Andersen, H V

Jorgensen, Damm

Klastrup, K S

Svendsen, O

English

Journals from University of Tartu

Scand. J. Lab. Anim. SGL No. 1. 1998 V01 25Haematology in miceafter weekly blood sampling for 7 weeksby Svendsen, 0. ‘1 Andersen. H. V. **, DammJargzmen, & K. Klastrup, SiScantox, DK-4623 Lille Skensved, DenmarkPresent address. * Department of Pharmacology and Pathobiology, The Royal Veterinary and Agricultural University,DK-1870 Frederiksbcrg C, Denmark, ** Novo Nordisk, Novo Nordisk PaIk, Byg G8.2.06, DK-2760 Mdlav,Denmai'k.*)IntroductionBlood sampling in rodents from the orbital veinplexus is a controversial procedure due to poten-tial animal welfare implications. As a consequen-ce the procedure is regulated differently in Euro-pean countries It is also controversial whether theanimals should be anaesthetised while the samp-ling takes place. Recommendations for removal ofblood from laboratory animals have recently beengiven in a report by a BVA/FRAME/RSPCA/UFAW (1993) working group. This wor-king group dealt specifically with retro-orbitalblood sampling and stated that “there are manypotential adverse effects to this technique andthese should be given full consideration beforeuse".The Danish Animal Experiments Inspectorate hasrecently conducted an in depth investigation ofblood sampling by puncture of orbital vein plexusin guinea pigs, rats and mice. Several members ofthe Inspectorate have had the opportunity at in-spections to supervise this sampling procedure innumerous animals and seen animals being sampledby this procedure weekly for seven weeks. Thepresent study was followed closely by the Inspec—torate. The procedure these members has seen hasnot been the course of remarks (Kjazrsgaard,1997). In a recent work by Herck et al. (1997) it isconcluded that orbital bleeding in rats while underdiethylether anaesthesia does not influence tele-metrically determined heart rate, body temperatu-re, locomotor and eating activity when comparedwith anaesthesia alone. The Danish Animal Expe-riments Inspectorate came up with the followingconclusion: Anaesthesia of mice is not required atblood sampling from the periorbital plexus.Sampling in conscious mice is only acceptable ifcarried out by trained and experienced technicians.Anaesthesia is required at blood sampling from theperiorbital plexus of rats and guinea pigs. Samp-ling from the periorbital plexus of rats withoutanaesthesia may be allowed by the Inspectorate inspecial cases in animals with a body weight below120 g (Kjtersgaard, 1997).In a series of immunisation studies in mice, samp-ling of 0.5 m1 of blood took place at a weeklyinterval for seven weeks. The mice were supposedto develop a cenain level of anaemia(BVA/FRAME:RSPCA/UFAW working group,1993; McGuilI and Rowan, 1989).For this reason haematological parameters weredetermined after the final blood sample for immu-nology was taken.*)Abbreviation: British Veterinary Association (EVA), Fund for the Replacement of Animals in Medical Experiments(FRAME), Royal Society for the Prevention of Cruelty to Animals (RSPCA) and Universities Federation for AnimalWelfare (UFAW13Scand. J. Lab. Anim. Sci No. 1. 1998‘ Vol 25Materials & MethodsHaematological parameters were determined in 24BALB/c Bom female mice (7-8 weeks of age). Themice were divided into three groups, each of 8animals. Group 1 was a blank control group andonly one blood sample for haematology was takensimultaneously with the sample taken for haema-tology from the other two groups. From the micein the other two groups, a blood sample of 0.5 mlfor immunology was taken weekly for six-sevenweeks before a blood sample for haematology wastaken 1 day (group 2) and 9 days (group 3) re-spectively after the final blood sample for immu-nology All blood samples were taken on the samedate.The immunological part of the study includedimmunisation on 4 occasions and subsequentblood sampling once weekly for seven weeks. Atthe blood sampling for haematology the bodyweight range was 20 - 27 g. 'The mice were kept in maerolone type III cageswith softwood sawdust bedding. A pelleted com-plete rodent diet "“Altromin 1314” (for growinganimals) and domestic quality drinking water wereavailable ad libitum. The room temperature was21°C i 3°C, relative humidity 55% i 15% and airchanges 10 times/hour, Light was on from 6 am to6 pmBlood samples for immunology were taken bypuncture of the retroorbital vein plexus on days 0,28, 35, 42, 49, 56, 63 and 72 (only group 2).For haematology a volume of 0.5 ml EDTA stabi-lised blood was taken simultaneously from thecontrol animals and the day 73 animals (group 2 -one day after the 7‘h weekly blood sampling) Onday 72 (9 days after the 6‘h weekly blood samp-ling) a similar blood sample was taken from group3. The haematological parameters were determinedusing Cobas Minos (Roche, Montpellier, France).Data were processed to give group mean valuesand standard deviations where appropriate Possi-ble outliers Were identified, too. Thereafier eachcontinuous variable was tested for homogeneity ofvariance with Bartlett’s test. If the variance washomogeneous, analysis of variance was carried outfor the variable. If any significant differences were14detected, possible intergroup differences wereassessed with least-squares means. If the variancewas heterogenuous each variable was tested fornormality by the Shapiro-Wilk method. In case ofnormal distribution, the possible intergroup diffe—rence was identified with Student’s t-test. Other-wise the possible intergroup difference was carriedout with Wilcoxon Rank-Sum test.ResultsOne day after the seventh blood sampling allparameters apart from platelet count were lowerthan control level (Table 1), Haemoglobin con-centration, red blood cell count and haematocritwere 15-18% lower whereas white blood cellcount was 35% lower than control level.Nine days after the seventh blood sampling thehaemoglobin, red blood cell and haematocritlevels were 18-22% higher than control level. Thewhite blood cell level was not different from con—trol level whereas the platelet level was 13% lowerthan control level.No macroscopic lesion to the eye was observed inany of the animals.DiscussionIt is rather unlikely that the results obtained shouldbe influenced by the immunological part of thestudy since this only included administration of anantigen. Only relatively slight anaemia was seenone day alter the final blood sampling when com-pared to the control group. However, the levelsobtained one day alter final sampling were 29-35% lower than the levels found nine days afierfinal sampling. At each blood sampling a volumeof 0.5 ml was taken. This corresponds to about30% 0f the total blood volume. Based on theresults from the present Study it is concluded thatmice are capable to compensate the blood lossinduced by weekly sampling of 0.5 ml per weekfor seven weeks.Removal of a volume ofthis magnitude shouldaccording to the literature (BVA/FRAME; RSPCA/UFAWwarking group, 1993; McGuz’ll and Rowan,1989) have more severe consequences than seen inthe present study,Scand. J Lab. Anim. Sci. No. l, 1998. Vol. 25 Table 1. Haematology in mice 1 and 9 days after weekly blood sampling (0.5 ml/sample) for 6 - 7weeksHaemoglobin Red blood cell Haematocrit White blood cell Platelet countGroup (mmol/l) count (m1/100 ml) count (109/1)(10% (109/1)Mean SD. Mean S.D. Mean SD Mean S.D. Mean S.D.1 - Control 9.5" 0.5 9.30|> 0.53 44.0b 2.1 7.4b 1.4 752;ab 65(n=8)2 - Day 1 7.9“ 1.0 7.643 0.95 37.43 4.7 4.3' 0.8 791b 110(F3)3 - Day 9 11.20 0.9 11.01n 1.13 535° 50 7.4b 1,5 660' 113(F3)            Mean = mean value of eight animalsMean values with different letters are statistically significantly different (p<0.05)S.D. = stande deviationReferences:BVA/FRAME/RSPCA/UFAW working group: Re-moval of blood from laboratory mammals andbirds. Laboratory Animals, 27, 1-22, 1993.chersgaard, C.J.: Personal communication.Chairman Danish Animal Experiments Inspecto-rate, 1997.McGuill, MW, & Rowan. A.N.: Biological effectsof blood loss: Implications for sampling volu-mes and techniques. lLAR News, 3], 5-18,1989.Van Herck, H, De Boer, S.F., Hesp, A.P.M.,Van Lith, HA, Baumans, V. & Beynen, A.C..'Orbital bleeding in rats while under diethylet-her anaesthesia does not influence telemetri-cally determined heart rate, body temperature,locomotor and eating activity when comparedwith anaesthesia alone. Laboratory Animals,3], 271-278, 1997.15

Haematology in mice after weekly blood sampling for 7 weeks

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Haematology in mice after weekly blood sampling for 7 weeks

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