Jul 13, 1992 - (Charles. River Technical. Bulletin, 1987), was developed by the Colorado ..... ST, Lucas A, Fields CD & Isner JM (1990) Atherosclerotic.
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Laboratory
Animals
(1993) 27, 368-373
Haematological and plasma biochemical values for healthy Yucatan micro pigs G. RISPATI,
M. SLAOUI1, D. WEBERl, P. SALEMINK2, C. BERTHOUX3
& R. SHRIVASTAVAI
IRL-CERM, Route de Marsat, 63203 RIOM CEDEX, France, 20rganon Int BV, PO Box 20,5340 BH 055, Holland, and 3Charles River France, 59 rue de la Paix, Saint-Aubin-Les-Elbeuf, 76410 Cleon, France Summary Blood samples (n = 135) were collected from 36 male and 35 female 20 ± 4-week-old Yucatan micro pigs and were analysed for 19 haematological and 18 plasma biochemical parameters at weeks 20, 24, 33, 46, 59, 71 and 80. For each parameter, the total number of analyses per sex, mean values, standard deviation, lowest and highest values, and 950/0 confidence intervals are presented as reference values for this breed. Ageand sex-related differences in these parameters are also discussed. Keywords: Yucatan Plasma biochemistry
micropig;
Haematology;
In the last few years, the increasing demand from animal protection groups to reduce the use of dogs and cats in biomedical research has led researchers in a variety of disciplines to consider the micro pig as an animal of choice. Swine species offer many similarities to man in terms of basic anatomy, physiology, biochemistry, pathology and pharmacology (Khan, 1984; Dantzer, 1986). The coronary artery system of the swine heart, which is similar to the human system, makes it suitable for cardiovascular research (Smith et al., 1990). The physiology of digestion and the length of intestinal tract of swine are also similar to that of man, which makes swine useful for gastroenterology studies. Other similarities are the
pathology of atherosclerosis (Gal et al., 1990; Reitman et al., 1982), anatomy of the kidney, arrangement of accessory sex glands, and structure of the skin (Panepinto et al., 1978). Despite the recognition of the potential usefulness of swine as experimental animals, there is a reluctance to use them instead of more traditional laboratory animals. This is probably due in part to the difficulties involved in handling relatively larger animals like pigs: the size-related problems in laboratory use of the domestic pig have stimulated development of miniature swine (Panepinto & Phillips, 1981), and there are a number of miniature breeds available today for use in biomedical research. The Yucatan micropig, which was derived from the Yucatan pig native to Mexico and Central America (Charles River Technical Bulletin, 1987), was developed by the Colorado State University in the early 1970s. This breed is becoming increasingly popular as an experimental animal because of its small size. At present, however, very limited information on a small number of animals is available regarding the haematological and plasma biochemical values for this breed (Hitz et al., 1987; Parsons & Wells, 1986; Radin et al., 1986). The aim of the present study was to establish reference values for 19 haematological and 18 plasma biochemical parameters using automated and manual techniques. Materials
and methods
Animals Correspondence to: Dr G Rispat. Received 13 July 1992; accepted 19 January 1993
Thirty-six male and 35 female Yucatan micropigs having a mean age of 20 ± 4 weeks with a mean body weight of 13, 8 (11 . 7-16' 0) kg for males
Blood analysis of Yucatan micropigs
and 13.7 (11 .0-16' 5) kg for females at the start, and 14,0 (12'0-16·8) kg for males and 16·1 (11' 9-18' 4) kg for females at the end of the study. All animals were purchased from Charles River Laboratories (Windham, ME, USA). The animals originated from an indoor facility whose breeding stock was negative for antibody titres to brucellosis and pseudo-rabies. The animals had never been exposed to other swine populations and were free of infection with vesicular exanthema, hog cholera and other communicable diseases. They were vaccinated against leptospirosis and erysipelas. After transportation by air to France, animals were found to be in good health and free of clinical signs of illness. They were not given any treatment throughout the study.
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Animals were restrained in a dorsoventral position, whilst blood was collected via the jugular vein with a needle (1' 2 x 40 mm) attached to a (5 ml) syringe. Immediately after collection, approximately 2 ml of blood was transferred into a tube containing 0·1 ml anticoagulant solution (10 g sodium citrate 2H20 + 2 g ammoniumoxalate in water per 100 ml) for haematological and coagulation studies. The remaining volume of blood was transferred into another tube containing lithium-heparinate and was centrifuged at 1500g for 10 min to obtain plasma for biochemical studies. All analyses were completed on the day of blood collection.
Analytical procedure Haematological parameters were determined using CELL-DYN® 900 semi-automated haemaHousing tology analyser (SequoIa-Turner Corporation, Mountain View, CA, USA). The animals were housed in stainless-steel, wirebottomed cages (1' 1xl, 4 xl, 6 m), within a Blood collected in citrate-oxalate anticoagulant room maintained at 21 ± 3°C on a 12-h lightl was used to prepare smears for differential white dark cycle with filtered air (986,10)renewed 8-10 cell counts. Smears were stained with May times per hour. Griinwald-Oiemsa stain, and 100 cells were examined microscopically (x 400, oil immersion). Diet and water Other smears were stained with cresyl blue stain The animals were given a pelleted maintenance for reticulocyte counts. Blood collected with ration diet 127C supplied by UAR (Villemoisson citrate-oxalate anticoagulant was used to detersur Orge, France), in the amount of 450 gl mine 1hand 2 h sedimentation rates (0' 08 ml animal/day up to week 40 and 500 g/animal/day glass pipettes; Preciss, France). After centrifugation (800 g for 10 min), citrate-oxalate from week 41 onwards. Water was supplied ad libitum by an automated watering system using plasma was separated for the determination of nipple-type mouthpieces. prothrombin time (PT) and activated cephalin time (APTT) by means of a Fibrometer (BioThe mean composition of the diet was as follows: water (11 . 16,10), proteins (15' 36,10), lipids Merieux, France). (2'86,10), glucides (53.56,10with 26.76,10carboPlasma biochemical analyses were performed hydrates), cellulose (10.96,10) total minerals (6.46,10) using an automated Olli C + D analyser (Kone, Espoo, Finland). Plasma potassium and sodium and vitamin A (5100 IV/kg). levels were determined by flame spectrophotoBlood sampling metry (IL 243, Instrumentation Laboratory, Blood samples were collected in the early Milan, Italy) and calcium by atomic absorption morning following a 16-18 h fast on weeks 20 (Spectra 10, Techtron Springvale, Varian, Australia). Alkaline phosphatase (ALP), (n = 36 males and 35 females), weeks 24, 33,46, 59 and 71 (n = 6 males and 6 females) and week aspartate aminotransferase (ASTlOOT), alanine 80 (n = 2 males and 2 females). Analyses were not aminotransferase (ALT IOPT) were measured performed when samples were haemolyzed; this according to the recommendations of the French was rare and less than 16,10of the samples. Society for Clinical Chemistry using Boehringer
Rispal et al.
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Table 1. Haematological values for 13Sanalyses (68 for males and 67 for females) conducted between 20 and 80 ± 4 weeks of age Parameter
Unit
Sex
Mean value
SD
Lowest value
Highest value
Erythrocytes
10'2/1
Haemoglobin
mmol/I
PCV (Packed Cell Volume)
ratio
MCV (Mean Corpuscular Volume)
f1
M+F M F M+F M F M+F M F M+F M F M+F M F M+F M F M+F M F M+F M F M+F M F M+F M F M+F M F M+F M F M+F M F M+F M F M+F M F M+F M F M+F M F M+F M F M+F M F
7,34 7·28 7,40 8·91 8·79 9,04 0,47 0·46 0,48 63,96 63·24 64,69 1·22 1'21 1·22 19·04 19'16 18·92 6'33 5,76 6·91 510,00 549,00 470'60 11·74 11'76 11'71 15·46
0·71 0'78 0,64 0,89 0,92 0,86 0,05 0,05 0,05 3,78 3,84 3,59 0'07 0'06 0,07 0'78 0'83 0·71 2·38 2·21 2·42 124·00 127·00 107,40 0,64 0,66 0·62 1·15
5,30 5,30 6,00 6,70 6,70 7,60 O' 36 0'36 0,39 54,00 54,00 56,00 1·05 1·05 1·06 17·20 17'60 17·00 3,00 3,00 3,00 215·00 335,00 215·00 9,30 10,10 9,30 12·30
9,04 9,04 8,70 11·20 10·80 11·20 0'61 0'57 0'61 73,00 73,00 72,00 1'39 I· 36 I· 39 22·20 22·20 20·40 14·00 13,00 14·00 898'00 898,00 770'00 13'30 13,30 12·80 17·80
12'30 0,50 0'50 0'50 0'50 0,50 0'50 7,40 8,60 7,40 0,00 0'00 0,00 18·00 28·00 18·00 0,00 0,00 0,00 0,00 0'00 0,00 14·00 14'00 17·00 0,00 0,00 0,00
17,80 34,00 19'00 34,00 57,00 39,00 57,00 26·40 25·80 26·40 3,00 3,00 3,00 80'00 80'00 78'00 7'()() 7,00 7,00 1·00 1'00 1·00 70,00 65'00 70,00 13,00 11·00 13·00
fmo] MCH (Mean Corpuscular Haemoglobin) MCHC mmol/I (Mean Corpuscular Haemoglobin Concentration) 10-3 RBC Reticulocytes Thrombocytes
109/1
Prothrombin time APTT (Activated Cephalin time) Sedimentation rate
mm/h
Sedimentation rate
mm/2h
Leucocytes
109/1
Band neutrophils
Olo
Segmented neutrophiIs
Olo
Eosinophils
Olo
Basophils
Olo
Lymphocytes
Olo
Monocytes
Olo
]5·35
15·57 3·25 2·97 3,53 7·14 6·72 7'57 14·94 15'52 14'36 0,55 0'65 0,45 53,96 56,38 51·51 1·56 1·62 1·49 0,06 0,04 0,08 39,08 36,46 41·75 4,69 4'85 4'52
1·15
1'15 4,54 3,50 5·41 9·51 8,08 10'81 3'57 3'79 3·26 0'74 0·77 0,70 12'02 11· 55 12·07 1·53 1·48 1·60 0·24 0'21 0·27 11·79 11·51 11·56 2·77 2· 59 2·96
12·30
17·80
Blood analysis
of Yucatan
371
micropigs
Table 2. Blood biochemical values for 127 analyses (64 for males and 63 for females) conducted between 20 and 80 ± 4 weeks of age Parameters
Unit
Sex
ALP
JLkat/l
AST/GOT
JLkat/l
ALT/GPT
JLkat/l
CK
JLkat/l
LDH
JLkat/l
Calcium
mmol/I
Inorganic phosphate
mmol/I
LDL
mmol/I
Triglycerides
mmol/l
Urea
mmol/l
M+F M F M+F M F M+F M F M+F M F M+F M F M+F M F M+F M F M+F M F M+F M F M+F M
F Creatinine
ILffiolll
Glucose
mmol/l
Tota] bilirubin
JLmol/1
Total cholesterol
mmol/l
M+F M
F M+F M F M+F M F M+F M
Total proteins
gil
Potassium
mmol/l
F M+F M F M+F M
Sodium
mmol/l
Chloride
mmol/I
F M+F M F M+F M F
SD
Lowest value
Highest value
0,99 0,92 1·05 0,68 0,67 0,69 1'00 1·00 1·00 11·84 11·95 11·74 12·89 12·66 13·13 2·72 2·72 2·71 2·15 2'15 2·14 0·16 0'16 0'15 0,33 0·31 0,36 6,84 6,94 6·74 77,40 75,30 79,40 3·71 3·62 3,80 5·71 5,65 5,78 2·01 1·89 2· ]2 73·87 72,86 74'90 5'48 5,50
0·42 0'33 0,48 0·41 0'37 0'45 0'18 0'20 0·16 17·93 16'59 19·31 2·99 3,00 2'98 0,17 0'18 0·16 0·24 0·24 0·24 0'09 0,10 0'08 0,10 0,10 0·1] 1·14 1·20 1·06 14-30 16·60 11'30 0·62 0-67 0-56 3,90 4,93 2'50 0,37 0,33 0,39 4·70 4,40 4,80 0'84 0·84
5·46
0·84
139,80 139'80 139,80 102·70 JO],80 103'60
2·80 2'80 2-90 5'60 3,60 6'90
0,33 0'33 0'51 0,30 0·31 0,30 0,63 0,63 0·71 1·75 1·75 2·20 7'70 7'70 9'80 2'15 2'15 2·40 1·62 1·74 1·62 0·02 0'02 0'02 0,05 0,05 0'18 4,30 4'30 4'50 42'00 42·00 57,00 2·40 2'50 2·40 1'80 2'10 ],80 1·25 1·35 1·25 60,00 60,00 62·00 3'50 3,50 3-80 132,00 132·00 132,00 94'00 96,00 94,00
3,07 2·08 3,07 2'59 2·17 2'59 1· 55 1· 55 1'38 99,99 86,00 99,99 28·50 21'50 28'50 3'15 3,10 3·15 2·90 2·90 2·77 0·5] 0·5] 0,36 0,76 0,56 0'76 9,80 9,80 9'10 109-00 109,00 104'00 7·40 7,40 6'10 40'20 40'20 15'40 3·07 2·76 3'07 88,00 81'00 88,00 7'40 7,40 7,30 146·00 146'00 146·00 140·00 I JO·OO 140·00
Mean value
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Mannheim reagents for AL T IGPT and AST I GOT determinations and Bio-Merieux reagent for ALP determinations. Creatine kinase (CK) and lactic acid dehydrogenase (LDH) were measured according to the recommendation of the German Society for Clinical Chemistry using Boehringer Mannheim reagents. All enzyme determinations were performed at 30°C. Inorganic phosphate was measured by the phosphomolybdate reaction; triglycerides, cholesterol, urea and glucose by enzymatic coupled reactions, and creatinine by the Jaffe reaction. Low-density lipoprotein cholesterol (LDL) was calculated from the difference between the total cholesterol before and after precipitation with polyvinyl sulphate. All of those reagent kits were purchased from Boehringer Mannheim, Germany. Bilirubin was measured according to the Jendrassik-Grof method. Total protein was determined by the biuret reaction using a kit supplied by Inside Garnall, and chloride by a reagent kit supplied by Bio-Merieux, France. Statistical analysis Values are expressed as the mean ± one standard deviation for the total number of animals and also separately for each sex. Results and discussion The values of the 19 haematological parameters and 18 plasma biochemical parameters obtained with 135 blood samples collected from 36 males and 35 females are presented in Tables I and 2, respectively: for each parameter the mean values, standard deviations, lowest and highest values are shown. There were no significant differences between males and females for the haematological and biochemical parameters, except for thrombocyte count which was slightly lower in females than in males. Most of the results presented in this paper were obtained from animals aged 20 ± 4 weeks. The
Rispat et al.
data from the small number of animals evaluated during the course of the experiment, nevertheless, provided a basis for comparing the effects of age. This comparison revealed minor intra-animal differences for erythrocytic parameters, reticulocyte, thrombocyte, total and differential leucocyte counts and sedimentation rates. From week 20 up to week 80, there were slight decreases in creatinine, inorganic phosphate, triglyceride and cholesterol levels and a slight increase of ALT values. Individual differences were seen in creatine kinase values in both males and females, and triglyceride levels in males while slight individual differences were observed for most of the enzymes. The reference values of earlier studies (Parsons & Wells, 1986; Radin et al., 1986) which were conducted on a smaller number of animals, in general are similar to those obtained in the present study. The only noteworthy change was seen for the differential eosinophilic leucocyte count, which was lower in our study compared to previously published data (Radin et al., 1986). Certain of our biochemical values were somewhat higher than those obtained by Radin et al., in particular AST/GOT, ALT/GPT, CK, urea and bilirubin. These minor differences for some biochemical values can be explained by individual variations (probably due to geneticheterogenicity), experimental conditions (environment, diet, housing, etc.), and analytical techniques used. Recent development in the selection of miniand micropigs and their anatomophysiological similarities to man make them a suitable species for pharmacotoxicological studies (Panepinto & Phillips, 1986; Tumbleson, 1986). However, despite continuous efforts from breeders, pigs are not yet widely used in scientific research and one possible reason is the lack of reference values based on a large sample size. The values obtained in the present study can serve as a basis for comparison and reference, and thereby facilitate the use of pigs in biomedical research.
Blood analysis of Yucatan micropigs
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Wilmington, Massachusetts: Charles River Laboratories, Inc. Dantzer R (1986) The pig as a model for behavioural research. Laboratory Animal Science 36, 362-365 Gal D, Rongione AJ, Geralyn BA, Siovenkai GA, Dejesus ST, Lucas A, Fields CD & Isner JM (1990) Atherosclerotic Yucatan microswine: An animal model with high grade, fibrocalcic, non fatty lesions suitable for testing catheterbased interventions. American Heart Journal 119, 291-300 Hitz J, Devaux F, Galli F, Siest G & Kiechel JR (1987) Valeurs de reference des constituants chimiques et des enzymes plasmatiques chez Ie miniporc. Annales de Biologie et Clinique 45, 532-536 Khan MA (1984) Minipig: advantages and disadvantages as a model in toxicity testing. Journal of the American College of Toxicology 3, 337-342 Panepinto LM, Phillips RW, Wheeler LR & Will DH (1978) The Yucatan miniature pig as a laboratory animal. Laboratory Animal Science 28, 308-313 Panepinto LM & Phillips RW (1981) Genetic selection for
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Animal Science 31, 403-404 Panepinto LM & Phillips RW (1986) The Yucatan miniature pig: characterization and utilization in biomedical research. Laboratory Animal Science 36, 344-347 Parsons AH & Wells RE (1986) Serum biochemistry of healthy Yucatan miniature pigs. Laboratory Animal Science 36, 428-430 Radin MJ, Weiser MG & Fettman MJ (1986) Hematologic and serum biochemical values for Yucatan miniature swine. Laboratory Animal Science 36, 425-427 Reitman JS, Mahley RW & Fry DL (1982) Yucatan miniature swine as a model for diet-induced atherosclerosis. Atherosclerosis 43, 119-132 Smith AC, Spinale FG & Swindle MM (1990) Cardiac function and morphology of Hanford miniature swine and Yucatan miniature and micro swine. Laboratory Animal Science 40, 47-50 Tumbleson ME (1986) Swine in biomedical research. New York: Plenum Press
