Clinical and anatomic pathology effects of serial blood sampling in rat toxicology studies, using conventional or microsampling methods.

UNLABELLED: As a general practice in rodent toxicology studies, satellite animals are used for toxicokinetic determinations, because of the potential impact of serial blood sampling on toxicological endpoints. Besides toxicological and toxicokinetic determinations, blood samples obtained longitudinally from a same animal may be used for the assessment of additional parameters (e.g., metabolism, pharmacodynamics, safety biomarkers) to maximize information that can be deduced from rodents. We investigated whether removal of up to 6 × 200 µL of blood over 24h can be applied in GLP rat toxicology studies without affecting the scientific outcome. METHODS: 8 week-old female rats (200-300 g) were dosed for up to 1 month with a standard vehicle and subjected or not (controls) to serial blood sampling for sham toxicokinetic/ancillary determinations, using miniaturized methods allowing collection of 6 × 50, 100 or 200 µL over 24h. In-life endpoints, clinical pathology parameters and histopathology of organs sensitive to blood volume reduction were evaluated at several time points after completion of sampling. RESULTS: In sampled rats, minimal and reversible changes in red blood cell mass (maximally 15%) and subtle variations in liver enzymes, fibrinogen and neutrophils were not associated with any organ/tissue macroscopic or microscopic correlate. CONCLUSION: Serial blood sampling (up to 6 × 200 µL over 24h) is compatible with the assessment of standard toxicity endpoints in adult rats.

Research Note: Irritating flashing light or poultry-friendly lighting-are flicker frequencies of LED luminaires a potential stress factor in the husbandry of male fattening turkeys?

Conventional fluorescent tubes are increasingly being replaced with innovative light-emitting diodes (LEDs) for lighting poultry houses. However, little is known about whether the flicker frequencies of LED luminaires are potential stressors in poultry husbandry. The term "light flicker" describes the fluctuations in the brightness of an electrically operated light source caused by the design and/or control of the light source. In this context, the critical flicker frequency (CFF) characterizes the frequency at which a sequence of light flashes is perceived as continuous light. It is known that CFF in birds is higher than that in humans and that light flicker can affect behavioral patterns and stress levels in several bird species. As there is a lack of knowledge about the impact of flicker frequency on fattening turkeys, this study aimed to investigate the effects of flicker frequency on the behavior, performance, and stress response in male turkeys. In 3 trials, a total of 1,646 male day-old turkey poults of the strain B.U.T. 6 with intact beaks were reared for 20 wk in 12 barn compartments of 18 m² each. Each barn compartment was illuminated using 2 full-spectrum LED lamps. Flicker frequencies of 165 Hz, 500 Hz, and 16 kHz were set in the luminaires to illuminate the compartments. Analyses of feather corticosterone concentration were performed on fully grown third-generation primaries (P 3) of 5 turkeys from each compartment. No significant differences were found in the development of live weight, feed consumption, or prevalence of injured or killed turkeys by conspecifics reared under the above flicker frequencies. The flicker frequencies also did not significantly influence feather corticosterone concentrations in the primaries of the turkeys. In conclusion, the present results indicate that flicker frequencies of 165 Hz or higher have no detrimental effect on growth performance, injurious pecking, or endocrine stress response in male turkeys and, thus, may be suitable for use as animal-friendly lighting.

Prevalence and severity of foot pad alterations in German turkey poults during the early rearing phase.

In the previously performed field study from 2007 to 2009, it became evident that foot pad alterations were already commonly found in turkeys at the age of 6 wk. At this early age, 45% of the clinically examined birds were diagnosed with epithelial necrosis. Therefore, it became important to specifically analyze the situation during the early rearing phase. The present study reflects the prevalence and severity of foot pad alterations of turkey poults up to the age of 35 d (5 wk), starting as early as the age of 3 d. From 24 turkey farms throughout Germany, in general 5,531 turkeys [3,131 male and 2,400 female] of the British United Turkeys 6 strain from 46 flocks, were examined to that effect. Prevalence and severity increased within the duration of stay in the stable, and the prevalence was higher (P < 0.001) during the second visit between d 22 to 35 (factor: 0.94). Therefore, 27.3% (d 3 to 5; male/female: 39.1/25.0%) and 63.3% (d 22 to 35: 61.3/65.7%) of the examined poults had alterations of the foot pads, such as hyperkeratosis (d 3 to 5: 20.4/14.2%; d 22 to 35: 17.6/17.1%), high-grade hyperkeratosis with adhesive dirt (d 3 to 5: 8.7/10.7%; d 22 to 35: 29.2/39.3%), and epithelial necrosis (d 3 to 5: 0.1/0.1%; d 22 to 35: 14.6/9.3%). Female poults showed a higher risk (P < 0.001) of developing food pad alterations (factor: 0.76) than male poults. Male poults developed a higher percentage of epithelial necrosis than hens shortly before relocation. A higher stocking density during the very early rearing phase (d 3 to 5) led to a worse foot pad health status (P < 0.001). Because even mild alterations in the foot pad condition can be indicators for suboptimal design of the rearing environment and are to be seen as a pre-state for severe cases of foot pad dermatitis, it is important to set the main focus on the early rearing phase.

Examinations on the prevalence of footpad lesions and breast skin lesions in British United Turkeys Big 6 fattening turkeys in Germany. Part I: prevalence of footpad lesions.

The present study demonstrates the prevalence of footpad lesions in turkeys and their level of expression in both live birds and in carcasses. In clinical investigations of 11,860 turkeys (5,740 males, 6,120 females) of the strain British United Turkeys Big 6, individuals of all the observed flocks showed alterations to the plantar skin. In general, the degree and severity of skin alterations were higher in the 16th week of life than in the 6th or 11th week of life. Epithelial necrosis, however, was already apparent in about 45% of individuals in the 6th week of life. At this point, deep lesions on the footpad surface were still rare findings. In the 11th week of life, such skin changes were apparent in 14.7% of males and 25.7% of hens. In general, the feet of male turkeys were less affected by footpad damage than those of females in the subsequent fattening period. In hens, the prevalence of lesions of the footpads at wk 16 of age was 60.0%, almost twice as high as in males of the same age group (33.8%). A comparison of the findings from the right and left feet showed that the state of the 2 extremities was correlated (Spearman correlation: r = 0.830). During the visual inspection of 16,200 slaughtered turkeys (7,800 males, 8,400 hens), almost all carcasses studied showed alterations to the footpad skin. Only 2.1% of males and 0.6% of females were without lesions. The most frequently observed lesions measured up to 2 cm in diameter (males: 59.2%; hens: 57.7%). Pronounced skin lesions of the footpad (males: 21.1%; females: 29.5%) and necrosis of the superficial epithelium (males: 17.5%; females: 12.1%) were less common. Foot abscesses were diagnosed only sporadically (males: 0.1%; females: 0.1%).

Examinations on the prevalence of footpad lesions and breast skin lesions in B.U.T. Big 6 fattening turkeys in Germany. Part II: Prevalence of breast skin lesions (breast buttons and breast blisters).

This project involves the prevalence of breast skin alterations (breast buttons, blisters, purulent bursitis) in male and female fattened turkeys (British United Turkeys Big 6) from conventional intensive farming. In this case, 24 turkey farms across Germany and 11,860 turkeys (5,740 toms, 6,120 hens) were clinically examined, and 16,200 birds (8,400 hens, 7,800 toms) were evaluated on slaughter lines at 7 turkey abattoirs. Each turkey farm included examination of at least 3 consecutive fattening cycles. Especially in the meat examinations, breast skin alterations were a frequent discovery, particularly in the form of breast buttons. The turkey toms exhibited a prevalence of 27.15%, and the toms were affected significantly more frequently (P < 0.001) by these variations than the turkey hens (7.77%). It is possible that the higher BW of the male specimens leads to increased lying times and is therefore connected with more severe strain on the breast region. The increased fattening period of the turkey toms (slaughtering at wk 21 to 22 of life) vs. the female specimens (slaughtering at wk 15 to 17 of life) must be discussed as a cause. Breast blisters and bursitis sternalis were discovered with a prevalence of 7.36 and 1.24% in toms and more seldom in hens, at 0.30% and 0.15%. Large differences in prevalence were partially recognizable between individual farms as well as between the 3 fattening cycles on a farm. Breast buttons and breast blisters are pathological skin variations for which the etiology is multifactorial. Clear correlations between the prevalence of breast skin alterations and the manner of rearing the birds were not able to be determined. An improvement in this situation, which is considered very problematic from the perspective of bird protection and economics, may be achieved only via cooperation to achieve optimized rearing methods, management, and breeding practices.

Research Note: It's not just stress-fecal contamination of plumage may affect feather corticosterone concentration.

The feather corticosterone concentration (fCORT) is increasingly used to assess long-term stress in birds as this indicator provides the potential to retrospectively evaluate the adrenocortical activity of a bird during the growth period of a feather over several weeks in one sample. However, there is still a lack of knowledge concerning external factors which can potentially influence fCORT in birds. The aim of the present study was to determine whether fCORT in laying hens is affected by previous fecal contamination of the plumage. Fully grown primaries 5 (P5s) of laying hens (n = 40) were used for the study. To test the effects of fecal contamination on fCORT, freshly defecated droppings from laying hens were collected and mixed manually. In order to simulate practical and at the same time standardized contamination, the upper surface of the previously determined middle third of the right P5 of each hen then was evenly coated with the paste prepared from fresh feces. The treated feathers were stored for 24 h protected from light at room temperature under a film cover to prevent evaporation. Thereafter, the applied layer of excreta was removed manually. Contralateral P5s of the same individuals were stored under identical conditions and served as controls. Both treatment and control feathers were washed in an aqueous soap solution, rinsed several times in pure water and air-dried subsequently. After pulverization and methanolic extraction, fCORT was analyzed by ELISA. The mean fCORT of treatment P5s (reference 'feather length': 12.88 ± 3.16 pg/mm; reference 'feather weight': 7.81 pg/mg ± 1.86 pg/mg) were significantly higher (P < 0.001) than those of control P5s (reference 'feather length': 9.76 ± 2.42 pg/mm; reference 'feather weight': 5.93 ± 1.44 pg/mg). Our results show that previous contamination with excreta can increase fCORT, which was detectable even after a washing procedure prior to analysis. In conclusion, fecal contamination of feathers is a significant influencing factor that has to be considered when applying fCORT measurements.

Research Note: A sip of stress. Effects of corticosterone supplementation in drinking water on feather corticosterone concentrations in layer pullets.

The measurement of feather corticosterone concentrations (fCORT) is a comparatively new method for the evaluation of stress in wild and captive birds and may be a useful indicator in animal welfare research. The aim of this study was to assess the suitability of fCORT as an indicator of stress, and for this purpose a prolonged stress period was experimentally simulated by oral intake of corticosterone via drinking water and corticosterone concentrations were analyzed in feathers grown during this period. Layer pullets of both a control group (n = 20) and a CORT group (n = 20) were offered drinking water ad libitum throughout the entire experimental phase. The drinking water of the CORT group was supplemented with corticosterone at a concentration of 20 mg/l from the 64th to the 114th day of life. The vaned parts of the primaries 5 (P5s) were clipped on d 114 and fCORT was analyzed by ELISA after extraction. Body weights increased from day 64 until d 114 in both groups, however, at the end of the experiment, mean body mass in the CORT group was significantly lower than in the control group (P < 0.001). Pullets of the CORT group also showed shorter and lighter P5s as well as a retarded molt of the primaries. The supplementation of drinking water with corticosterone increased the average fCORT in the P5s of the CORT pullets compared with the control group (median: 110.3 pg/mm [interquartile range (IQR): 47.2] vs. 10.0 pg/mm [IQR: 2.5], P < 0.001). The results show that experimentally increased systemic corticosterone concentrations over a period of seven weeks in layer pullets are reflected in corticosterone concentrations of feathers grown during that time. This indicates that the measurement of fCORT may be a useful and minimally invasive tool for the evaluation of long-term stress in chicken and provides the basis for further investigations on its use in animal welfare research.

The dark side of white hair? Artificial light irradiation reduces cortisol concentrations in white but not black hairs of cattle and pigs.

Analysing hair cortisol concentrations (HCCs) is a minimally invasive way to retrospectively assess long-term stress, and its application in studies of animal welfare and stress has attracted considerable interest. However, not only stress-related effects but also hair-specific characteristics and external influences can affect HCCs and interfere with the interpretation of results. Thus, it was the aim of this study to investigate the impact of daylight and UV irradiation on cortisol concentrations in the hairs of pigs and cattle. We also examined whether a potential irradiation effect on HCCs depended on the colour of the hair. For this purpose, black and white hair samples from 18 Saddleback pigs and 18 Holstein Friesian cattle were exposed to artificial light (both visible and UV) and compared with control hair samples from the same animals kept in the dark. Exposure to artificial light significantly decreased HCCs in both pigs (P < 0.05) and cattle (P < 0.001), and hair colour had an influence on HCCs, with black hair showing higher cortisol levels than white hair (cattle: P < 0.001, pigs: P = 0.07). The interaction between light exposure and hair colour was significant in both pigs (P < 0.01) and cattle (P < 0.001), so light exposure reduced HCCs in porcine white hair but not black hair. In cattle, light-exposed white hair exhibited lower hair cortisol levels than control white hair or black hair. These results demonstrate that artificial light irradiation degrades hair cortisol or favours its elimination by structural changes of the hair matrix. However, this effect was only detectable in white hair, indicating that the melanin pigments in black hair absorbed radiation, thereby reducing the effects of photodegradation. Compared with other known influencing factors on HCCs, such as age and body region, the influence of light irradiation was relatively low in this in vitro experiment. However, further studies should investigate this influence under real-life animal conditions, such as outdoor and indoor housing.

Research Note: Injurious pecking in fattening turkeys (Meleagris gallopavo f. dom.)-video analyses of triggering factors and behavioral sequences in small flocks of male turkeys.

Injurious pecking is one of the main welfare issues in fattening turkey husbandry. Birds pecked by conspecifics can suffer from serious injuries that may even lead to the death of the victimized animals or require their culling. In the present study, the behavior of male turkeys was documented day and night using video recordings throughout the entire fattening period of 19 wk. Thus, when a turkey was found injured or dead in the barn, a retrospective analysis of video recordings was carried out to clarify the circumstances surrounding the death or injuries of the bird in the present study. In 3 fattening trials with a total of 1,620 male turkeys with intact beaks, 41 birds (2.5%) were found either seriously injured (n = 24) or dead (n = 17) in the barn as a result from conspecific pecking. The detailed evaluation of the video recordings showed that the onset of injurious pecking was mainly in the afternoon after the last daily visual controls of animals and that one third of the dead found animals died a natural death without any pecking incidents. The duration of injurious pecking directed against a certain conspecific was on average 794 min, ranging from 84 min to 1,437 min, that is up to almost an entire day. Pecking activities lasting more than 10 h were interrupted from the light regime between dusk and dawn but continued with the onset of light in the morning. Duration of injurious pecking events decreased with age (P = 0.031). If the victims laid down or were impaired in their mobility, they would be surrounded by up to 10 turkeys that would still be forcefully pecking at the occipital and neck area of the badly injured or moribund birds.

Investigation of the prevalence and severity of foot pad dermatitis at the slaughterhouse in fattening turkeys reared in organic production systems in Germany.

The present study shows the prevalence and severity of foot pad dermatitis (FPD) in turkeys reared in organic production systems assessed at slaughterhouses in Germany. The investigations of altogether 1,860 turkeys of the strains Kelly Broad Breasted Bronze (Kelly BBB; 540 toms, 540 hens) and British United Turkeys (B.U.T.) 6 and the Test Product 7 (TP 7; 780 hens) showed that 97.7% of the examined turkeys were diagnosed with different degrees of FPD. Only 4.6% of the toms and 1.3% of the hens had feet without lesions. Most frequent were necrotic lesions measuring up to 2 cm in diameter (64.3% of all turkeys). Extensive necrotic lesions of the foot pads (toms: 29.8%; hens: 12.4%) and necrosis of superficial scales (toms: 11.3%; hens: 7.6%) were less frequent. Plantar abscesses were rare findings (1.9%). In general, the feet of the Kelly BBB hens were more affected by foot pad lesions than those of the Kelly BBB toms. There were significant differences between the investigated flocks concerning the occurrence of foot pad lesions. The aim in rearing turkeys must be the reduction of FPD.

[Assessment of animal welfare aspects in extreme breeds of rabbits, poultry and pigeons]. / Zur Beurteilung von Tierschutzaspekten bei Extremzuchten von Rassekaninchen, Rassegeflügel und Rassetauben.

In a review based on literature searches animal welfare related characteristics in extreme breed types of rabbits, poultry and pigeons are summarized. Animal welfare aspects are assessed and measures for improvement are described.

[Assessment of animal welfare aspects in extreme breeds of pet animals]. / Zur Beurteilung von Tierschutzaspekten bei Extremzuchten von kleinen Heimtieren.

In a review based on literature searches animal welfare related characteristics in extreme breed types of small mammals, ornamental birds, reptiles and ornamental fish are summarized. Animal welfare aspects are assessed and measures for improvement are described.

[3D-X-ray microcomputer tomography and optical coherence tomography as methods for the localization of the blastoderm in the newly laid unincubated chicken egg]. / 3D-Röntgen-Mikrocomputertomographie und Optische Kohärenztomographie als Methoden zur Lagebestimmung des Blastoderms im unbebrüteten Hühnerei.

The routine culling of the male offspring of hybrid layer type chickens is met with increasing public disapproval for both ethical and legal reasons. Until now practice-oriented methods for reliable sex diagnosis prior to hatch could not be developed. Molecular genetical analysis of blastodermic cells can be used for sex determination in unincubated eggs; however, knowledge of the precise localization of the germinal disc is crucial for the extraction of a carefully directed cell biopsy. In principle, 3D-X-ray micro computed tomography (3D-CT) has been proven a suitable method to localize the germinal disk in the unincubated egg without damaging the egg shell. No negative effects on embryogenesis and hatching rate of irradiated hatching eggs were established. The pictorial representation of the germinal disk using optical coherence tomography (OCT) failed in the unopened egg. The egg shell formed an impenetrable barrier for the currently available measuring method which utilized near infrared (NIR) wavelength regions. After opening the egg shell, the germinal disk could be visualized without any difficulties. In conclusion, technical possibilities for localization of the germinal disk in the unincubated egg already exist, but regarding technical parameters, the procedures have to be adapted to the specific purpose.

Current approaches to avoid the culling of day-old male chicks in the layer industry, with special reference to spectroscopic methods.

The negative correlation between fattening and laying performance prevents breeding improvement in both laying performance and meat yield. Therefore, specialized chicken lines have been bred in order to achieve either an efficient production of high-quality eggs or high growth rates. As a result, day-old male chicks are culled in the layer hatchery, which poses animal welfare and ethical problems. Breeding companies, scientific groups, and hatcheries are attempting to resolve this issue, with a common aim to find feasible alternatives for the routine killing of male layer chicks. Some approaches aim to influence the sex ratio, while others target at the economically feasible use of the male layer offspring, such as the fattening of "laying hen brothers" or crossbreedings of layers and broilers to create "dual-purpose chickens." Another approach is the sex determination prior to hatch. One of the prerequisites of in ovo sex determination is a practicable method that can be used in industry. The analysis needs to be rapid, cost-efficient, and highly precise; in addition, negative impacts on hatching rate, animal health, and/or performance parameters should be limited. Furthermore, sex determination should be performed before the sensory nervous system's response of the chick embryo to certain or potentially harmful stimuli is developed, which according to current knowledge is before the d 7 of incubation.

Vitamin A requirements of alipochromatic ('recessive-white') and coloured canaries (Serinus canaria) during the breeding season.

Six pairs of alipochromatic ('recessive-white') canaries (Serinus canaria) and six pairs of coloured canaries were kept through a complete breeding cycle while being fed a diet providing 12,000 iu vitamin A/kg. The eggs of three pairs (one recessive-white and two coloured) were all unfertilised and there were only 23 hatchlings (14 recessive-white and nine coloured), of which 14 (10 recessive-white and four coloured) were alive after the first moult. However, there was no clinical, biochemical or pathological evidence that the recessive-white canaries were suffering from vitamin A deficiency or that the coloured canaries were suffering from vitamin A toxicity, suggesting that the diet met the vitamin A requirements of both groups.

UV reflection properties of plumage and skin of domesticated turkeys (Meleagris gallopavo f. dom.) as revealed by UV photography.

Reflection and fluorescence properties of feathered and non-feathered body regions of white- and bronze-colored fattening turkeys of various ages were examined by ultraviolet (UV) photography. The examinations were carried out on 20 white-feathered fattening turkeys (B.U.T. 6; 10 males, 10 females) and 20 bronze-feathered fattening turkeys (Grelier 708; 10 males, 10 females) over a period of 21 weeks. The turkeys were photographed once a wk under long-wave UV (UVA) radiation illumination (λ = 344-407 nm) using a digital camera. A bandpass filter was used for UV reflectography to filter out the visible components of the used light source. A longpass filter was used for UV fluorescence photography to avoid blurring in the image due to chromatic aberration as a result of UV illumination. We found that natal down feathers of white-feathered turkeys showed an intense yellowish-green fluorescence under UVA light. UVA fluorescence also was shown by the natal downs of the slightly melanized plumage areas of bronze turkeys. Vaned feathers of white fattening turkeys reflected UVA radiation. Freshly molted feathers were optically distinguishable from the previous feather generation due to their more intense UVA reflection. In bronze turkeys, both the bright end seams of the dark pennaceous feathers and rectrices and the bright banding of primary and secondary remiges reflected UVA radiation. Intense UVA fluorescence was recognizable in day-old chicks of both color variants on the scutellate scales of the legs and toes. In male turkeys of both color variants, UVA-reflecting parts were recognizable with increasing age on the featherless head region. The UVA-fluorescent and UVA-reflective characteristics of the plumage of fattening turkeys were closely related to the plumage color, the feather type, the molting state, and the age of the birds. Further research is needed regarding the UVA-reflecting properties of the turkey plumage and the effects of full-spectrum illumination, including the UVA spectrum, on the behavior and health of fattening turkeys.

[Investigations on the influence of selected compulsory measures on clinically relevant haematological and blood-chemical parameters of racing pigeons (Columba livia f. dom.)]. / Untersuchungen zum Einfluss ausgewählter Zwangsmassnahmen auf hämatologische und blutchemische Parameter von Brieftauben (Columba livia f. dom.).

In the presented study the influence of stress and environmental factors on selected haematological and blood-chemical parameters in racing pigeons was examined. Blood was taken at three defined days and haematological as well as blood-chemical parameters of clinical relevance were determined. In comparison to reference values published for pigeons, the majority of the values obtained in this study were within physiological borders. The daily handling of the pigeons did not have any significant effect on the examined parameters. Also the heterophile/lymphocyte ratio did not show any changes characteristic of a stress reaction. In contrast, after change of the housing dies with pigeons originating normally from a flock, the first blood sampling should be performed after a 4 or 5-day lasting period of acclimatisation to the the individual housing conditions.

[Fast Response Training in Youth Soccer Players]. / Hochreaktives Training bei jugendlichen Fußballspielern.

BACKGROUND: Injuries of the knee and ankle joint are a serious ongoing problem in soccer. Although there is a variety of prevention programmes, a significant reduction of severe knee injuries has not been observed. Therefore, current strategies for diagnostics and training need to be re-evaluated. OBJECTIVE: Our study aimed to test a totally new intervention strategy (fast response training on the SpeedCourt). The efficiency of this method was evaluated with youth soccer athletes. MATERIALS AND METHODS: 24 young male class A athletes (mean age 18.0 ±â€Š0.7 years) of a local soccer team underwent SpeedCourt training for 7 weeks (1 training session per week = TS) during the regular season of competition. TS contained life-kinetic elements (time of exercise: 15 - 30 seconds, break: 2 minutes) and included a warm-up phase (15 minutes) and fast response training on the SpeedCourt (30 minutes). The players were tested on the SpeedCourt with different tests (Counter Movement Jump (CMJ), shuttle run, tapping, 10-second sprint) before and after the training programme. RESULTS: The univariate single-factor analysis of variance showed significant improvements in all test parameters (η(2)> 0.10). The range varied between η(2) = 0.106 (time of ground contact right leg) und η(2) = 0.730 (reaction time right leg). We did not find any relevant correlations between the tests and parameters. The number of injury-related accidents involving the lower extremities was reduced by about 50 % during defined periods of time. CONCLUSION: Our data revealed that training with fast responses at the SpeedCourt system clearly improved speed and speed strength performance of young soccer athletes, which is remarkable given the low intensity of influences (one TS per week). The increase in performance was accompanied by a significant reduction of the injury rate.

The corpus fibulae in sheep (Ovis ammon f. aries)--ontogeny, persistence, size and shape from the fetal period to adulthood.

Osteological characteristics are often used to identify animal species. In this regard it is believed that the corpus fibulae of the sheep is represented by a tight cord of connective tissue--the so-called "ligamentum fibulare". The results of the present morphometric study indicate, however, that in 67% of the fetuses, 61% of the lambs or 48% of the adult sheep out of the 160 examined animals, the corpus fibulae is represented by a pin-shaped flattened piece of bone. The length of the corpus fibulae increases with age and varies greatly between individuals but in lambs and adult animals its centre is located close to the border between the proximal and intermediate third of the tibia (distal type) or closely attached to the caput fibulae by connective tissue fibres (proximal type). The cartilaginous fetal anlage is either transformed to bone or catabolised during fetal life. Desmal ossification should play an important role in the growth of the corpus fibulae since no cartilage could be detected in juvenile and adult sheep. Since there are many similarities in the expression of the corpus fibulae between sheep, goat and fallow deer, this osteological characteristic may not be used for a rapid specific and simple animal species identification procedure.

Liver-associated toxicity of the HSV-tk/GCV approach and adenoviral vectors.

Intravenous injection of immunodeficient mice with adenoviral vectors carrying the HSV-tk gene led to preferential transduction of liver tissue. Subsequent application of ganciclovir (GCV) resulted in extremely high toxicity with negligible survival rates. This toxicity was only seen when GCV was applied in addition to the adenoviral vector and not if combined with the Ad-beta gal control vector. This indicates a specific Ad-tk/GCV-related toxicity. Low survival rates were seen not only after intravenous administration but also after injection of the vectors into the portal vein, the liver tissue and liver tumors, but not during the treatment of subcutaneous tumors. This, together with extensive signs of liver degeneration occurring as early as one day after the initiation of GCV treatment, strongly suggests a specific liver-associated toxicity. Severe toxicity was observed when animals received GCV treatment as late as 7 weeks after vector administration. Moreover, in vitro survival rates of resting primary hepatocytes treated with Ad-tk and GCV were very low. We therefore suppose a mechanism of toxicity of phosphorylated GCV which is independent from cellular proliferation. Since our results indicate that the Ad-HSV-tk/GCV approach is toxic for the resting liver, additional safety features such as tumor-restricted transgene expression should be included in adenoviral vectors.