dromaius novaehollandiae) and of ratites in general. This situation is further compounded by the lack of a uniform system for the morphological classification of avian sperm defects. Considering the important role that sperm morphology plays in the assessment of semen quality, a detailed description of avian sperm defects is of paramount importance. Based on morphological data provided by light and electron microscopy, a mean of 17.3% abnormal sperm was recorded in semen samples collected from the distal deferent duct of four adult emus during the middle of the breeding season. Four categories of defects were identified. Head defects (57.2% of total defects) consisted of bent heads, macrocephalic heads, round heads and acephalic sperm. Zones of incomplete chromatin condensation and retained cytoplasmic droplets appeared to be implicated in head bending, while giant heads were often associated with multiple tails. Acephalic sperm revealed a complete tail devoid of a head which was replaced by a small spherical structure. Tail defects (22.6% of total defects) were subdivided into neck/midpiece defects and principal piece defects. In the neck/midpiece region disjointed sperm were the exclusive defect noted and were characterized by the complete separation of the head and midpiece in the neck region but within the confines of the plasmalemma. Defects observed in the principal piece were subdivided into short tails, coiled tails and multiple tails. No conclusive evidence was obtained that tail coiling represented the 'Dag' defect. Biflagellate sperm were the most common form of multiple tails, demonstrating two complete tails with all the normal structural elements. Cytoplasmic droplets (13.9% of total defects) were classified as a separate defect. The location and eccentric positioning of retained cytoplasmic droplets was similar to that described in ostrich sperm although the composition of the droplets differed markedly between the two species. A small percentage of sperm (6.3% of total sperm defects) displayed multiple abnormalities. Based on these findings we propose a morphological classification for abnormal ratite sperm identifying head and tail defects, with additional categories for cytoplasmic droplets and multiple defects. Each category is further subdivided to reflect a range of specific defects within the category. It is envisaged that additional defects will be added to each category or that new categories may be added as future studies on the detailed morphology of avian sperm defects are completed.