There is widespread concern about the quality, reproducibility and translatability of studies involving research animals. Although there are a number of reporting guidelines available, there is very little overarching guidance on how to plan animal experiments, despite the fact that this is the logical place to start ensuring quality. In this paper we present the PREPARE guidelines: Planning Research and Experimental Procedures on Animals: Recommendations for Excellence. PREPARE covers the three broad areas which determine the quality of the preparation for animal studies: formulation, dialogue between scientists and the animal facility, and quality control of the various components in the study. Some topics overlap and the PREPARE checklist should be adapted to suit specific needs, for example in field research. Advice on use of the checklist is available on the Norecopa website, with links to guidelines for animal research and testing, at https://norecopa.no/PREPARE.

A method is described for blood collection from the lateral saphenous vein. This enables rapid sampling, which if necessary can be repeated from the same site without a need for new puncture wounds. The method is a humane and practical alternative to cardiac and retro-orbital puncture, in species where venepuncture has traditionally been regarded as problematic.

SummaryThe aim of this paper is to provide background material necessary for the development of international guidelines for the health and welfare monitoring of fish used in research. It provides an overview of present guidelines and discusses why more detailed and speciesspecific guidelines are needed. A major issue within fish research is to document the situation today and point out areas where improvements are needed.

There are important scientific, legal and ethical reasons for optimising the quality of animal research and testing. Concerns about the reproducibility and translatability of animal studies are now being voiced not only by those opposed to animal use, but also by scientists themselves. Many of the attempts to improve reproducibility have, until recently, focused on ways in which the reporting of animal studies can be improved. Many reporting guidelines have been written. Better reporting cannot, however, improve the quality of work that has already been carried out-for this purpose better planning is required. Planning animal studies should involve close collaboration with the animal facility where the work is to be performed, from as early a stage as possible. In this way, weaknesses in the protocol will be detected and changes can be made before it is too late. Improved planning must focus on more than the "mathematical" elements of experimental design such as randomisation, blinding and statistical methods. This should include focus on practical details such as the standard of the facility, any need for education and training, and all the factors which can improve animal welfare. The PREPARE (Planning Research and Experimental Procedures on Animals: Recommendations for Excellence) checklist was developed to help scientists be more aware of all the issues which may affect their experiments. The checklist is supported by comprehensive webpages containing more information, with links to the latest resources that have been developed for each topic on the list.

The severity classification of procedures using animals is an important tool to help focus the implementation of refinement and to assist in reporting the application of the 3Rs (replacement, reduction and refinement). The recently revised Directive that regulates animal research and testing within the European Union requires Member States to ensure that all procedures are classified as ‘non-recovery’, ‘mild’, ‘moderate’ or ‘severe’, using assignment criteria set out by the European Commission (EC). However, these are focused upon terrestrial species, so are of limited relevance to fish users. A Working Group set up by the Norwegian Consensus-Platform for the 3Rs (Norecopa) has produced guidance on the classification of severity in scientific procedures involving fish, including examples of ‘subthreshold’, ‘mild’, ‘moderate’, ‘severe’ and ‘upper threshold’ procedures. The aims are to complement the EC guidelines and help to ensure that suffering in fish is effectively predicted and minimized. Norecopa has established a website () where more information on severity classification for procedures using fish, including field research, will be made available.

BackgroundThis study describes the development of Response Surface Pathway (RSP) design, assesses its performance and effectiveness in estimating LD50, and compares RSP with Up and Down Procedures (UDPs) and Random Walk (RW) design.MethodsA basic 4-level RSP design was used on 36 male ICR mice given intraperitoneal doses of Yessotoxin. Simulations were performed to optimise the design. A k-adjustment factor was introduced to ensure coverage of the dose window and calculate the dose steps. Instead of using equal numbers of mice on all levels, the number of mice was increased at each design level. Additionally, the binomial outcome variable was changed to multinomial. The performance of the RSP designs and a comparison of UDPs and RW were assessed by simulations. The optimised 4-level RSP design was used on 24 female NMRI mice given Azaspiracid-1 intraperitoneally.ResultsThe in vivo experiment with basic 4-level RSP design estimated the LD50 of Yessotoxin to be 463 μg/kgBW (95% CI: 383–535). By inclusion of the k-adjustment factor with equal or increasing numbers of mice on increasing dose levels, the estimate changed to 481 μg/kgBW (95% CI: 362–566) and 447 μg/kgBW (95% CI: 378–504 μg/kgBW), respectively. The optimised 4-level RSP estimated the LD50 to be 473 μg/kgBW (95% CI: 442–517). A similar increase in power was demonstrated using the optimised RSP design on real Azaspiracid-1 data. The simulations showed that the inclusion of the k-adjustment factor, reduction in sample size by increasing the number of mice on higher design levels and incorporation of a multinomial outcome gave estimates of the LD50 that were as good as those with the basic RSP design. Furthermore, optimised RSP design performed on just three levels reduced the number of animals from 36 to 15 without loss of information, when compared with the 4-level designs. Simulated comparison of the RSP design with UDPs and RW design demonstrated the superiority of RSP.ConclusionOptimised RSP design reduces the number of animals needed. The design converges rapidly on the area of interest and is at least as efficient as both the UDPs and RW design.

An experiment was conducted to examine the effects of body condition at calving and feeding level post partum on the duration of the post-partum anoestrous period in beef cows. Forty-seven cows were used in each of 2 years. Cows were fed differentially before calving to achieve two levels of body condition at calving in November and December and were then fed at one of two levels of feeding post partum in a 2 X 2 factorial experiment. At calving, mean body condition scores were 2-85 (F) and 2-23 (T) (P < 0-001) while live weights were 521 kg and 457 kg (P < 0-001) for F and T cows respectively. After calving metabolizable energy intakes were 111 and 118 M]/ day (H) or 56 and 64 M]/day (L) in years 1 and 2 respectively. The H cows maintained live weight during the first 61 days of lactation while the L cows lost live weight (0-0 v. -1-1 kg/day, P < 0-001). The number of cows still acyclic on 1 May each year was higher (P < 0-01) on the TL treatment (10/24) compared with the other treatment combinations (FH: 2/22; FL: 5/22; TH: 2/24). The effect of level of feeding on the duration of the anoestrous period was significant in the T cows (89 v. 116 days for TH and TL cows respectively; P < 0-01) but not in the F cows (80 v. 89 days for the FH and FL cows respectively; P > 0-05). At 3 weeks post partum LH pulse frequency was higher in F than in T cows and tended to be higher in H than in L cows. At 6 and 9 weeks post partum LH pulse frequency was higher in H than L cows. LH pulse amplitude was higher in F than T cows at 3 weeks post partum and tended to be higher at 6 weeks post partum. It is concluded that both body condition at calving and feeding level after calving influence the duration of the post-partum anoestrous period with the effect of feeding level being particularly pronounced in thin cows.

Summary. The seasonal changes in testicular weight in the blue fox were associated with considerable variations in plasma concentrations of LH, prolactin, androstenedione and testosterone and in FSH-binding capacity of the testis. An increase in LH secretion and a 5-fold increase in FSH-binding capacity were observed during December and January, as testis weight increased rapidly. LH levels fell during March when testicular weight was maximal. Plasma androgen concentrations reached their peak values in the second half of March (androstenedione: 0\m=.\9\ m=+-\0\m=.\1ng/ml; testosterone: 3\m=.\6 \ m=+-\ 0\m=.\6 ng/ml). A small temporary increase in LH was seen in May and June after the breeding season as testicular weight declined rapidly before levels returned to the basal state (0\ m=. \ 5\ p=n-\ 7ng/ml) that lasted until December. There were clear seasonal variations in the androgenic response of the testis to LH challenge. Plasma prolactin concentrations (2\p=n-\3ng/ml) were basal from August until the end of March when levels rose steadily to reach peak values (up to 13 ng/ml) in May and June just before maximum daylength and temperature. The circannual variations in plasma prolactin after castration were indistinguishable from those in intact animals, but LH concentrations were higher than normal for at least 1 year after castration.