The reporting of clinical signs in laboratory animals: FELASA Working Group Report.

Observing and reporting clinical signs in laboratory animals is necessary for many reasons: the assessment of animal welfare, compliance with the principle of refinement (e.g. humane endpoints), regulatory compliance (e.g. reporting severity) and, importantly, as a scientific outcome, e.g. in animal models of disease or safety studies. Developments in the reporting of clinical signs will enhance the scientific value gained from animal experiments and further address the ethical cost. This paper discusses systematic approaches to the observation and reporting of clinical signs in animals (to be) used for research. Glossaries from public and corporate institutions have been consulted and a reference glossary has been set up, providing terminology to be tailored for institutional or project-specific use. The clinical examination of animals must be carried out by competent and specifically trained staff in a systematic way and repeated at adequate intervals and clinical observations must be registered effectively to allow this information to be used. The development of institutional or project-specific glossaries and the use of handwritten records or automated databases are discussed in detail. Among the users are animal care staff, veterinarians and researchers who will need to agree on a given set of clinical signs to be monitored routinely or as a scientific read-out and to train for the proper application. The paper introduces a long list of clinical signs with scientific terminology, descriptions and explanations as a reference glossary to be published and maintained online as a living document supported by the authors as an editorial committee.

ESLAV/ECLAM/LAVA/EVERI recommendations for the roles, responsibilities and training of the laboratory animal veterinarian and the designated veterinarian under Directive 2010/63/EU.

Directive 2010/63/EU was adopted in September 2010 by the European Parliament and Council, and became effective in January 2013. It replaces Directive 86/609/EEC and introduces new requirements for the protection of animals used for scientific purposes. In particular, it requires that establishments that breed, supply or use laboratory animals have a designated veterinarian (DV) with expertise in laboratory animal medicine, or a suitably qualified expert where more appropriate, charged with advisory duties in relation to the well-being and treatment of the animals. This paper is a report of an ESLAV/ECLAM/LAVA/EVERI working group that provides professional guidance on the role and postgraduate training of laboratory animal veterinarians (LAVs), who may be working as DVs under Directive 2010/63/EU. It is also aimed at advising employers, regulators and other persons working under the Directive on the role of the DV. The role and responsibilities of the DV include the development, implementation and continuing review of an adequate programme for veterinary care at establishments breeding and/or using animals for scientific purposes. The programme should be tailored to the needs of the establishment and based on the Directive's requirements, other legislations, and current guidelines in laboratory animal medicine. Postgraduate laboratory animal veterinary training should include a basic task-specific training module for DVs to complement veterinary competences from graduation, and continuing professional development on the basis of a gap analysis. A tiered approach to further training in laboratory animal veterinary medicine and science offers career development pathways that are mutually beneficial to LAVs and establishments.

Effect of recombinant porcine somatotropin and monoclonal antibody directed to ovine somatotrophic hormone on nitrogen retention and immune parameters in pigs.

Single and combined effects of administration and withdrawal of recombinant porcine somatotropin (rpST) and an enhancing murine antiovine growth hormone monoclonal antibody (OA15) on nitrogen retention, and serological and immunological measurements in pigs were examined in a placebo-controlled experiment. Thirty-six barrows were allotted to one of four treatments: control, rpST, OA15, and OA15+rpST. The trial phase was four balance periods: a preperiod, two periods of treatment, and a postperiod. Weight- and nitrogen gain were higher for the rpST group by 13% (P < .01) and 15% (P < .001), for the OA15 group by 8% (P < .05) and 9% (P < .05), and for the OA15+rpST group by 25% (P < .001) and 20% (P < .001), respectively compared with the control group. During the postperiod, weight gain of the OA15- and the OA15+rpST group was 23% (P < .001) and 22% (P < .001) lower than that of the control group. Nitrogen gain during the postperiod was decreased by 19% (P < .01) for the OA15 group compared with the control group. Single or combined administration of rpST or OA15 did not affect (P > .10) cellular constituents in the blood of all groups during the periods of observation. Animals treated solely with rpST mounted a humoral immune response directed to rpST. This anti-rpST antibody response was, however, decreased (P < .01) in barrows treated with rpST and OA15 simultaneously. Also, a slight anti-rpST antibody response was noticed in barrows solely treated with OA15.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparison of renal toxicity after long-term oral administration of cadmium chloride and cadmium-metallothionein in rats.

There is a clear lack of information on the toxicological risk of dietary intake of cadmium-metallothionein (CdMt). The present study aimed at establishing dose-dependent cadmium (Cd) disposition and to investigate differences in renal toxicity after long-term dietary exposure to CdMt or cadmium chloride (CdCl2) in rats. Male Wistar rats were fed diets containing 0.3, 3, 30, or 90 mg Cd/kg either as CdMt or as CdCl2 for 10 months. In rats fed 30 and 90 mg/kg Cd as CdCl2 the Cd concentrations in intestine, liver, and kidneys were all higher than in rats fed the same doses in the form of CdMt. The kidney/liver Cd concentration ratio was higher with CdMt than with CdCl2. At the lower Cd concentrations (0.3 and 3 mg/kg), no differences in Cd accumulation between CdMt and CdCl2 groups were observed and the kidney/liver Cd ratio was also similar. When based on the amount of CdMt per milligram Cd in the tissue, rats fed CdMt and those fed CdCl2 had a similar relative CdMt concentration in liver and kidney. First signs of renal injury, indicated by an increase of urinary lactate dehydrogenase (LDH) activity, were seen 4 months after exposure to 90 mg/kg Cd as CdCl2. After 8 and 10 months the renal effect of 90 mg/kg Cd as CdCl2 became more pronounced and urinary enzyme activities of LDH, N-acetyl-beta-D-glucosaminidase and alkaline phosphatase were all elevated. The only clinical effect of CdMt at the dose level of 90 mg/kg was a slight increase in urinary gamma-glutamyl transpeptidase activity at 8 and 10 months.(ABSTRACT TRUNCATED AT 250 WORDS)

Growth and differentiation of the gubernaculum testis during testicular descent in the pig: changes in the extracellular matrix, DNA content, and hyaluronidase, beta-glucuronidase, and beta-N-acetylglucosaminidase activities.

The gubernaculum testis is a loose connective tissue organ that plays an essential mechanical role in testicular descent. In the pig, the first phase of descent (transabdominal migration) is brought about by growth of the gubernaculum through the inguinal canal into the scrotum and simultaneous somatic growth of the fetus. During the second phase the gubernaculum condenses, thus allowing the testis to descend into the scrotum. The nature of gubernaculum development (growth and differentiation) was investigated with respect to cell proliferation, extracellular matrix (ECM) composition, and acid hydrolases. Deoxyribonucleic acid (DNA) was used as a measure of cell number and hydroxyproline (HYP) was an estimate of interstitial collagen. The first phase of gubernaculum development was characterized by rapid cell proliferation and concomitant synthesis of sulphated glycosaminoglycans (S-GAG), hyaluronic acid (HA) and collagen. During the second phase cell proliferation ceased and DNA concentration increased. The amount of S-GAG remained closely related to the amount of DNA while HYP increased further. However, HA decreased during the second phase and thus HA metabolism seems to play a crucial role in biphasic development of the gubernaculum. The activities of the enzymes that are needed for biodegradation of HA (hyaluronidase, beta-glucuronidase and beta-N-acetylglucosaminidase) were measured in gubernaculum homogenate from animals during the first and second phase of testicular descent. These enzymes were detectable in gubernaculum and rose during the second phase of testicular descent. It was concluded that a very distinct dichotomy in the nature of gubernaculum development during the first and second phase could be discerned with respect to cell proliferation rate and ECM synthesis and degradation. These observations provide useful tools for future in vivo and in vitro investigations into the process and regulation of testicular descent.

Familial male pseudohermaphroditism and testicular descent in the racoon dog (Nyctereutes).

Sexual differentiation was investigated in familial male pseudohermaphroditism in Nyctereutes procyonoides (Canidae). In intersex males, development of external genital organs and prostate glandular tissue was severely disturbed; Wolffian (mesonephric) duct derivatives developed prepubertally but were absent in some adults. Müllerian (paramesonephric) duct regression was complete. Testicular descent was undisturbed. Male/female sex differences in plasma testosterone, 5 alpha-dihydrotestosterone, and luteinizing hormone concentrations were present. Intersex plasma hormone concentrations were within the normal male range. The concentration of androgen receptors in pubic skin was similar in male, female, and intersex animals and no significant differences in affinity for the ligand were detected. It was concluded that in intersex animals androgen-dependent virilisation was deficient despite the presence of androgens and androgen receptors and that this condition had not affected gubernaculum development and testicular descent.

In vitro model of the first phase of testicular descent: identification of a low molecular weight factor from fetal testis involved in proliferation of gubernaculum testis cells and distinct from specified polypeptide growth factors and fetal gonadal hormones.

The gubernaculum testis is the connective tissue organ that causes the testis to descend. How the process of testicular descent is regulated is not fully understood. Current hypotheses postulate that a nonandrogenic fetal testicular factor controls the first phase of descent, that is characterized by growth of the gubernaculum and transabdominal migration of the testis. When gonadal extracts from fetuses with ages corresponding to the first phase of testicular descent (50, 60, and 75 days) were tested on gubernacular cells, the growth stimulatory effect of testicular extracts exceeded the effect of both ovarian extract and fetal calf serum. Gonadal extracts from 80-, 90-, and 100-day-old fetuses showed only a minor sex difference. No sex difference or age-dependent changes were detected when fetal gonadal extracts were tested on murine 3T3 cells. Polypeptide growth factors (epidermal growth factor, insulin, fibroblast growth factor, platelet-derived growth factor, and transforming growth factor-beta) were tested for growth stimulatory activity and had only minor effects on gubernaculum cells. Fetal testicular hormones (anti-Müllerian hormone, inhibin, and androgenic steroids) did not induce initiation of DNA synthesis at concentrations that are highly bioactive in typical target systems. When testicular samples were dialyzed, the high mol wt fraction (greater than 3500) had lower growth stimulatory activity in gubernaculum cells, but not 3T3 cells. Bioactivity of ovarian extracts and fetal calf serum was not diminished after dialysis. The low mol wt fraction (less than 3500) of testicular extract was distinctly stimulatory to gubernaculum cells but not 3T3 cells, and the low mol wt fraction of ovarian extracts did not stimulate growth in either cell type. It was concluded that the fetal porcine testis during the first phase of testicular descent contains low mol wt factor(s) to which gubernaculum cells and not 3T3 cells are responsive. The bioactive fraction probably contains the factor(s) that initiate testicular descent. We suggest the name descendin for this new activity.

Testicular descent: androgen receptors in cultured porcine gubernaculum cells.

We have adapted an oil microcentrifuge assay for evaluating the binding of tritiated methyltrienolone ([3H]R1881) in metabolically active primary cultured porcine gubernaculum fibroblasts. Almost complete separation of cells with the bound hormone and medium with the unbound hormone can be achieved within 30 s of centrifugation at 10,000 g. Specific [3H]R1881 binding was found in four different gubernaculum fibroblast cultures (9,000-42,000 sites/cell) and MCF-7 human breast cancer cells (34,000 sites/cell) which were used as controls. Binding was inhibited by radio-inert R1881 and 5 alpha-dihydrotestosterone but not by estradiol and cortisol. The results indicate that androgen receptors are present in fibroblasts from the gubernaculum during both the first and second phase of testicular descent and that they may play a role in the regulation of that process.

Morphological development of the thyroid gland and serum T4-concentration in the intact and decapitated pig fetus.

The morphological and functional development of the fetal pig thyroid gland between 50 and 110 days post coitum have been examined in the normal pig fetus and after fetal decapitation at 42 days postcoitum. Body length and body weight developed at the same rate, comparing control and decapitated animals. Thyroid gland weight increased between 50 days and 110 days from 1.3 +/- 0.5 mg (SD) to 130.0 +/- 35.0 mg in control fetuses and from 0.9 +/- 0.2 mg to 113.4 +/- 23.0 mg in decapitated fetuses. A number of histomorphometrical parameters in thyroid tissue were measured. No significant differences in follicular epithelial height were observed between decapitated and control animals at 75 and 110 days. In control animals follicles increased both in size and number. In decapitated animals the follicles increased strikingly in number, but only slightly in size. Although colloid was formed in glands of decapitated animals, it was much less than in control fetuses. The gland of decapitated animals of 110 days resembled histologically the gland of much younger control animals (60 to 75 days). Gland development after fetal decapitation at 42 days may represent autonomous development when Thyroid Stimulating Hormone (TSH) is depleted. Serum thyroxine concentration (T4) was determined by radioimmunoassay and increased in control animals from 0.06 +/- 0.01 micrograms/100 ml to 4.18 +/- 0.87 micrograms/100 ml at 110 days, the greatest rate of increase being observed between 64 and 90 days. In decapitated fetuses serum T4 remained very low, namely less than 0.20 micrograms/100 ml. It is very unlikely that any significant transfer of T4 from mother to fetus or from one fetus to another occurred. Both the rise in serum T4 and the enlargement of the follicles may be TSH dependent events in fetal pig thyroid gland development, whereby the sudden rise is serum T4 precedes the greatest rate of increase in follicle size.