Different narcotic gases and concentrations for immobilization of ostrich embryos for in-ovo imaging.

In-ovo imaging using avian eggs has been described as a potential alternative to animal testing using rodents. However, imaging studies are hampered by embryonal motion producing artifacts. This study aims at systematically comparing isoflurane, desflurane and sevoflurane in three different concentrations in ostrich embryos. Biomagnetic signals of ostrich embryos were recorded analyzing cardiac action and motion. Ten groups comprising eight ostrich embryos each were investigated: Control, isoflurane (2%, 4%, and 6%), desflurane (6%, 12%, and 18%) and sevoflurane (3%, 5%, and 8%). Each ostrich egg was exposed to the same narcotic gas and concentration on development day (DD) 31 and 34. Narcotic gas exposure was upheld for 90 min and embryos were monitored for additional 75 min. Toxicity was evaluated by verifying embryo viability 24 h after the experiments. Initial heart rate of mean 148 beats/min (DD 31) and 136 beats/min (DD 34) decreased over time by 44-48 beats/minute. No significant differences were observed between groups. All narcotic gases led to distinct movement reduction after mean 8 min. Embryos exposed to desflurane 6% showed residual movements. Isoflurane 6% and sevoflurane 8% produced motion-free time intervals of mean 70 min after discontinuation of narcotic gas exposure. Only one embryo death occurred after narcotic gas exposure with desflurane 6%. This study shows that isoflurane, desflurane and sevoflurane are suitable for ostrich embryo immobilization, which is a prerequisite for motion-artifact free imaging. Application of isoflurane 6% and sevoflurane 8% is a) safe as no embryonal deaths occurred after exposure and b) effective as immobilization was observed for approx. 70 min after the end of narcotic gas exposure. These results should be interpreted with caution regarding transferability to other avian species as differences in embryo size and incubation duration exist.

Timed Collinear Activation of Hox Genes during Gastrulation Controls the Avian Forelimb Position.

Limb position along the body is highly consistent within one species but very variable among vertebrates. Despite major advances in our understanding of limb patterning in three dimensions, how limbs reproducibly form along the antero-posterior axis remains largely unknown. Hox genes have long been suspected to control limb position; however, supporting evidences are mostly correlative and their role in this process is unclear. Here, we show that limb position is determined early in development through the action of Hox genes. Dynamic lineage analysis revealed that, during gastrulation, the forelimb, interlimb, and hindlimb fields are progressively generated and concomitantly patterned by the collinear activation of Hox genes in a two-step process. First, the sequential activation of Hoxb genes controls the relative position of their own collinear domains of expression in the forming lateral plate mesoderm, as demonstrated by functional perturbations during gastrulation. Then, within these collinear domains, we show that Hoxb4 anteriorly and Hox9 genes posteriorly, respectively, activate and repress the expression of the forelimb initiation gene Tbx5 and instruct the definitive position of the forelimb. Furthermore, by comparing the dynamics of Hoxb genes activation during zebra finch, chicken, and ostrich gastrulation, we provide evidences that changes in the timing of collinear Hox gene activation might underlie natural variation in forelimb position between different birds. Altogether, our results that characterize the cellular and molecular mechanisms underlying the regulation and natural variation of forelimb positioning in avians show a direct and early role for Hox genes in this process.

The Use of Ostrich Eggs for In Ovo Research: Making Preclinical Imaging Research Affordable and Available.

In ovo studies are a valuable option in preclinical research, but imaging studies are severely limited by the costs of dedicated equipment needed for small-sized eggs. We sought to verify the feasibility of using larger, ostrich, eggs (Struthio camelus) for imaging on the PET/CT scanners used for routine clinical investigations. Methods: Ostrich eggs were incubated until shortly before hatching, prepared for intravitelline venous injection of contrast medium or radiotracer, and imaged using native CT, contrast-enhanced CT, and PET/CT. Any technical adaptations that were needed to improve the outcome were noted. Results: Of the 34 eggs initially incubated, 12 became fully available for imaging of embryonal development. In ovo imaging with conventional PET/CT not only was feasible but also provided images of good quality, including on dynamic PET imaging. Conclusion: In ovo imaging with ostrich eggs and routine clinical scanners may allow broader application of this field of preclinical research, obviating costly dedicated equipment and reducing the number of animals needed for classic animal research. Further experiments are warranted to refine this novel approach, especially to reduce motion artifacts and improve monitoring of viability.

Morphology of the extraocular muscles (m. bulbi) in the pre-hatchling and post-hatchling african black ostriches (struthio camelus domesticus L., 1758) (Aves: Struthioniformes).

The aim of the study was to describe the morphology and the development of the extraocular muscles (EOMs) in the pre-hatchling and post-hatchling African black ostrich. The study involved 50 birds aged between 28 days and 3 years. The EOMs were analyzed morphologically with respect to the location and length of the straight and oblique muscles and the third eyelid muscles, the length and breadth of their tendons as well as the distance and shape of the muscle tendon insertions at the corneal limbus. A histological and histometric analysis were also carried out. The greatest increase in the length of the EOMs was noted in groups III-V. A marked increase in the length of the tendons of the dorsal straight muscle was found in groups II and III, in the tendons of the nasal straight muscle in groups IV and V, in the tendons of the dorsal oblique muscle in groups III to V and in the tendons of the ventral oblique muscle in groups IV and V. There was a significant increase in the breadth of the dorsal straight and ventral oblique muscle tendons in groups IV and V and the tendons of the pyramidal muscle in groups III and V. The distance of the distal insertion of the tendon at the corneal limbus increased steadily with age in all the examined groups. The number of fascicles and muscle fibres, their diameter and length in all the studied EOMs were different in the different groups.

Ostrich (Struthio camelus) embryonic development from 7 to 42 days of incubation.

1. Hatching success of ostrich eggs is poor (50-60% of fertile eggs). The current study was designed to identify the timing of key stages in the development of the ostrich embryo. 2. Growth of both embryo and wing length during 42 d of incubation was comparable and approximately linear, with a more or less weekly doubling in size up to 35 d of incubation. 3. The embryo eye size increased more rapidly than beak length and reached a maximum of ~16.2 mm by 28 d of incubation, whereas beak length increased continuously until hatching at 42 d. 4. Linear regression equations were derived from morphometric measurements of embryos between 7 and 42 d. 5. Information stemming from these results can be used to estimate the age of dead-in-shell embryos in an attempt to identify timing of incubation problems that potentially result in low hatchability of fertile eggs.

Respiratory Pores on Ostrich Struthio camelus (Aves: Struthionidae) Eggshells.

Respiratory pores are essential for the survival of the embryo within the eggshell. Distribution patterns of such pores on ostrich (Struthio camelus) eggshells show remarkable variations in bird group. Eggshells preserved in the museum of New Zealand have long, superficial, winding grooves and ridges, with pores distributed densely in the bottom of grooves. Both the grooves and ridges that separate them are twisted. By contrast, the surfaces of eggs from farmed ostriches are mostly smooth, with only occasional, short grooves, and respiratory pores distributed more evenly. The cause of ridging and grooving of the surface of eggs from wild birds is unclear but may be due to the need for stronger shells and effects of environmental stresses. It appears that the arrangement of respiratory pores on ostrich eggshells seems to be changeable by surrounding stresses.

Microstructure, crystallography and diagenetic alteration in fossil ostrich eggshells from Upper Palaeolithic sites of Indian peninsular region.

Biominerals studies are of importance as they provide an understanding of natural evolutionary processes. In this study we have investigated the fossil ostrich eggshells using Scanning Electron Microscopy (SEM), X-ray Diffraction (XRD) and Electron Backscatter Diffraction (EBSD). SEM studies demonstrated the ultrastructure of fossil eggshells and formation of calcified cuticular layer. The presence of calcified cuticle layer in eggshell is the basis for ancient DNA studies as it contains preserved biomolecules. EBSD accentuates the crystallographic structure of the ostrich eggshells with sub-micrometer resolution. It is a non-destructive tool for evaluating the extent of diagenesis in a biomineral. EBSD analysis revealed the presence of dolomite in the eggshells. This research resulted in the complete recognition of the structure of ostrich eggshells as well as the nature and extent of diagenesis in these eggshells which is vital for genetic and paleoenvironmental studies.

Distribution and Characterisation of Goblet Cells in the Large Intestine of Ostriches during the Pre- and Post-Hatch Period.

The role of goblet cell secretion, containing mucopolysaccharides, in the formation of a protective barrier of intestinal mucosa and transportation of the intestinal content has been described quite extensively. However, information on the quality composition of mucopolysaccharides and its changes in the intestinal tract of ostrich chicks, especially in the large intestinal segments, is unavailable. In the current study, ostrich embryos/chicks (n = 6/36) of both sexes were used shortly before hatching and during the first months of the post-hatch period. Tissues for histology were taken from the large intestine: the medium segments of the caecum, proximal and distal parts of colon. By using histochemical reactions, the differentiation of goblet cells as well as chemical composition of mucopolysaccharides was carried out. The cells contained acid (AB+), neutral (PAS+) and mixed (AB/PAS+) mucopolysaccharides. The number of goblet cells in the large intestine per unit area of mucosa increased towards the cloaca, and it was the highest in the distal part of the colon. The qualitative goblet cell composition in different large intestinal parts was different in all ages. In the caecum, goblet cells containing acid and mixed mucopolysaccharides dominate post-hatch, whereas in the colon, goblet cells containing acid mucopolysaccharides predominated. The most rapid changes in the qualitative goblet cell composition occur during the first week post-hatch when in all the intestinal segments the proportion of cells containing acid mucopolysaccharides continuously increased.

Functional anatomy of the lacrimal gland in African black ostrich Struthio camelus domesticus in the embryonic and postnatal period.

The aim of the present study was morphological and histochemical analysis of the lacrimalgland (LG) in African black ostrich Struthio camelus domesticus in the embryonic and postnatalperiod. Studies were conducted on 50 ostriches aged between the 28th day of incubation until7 months old. Tissue sections were stained with haematoxylin and eosin, Azan trichrome,periodic acid-Schiff, Alcian blue pH 2.5, aldehyde fuchsin and Hale's dialysed iron. The LGin ostrich was classified as a tubulo-acinar type. The primordia of the lobes were determinedin the LG structure on the 28th day of incubation, whilst the weakly visible lobes with aciniand tubules were observed on the 40th day of incubation. Morphometric studies of the LGshowed steady growth, characterised by an increase in both length and width. Histometricmeasurements of lobe size showed little difference between the first, second and third agegroups, whilst in the fourth age group a marked increase in size of lobes was observed.The study showed that, apart from morphological changes, during the growth of the LGthe character of acid mucopolysaccharides changed. Sulphated acid mucopolysaccharideswere indicated, particularly with aldehyde fuchsin (AF) staining in the fourth age group.The Hale's dialysed iron (HDI) staining showed a low concentration of carboxylated acidmucopolysaccharides in the first and second age groups and a higher concentration in thethird and fourth age groups. Periodic acid-Schiff staining (PAS)-positive cells were observedin each age group, but only a small number of cells with a weakly PAS-positive reaction weredemonstrated in the first age group.

Morphological studies on the harderian gland in the ostrich (Struthio camelus domesticus) on the embryonic and post-natal period.

The present investigation was performed on 50 ostriches from 28th day of incubation until the 7th month of life. The morphological (morphometric, histological, histometric and histochemical) studies were conducted. Tissue sections were stained with haematoxylin-eosin, methyl green-pyronin Y, periodic acid-Schiff, alcian blue pH 2.5, aldehyde fuchsin and Hale's dialyzed iron studies. The Harderian gland becomes macroscopically visible on the 28th day of incubation. It is situated in the ventronasal angle of the orbit near inter-orbital septum, between medial rectus muscle, pyramidal and ventral oblique muscles. The Harderian gland of ostrich is a tubulo-acinar gland. The acini were composed of tall conical cells which formed a small lumen and were surrounded by myoepithelial cells. These cells had a granular basophilic, vacuolated cytoplasm. Each of the lobes has a system of complex branching ducts - tertiary, secondary and primary. In the III of research group (3rd week of life), the presence of few plasma cells was demonstrated, which were located within acini and tertiary and secondary ducts, whereas the biggest concentration of plasma cells was observed in group IV of research tissue (4th month of life). The dark cells were observed first time in main ducts 72 h after hatching of nestlings (group II). The morphometric and histometric studies showed that the most intensive growth of Harderian gland occurred between the third week and the seventh month of birds' life. The histochemical study indicated the presence of neutral and acidic mucins, glycoproteins and carboxylated acid mucopolysaccharides.

Reconstruction and in vivo analysis of the extinct tbx5 gene from ancient wingless moa (Aves: Dinornithiformes).

BACKGROUND: The forelimb-specific gene tbx5 is highly conserved and essential for the development of forelimbs in zebrafish, mice, and humans. Amongst birds, a single order, Dinornithiformes, comprising the extinct wingless moa of New Zealand, are unique in having no skeletal evidence of forelimb-like structures. RESULTS: To determine the sequence of tbx5 in moa, we used a range of PCR-based techniques on ancient DNA to retrieve all nine tbx5 exons and splice sites from the giant moa, Dinornis. Moa Tbx5 is identical to chicken Tbx5 in being able to activate the downstream promotors of fgf10 and ANF. In addition we show that missexpression of moa tbx5 in the hindlimb of chicken embryos results in the formation of forelimb features, suggesting that Tbx5 was fully functional in wingless moa. An alternatively spliced exon 1 for tbx5 that is expressed specifically in the forelimb region was shown to be almost identical between moa and ostrich, suggesting that, as well as being fully functional, tbx5 is likely to have been expressed normally in moa since divergence from their flighted ancestors, approximately 60 mya. CONCLUSIONS: The results suggests that, as in mice, moa tbx5 is necessary for the induction of forelimbs, but is not sufficient for their outgrowth. Moa Tbx5 may have played an important role in the development of moa's remnant forelimb girdle, and may be required for the formation of this structure. Our results further show that genetic changes affecting genes other than tbx5 must be responsible for the complete loss of forelimbs in moa.

Embryonic development in the ostrich (Struthio camelus) during the first 7 days of artificial incubation.

1. Early development of ostrich embryos was investigated in relation to time of egg collection and genotype. 2. A total of 321 ostrich eggs were collected during the 2008 and 2009 breeding seasons and the development of the embryo for up to the first 168 h of incubation was described and analysed. A sample of the incubated eggs was weighed and opened daily to investigate developmental changes. 3. In fresh eggs, the blastoderm contained a round, translucent dark area pellucida (AP) in the centre, with a surrounding thin white ring, likely to be the beginning of the area opaca (AO). Fresh eggs were considered infertile if the blastoderm was absent and instead numerous white droplets were present surrounded by vacuoles. 4. The average blastoderm area of a fresh fertile egg was 15.8 mm(2), increasing to 143.3 mm(2) after 2 d of incubation. By 72 h of incubation the area vasculosa (AV) was discernible in the posterior half of the blastoderm. 5. At 48 h of incubation the blastoderm area in eggs from the South African Black genotype (SAB) × Zimbabwean Blue genotype (ZB) crosses (104.5 ± 18.6 mm(2)) was lower than the pure SAB (141.0 ± 10.5 mm(2)), ZB (161.7 ± 13.5 mm(2)) and ZB × SAB crosses (166.1 ± 14.2 mm(2)). 6. Embryo length was 5.01 mm after 72 h of incubation and 14.5 mm after 168 h of incubation. At 168 h of incubation AV lengths for both ZB × SAB (53.2 mm) and SAB × ZB crosses (54.1 mm) were longer than in embryos from the pure breeds. 7. Results from this study can be put to practical use when determining whether eggs are infertile or fertile and also in investigating the age of early embryonic mortalities.

Morphological development of testes in ostrich (Struthio camelus) embryo.

Although the histological structure of ostrich testis has been studied, very little information is currently available on the embryonic development of this organ. The aim of this study was to determine the sequence of the histological changes in diverse components of the testis in ostrich embryo from embryonic day (E) 20 to E42. The main findings were categorized into four histological features, i.e., development of sex cords, interstitial tissue and rete ducts, and the appearance of defective septa. While the lumen of sex cords, tunica albuginea, capsular rete ducts and Leydig cell precursors appeared at E26, the filum-shaped defective septa were visible at E36. The emersion of the lumen in the primary sex cords and formation of capsular rete ducts in the ostrich embryo is considerably different from that in other birds. However, tunica albuginea and Leydig cell precursors appeared in a similar pattern to those of other birds. An interesting observation was that the primordial germ cell (PGC)-like cells were completely distinct, while the capsular rete ducts were formed by trapping of some Sertoli cell aggregations in the tunica albuginea. This suggests that similar to the primary sex cords, the capsular rete ducts may originate from the Sertoli cell aggregations which had corralled some PGCs. Stereological estimations in the ostrich embryo testis showed the major proportion of testis is occupied by the seminiferous tubules, which is unlike the fowl embryo testis.

Atividade folicular ovariana em avestruz (Struthio camelus) avaliada por ultrassonografia e sua relação com fotoperíodo e postura / Follicular activity in ostriches (Struthio camelus) evaluated by ultrasonography, and its relation with the photoperiod and egg laying

The objective of the experiment was to verify, through ultrasonography, the follicular activity of ostriches in different seasons of the year, correlating them with photoperiod, number of rainy days in each month and egg laying. Eight females were evaluated monthly, during 12 consecutive months in an ostrich farm located in the Center-West of Minas Gerais, Brazil. It was found that the time of the year for egg laying lasts for eight months, from June to January. The egg laying was positively correlated (r = 0.48; P<0.01) with photoperiod. However, there was no correlation between the egg laying and the follicular activity with the amount of rain. Considering the technique adopted, the right antimere offered better ultrasonographic access. The method was efficient for ovary follicular evaluation in the ostrich, offering adequate subsidies for the evaluation of the reproductive activity of the female.

The distribution of gastrin, somatostatin and glucagon immunoreactive (IR) cells in ostrich stomach during the pre- and post-hatching period.

Peptides of the gastrointestinal tract play a significant role in the digestive processes and the development of the body; therefore, it is important to have an understanding of location and distribution of gastrin, somatostatin and glucagon immunoreactive (IR) cells in the stomach mucosa of growing birds. For this purpose, 6 embryos and 37 chicks from an ostrich farm in Latvia were used. Tissue samples were collected from the proventriculus - superficial and deep glandular region and from the ventriculus - side wall and pyloric region. The number of cells was determined in 10 mucosal fields of each tissue sample. For statistical analysis, the one-way anova method was used. Gastrin IR cells regarding the stomach mucosa were found only in the pyloric region. Somatostatin IR cells were most densely located in the pyloric region too, but some cells were also discovered in the mucosa of proventriculus and ventriculus. Glucagon IR cells were found in the epithelium of the deep glands of the proventriculus and only some cells of the superficial glands of the proventriculus, and the ventriculus side wall mucosa. Gastrin and somatostatin IR cells were present in a comparatively large quantity in the ostrich chicks' ventriculus - pyloric region yet not long before hatching. They were located deep in the mucosa of pyloric glands, and their number tended to increase with birds advancing in age.

The composition of egg yolk absorbed by fasted ostrich (Struthio camelus L.) chicks from 1 to 7 days posthatching and for ostrich (Struthio camelus L.) chicks from 1 to 16 days posthatching on a prestarter broiler diet.

This study was performed to obtain information on yolk utilization in fasted and fed ostrich chicks posthatching. The fasted trial lasted for 7 d, whereas the fed trial continued for 16 d. Fasted ostrich chicks showed a decrease of 31.3 g of BW, with yolk weight decreasing by 28.9 g daily after hatching. Yolk weight comprised 28% of 1-d-old ostrich chick BW and decreased to 12% at 7 d of age. Only 44.4% of the fasted ostrich chick yolk was assimilated over the trial period. Crude protein content of the yolk decreased by 13.2 g daily. Fat content increased by 1.77% daily, whereas total yolk fat weight decreased with 8.91 g daily. Slaughter weight of fed ostrich chicks increased, with yolk weight decreasing by 16.3 g daily. Yolk content for fed ostrich chicks was 26% of BW at 2 d of age. Ostrich chicks absorb 30% of yolk over the first 4 d, 67% after 8 d, and only deplete the yolk after 14 d posthatch. Fasted ostrich chicks absorbed the yolk content at a rate of 28.9 g/d, compared with 22.3 g/d over the first 8 d and 16.3 g/d over the 16 d for fed ostrich chicks. The CP content of the yolk decreased by 6.84 g daily in fed ostrich chicks, whereas fat content of the yolk increased by 1.39% daily, although total yolk fat weight decreased by 6.61 g daily. Yolk weight and total CP decreased faster over the first 7 d in the fasted ostrich chicks compared with the fed ostrich chicks, which indicated that the decrease in yolk weight could be attributed to absorption of protein from the yolk. Fat content decreased faster over the first 8 d from the yolk of the fed ostrich chicks compared with that from the yolk of the fasted ostrich chicks, which could indicate that external feed has a positive influence on the absorption of fat from the yolk content.

Heterochronic shift between early organogenesis and migration of cephalic neural crest cells in two divergent evolutionary phenotypes of archosaurs: crocodile and ostrich.

Living archosaurs (crocodiles and birds) represent an intriguing evo-devo model system. Although close in phylogenetic relationship, the two lineages show considerable divergence in trends of phenotypic evolution. The head anatomy of recent crocodilians has changed little in comparison with that of their crocodylomorph ancestors. The head phenotype of the avians (birds), as well as some non-avian theropods, shows numerous evolutionary innovations that differ considerably from the crocodylomorph pattern. Most of the novel head structures, such as features of the craniofacial skeleton, cranial nerves, head muscles, and integument are derived from the same cellular source common to all archosaurs, the cephalic neural crest (CNC). Therefore, other factors must be involved in the developmental disparity of homologous structures in the aforementioned lineages. The present study analyzes the earliest developmental events that are associated with the appearance of the neural crest cells in the two archosaur models: Crocodylus niloticus and Struthio camelus. I found that both models share unique developmental features, the presence of an unpaired, rostrally migrating population of CNC cells, showing that the two are closely related to each other. On the other hand, the crocodile and the ostrich differ substantially in (1) timing, (2) duration, and (3) expression patterns of the CNC. Compared with the crocodile, the CNC cells in the ostrich (1) migrate much later into the embryonic head, (2) but relocate to their terminal positions faster, and (3) take specifically directed migratory routes in the mandibular/oral region and head/trunk-interface regions. I suggest that accelerated relocation of CNC cells combined with delayed head organogenesis may represent important innovative conditions in the developmental evolution of a new archosaur head phenotype.

Primary chondrification foci in the wing basipodium of Struthio camelus with comments on interpretation of autopodial elements in Crocodilia and Aves.

UNLABELLED: The present analysis consists of (1) description of the primary chondrification patterns and their transformation into ossified elements in the basipodium of Struthio camelus; (2) comparison of these with the conditions found in Alligator and Gallus; and (3) interpretation of the autopodial elements of Archaeopteryx. CONCLUSIONS: (1) The existence of five discrete metacarpal condensations in the 16-day embryo of Struthio argues for unique linear patterning process for each, and these are interpreted as digits 2,3,4 originating from metacarpal condensations 2,3,4. Nine chondrogenic foci appear in the Struthio carpus: radiale, centrale, intermedium, ulnare, pseudoulnare, pisiform, distal carpal 2+3, distal carpal 4, and distal carpal 5. It is evident that: (a) the avian "radiale" represents fused chondrogenic foci of the intermedium plus the radiale; (b) a neomorph carpal element, the pseudoulnare (probably avian autapomorphy), replaces the ulnare cartilage in Struthio; (c) the pseudoulnare in Struthio and Hinchliffe's element "X" are not identical to each other. (2) Spatio-temporal conditions of the autopodium are less constrained in the development of Struthio than they are in Gallus. This favors the ostrich model as the more appropriate for interpretation of the autopodial skeleton in the oldest birds and their ancestors. (3) An interpretation of the elements of the hand skeleton of Archaeopteryx is as follows: (a) digits 2,3,4; (b) distal carpal 2+3 (the semilunate); (c) distal carpal 4 (a missing element filling the gap between the semilunate and metacarpal IV); (d) the radiale+intermedium complex (the proximal carpal bone); (e) the pseudoulnare (the proximal carpal bone).

A case of conjoined cephalopagus twinning in an ostrich (Struthio camelus).

Conjoined twinnings have been reported in most domestic animal species and in some avian species. Cases of conjoined twins have not been reported in the ostrich so far. A hybrid Blue neck x African black male ostrich conjoined twinning was born at the end of artificial egg incubation and died spontaneously 24 h after the hatching. It was frozen and sent to the Unit of Veterinary Pathology of the University of Messina for gross examination. The most important gross findings involved the external body and most of the internal organs. On the basis of the duplication, the conjoined twins were classified as a cephalopagus. Radiological features included: development of one head containing a single brain, two spinal cords, deviated vertebral columnae with fusion of the two first cervical vertebrae. In one twin, the synsacrum was absent as well as portions of the vertebral column. Grossly, both twins showed two upper and lower limbs each. The gastro-enteric apparatuses of the twins were not completely developed and fused at different levels. One liver and one heart localized in the centre of the conjoined twins were observed. The authors conclude that the possible causes of the malformation could be related to a genetic factor.

Factors related to shell deaths during artificial incubation of ostrich eggs.

The ostrich industry experiences a high rate of embryonic mortalities during artificial incubation of eggs. Embryonic deaths were studied from data recorded on 37,740 fertile eggs incubated artificially during the 1998-2005 breeding seasons. Roughly 10,000 eggs that sustained embryonic mortalities were classified according to the stage and nature of death, i.e. before 21 days of incubation, after 21 days of incubation, deaths after pipping and rotten eggs. Although infection may have played a role in approximately 1300 rotten eggs, no detailed knowledge of the pathogens involved was available. The remainder of deaths could not be related to pathogens and the deaths were thus generally referred to as non-infectious. The overall level of embryonic mortality in all the eggs studied was 28.5 %. Overall embryonic mortality was affected by incubator, with higher levels (57.0 %) found in eggs incubated in an African Incubator and also in eggs that were transferred between incubators during incubation (38.1%). Overall embryonic mortality also increased in eggs produced by older females. Eggs produced in the autumn had the highest level of embryonic mortality at 53.6 %, whereas eggs produced in the winter had a marginally higher level of embryonic mortalities of 29.2 % compared with eggs produced during summer (27.4 %). Eggs produced by South African (SA) Black males crossed to Zimbabwean Blue females had high levels of embryonic losses of 45.7 %. The embryonic mortality of eggs produced by SA Blacks or Zimbabwean Blue breeding birds subjected to pure breeding was similar at approximately 33-34 %, but embryonic mortality was improved in eggs produced by Zimbabwean Blue males crossed to SA Black females (27 %). Embryonic mortality was increased in eggs that were set directly (32.0 %) or subjected to longer than 6 days of storage (43.5 %). Embryonic mortality was affected by year. The results that were obtained will assist in determining non-infectious factors that have a negative effect on hatching success. Steps can thus be taken to eliminate such factors that may compromise hatching success.