Impact of venipuncture on physiological research conducted in conscious macaques

Einfluß der Blutprobenentnahmetechnik auf Rhesusaffen auf physiologische Daten von Rhesusaffen

VIKTOR REINHARDT

Wisconsin Regional Primate Research Center, Madison, U.S.A.

(Received August 28, 1991; accepted December 8, 1991)

Key words: Venipuncture, stress, methodology, macaques

Summary

A survey of 397 publications dealing with macaques was conducted. Strcss-sensitive physiological data collected during venipuncture were evaluated in 58 reports. Despite of the fact that venipuncture often is a stressful event for research anirnals, 81% of the studies did not account for this circumstance by providing no information as to how tile subjects were caught and how they were immobilized during venipuncture. Common practices suggest that the animals were physically restrained with squeeze-backs or forced with fear-inducing techniques to leave their home areas and enter a transport cage in most of these studies, and that they experienced additional stress, triggered by manual or mechanical immobilization during actual venipuncture.
It was concluded that the description of the experimental animal's handling prior to and during venipuncture is a methodological issue which needs to. be clarified in order to account for a dependent possibly data-biasing variable. It was recommended that experimental animals should be trained to cooperate during in-homecage venipuncture whenever Stress-sensitive blood parameters are examined.

Introduction

Laboratory macaques are often fearful of personnel and resist being handled. This reaction is understandable because interactions with humans, are all too often associated with discomfort or pain. Aversive reactions, however, indicate that the research animal no longer is in a state of case but rather subjected to a distressing situation. Scientific data collected from such an animal must be evaluated with particular caution, taking into consideration possible stress-related deviations from expected normal physiological functions.

Venipuncture is a handling procedure to which laboratory macaques are subjected to commonly. During venipuncture, data are often collected that may be confounded by stress. This issue has been raised as a methodological concern by numerous authors, Loomis et al. (1980) cautioned that "restraining a monkey in its cage [to facilitate catching or venipuncture] represents a stressful situation", which "may result in a physiological leukocytosis and. hemoconcentration in the sample collected at time zero". IVES and DACK (1956) observed an elevated White Blood Cell Count as "alarm reaction" to physical restraint in rhesus monkeys. REINHARDT (unpublished finding) made a retrospective comparative evaluation of hematograms from 6 adult female rhesus macaques that showed fear and resistance during venipuncture in a restraint apparatus, and from 6 adult female rhesus macaques who showed no fear and no resistance during venipuncture in their familiar home cages. White Blood Cell Counts of the resisting subjects were markedly higher than those of non-resisting subjects (10,000±3,500 mm3 vs. 7,400±800/mm3; U=7, p<0.05, Mann Whitney test). This observation is congruent with findings of KING and GARGUS (1967) and ROSENBLUM and CLOUSTON (1981). The first two authors reported relatively high White Blood Cell Counts (12,800±4,800/mm3) in adult female rhesus macaques that supposedly resisted during venipuncture ("The blood samples used were obtained principally by femoral vein puncture".). The second two authors reported much lower values (8,000 ± 1,800 /mm3) in adult female rhesus macaques that presumably showed no resistance during venipuncture ("All animals were trained to present an arm through a small cage opening".). ROBINSON et al. (1964) concluded from their own studies and those from COPE and POLIS (1959) "that there can be a rise in SGO-T [serum glutamic-oxalecetic transaminase] in monkeys due to nonspecific stresses such as fright, handling or clinical procedures". VERLANGIERI et al. (1985) underscored undesirable variations in serum biochemical and hematological parameters in macaques (cf. MIGEON et al. 1955; ROLLINS et al. 1970; ALTSHULER et al. 1971; McCLURE, 1975) and suggested that incongruities between values presented in different reports may be due to a variety of factors including the method of restraint [during venipuncture]. MICHAEL et al. (1974) evaluated plasma corticosteroid concentrations in male rhesus macaques that "were trained to enter voluntarily a restraining apparatus" and permitted venipuncture without showing signs of stress. The authors pointed out that basal 08.00 h values (22.4±6.0µg/100ml) were 30% lower than previously reported" for different, untrained animals. ELVIDGE et al. (1976) showed "that significant differences [p<0.05, analysis of variance] may be found in the values for plasma cortisol between rhesus monkeys sampled under different 'basal' conditions". MASON et al. (1957) suggested that "increased pituitary-adrenal cortical activity may be related to a broad range of emotional responses or, perhaps, to a relatively undifferentiated emotional state reflecting active involvement of the animal [rhesus monkey] with a threatening environment". PURI et al. (1981) speculated "that the decline in serum testosterone levels [following cage restraint] is perhaps a result of stress induced by handling conscious monkeys as evidenced by the concomitant rise in serum cortisol levels". HAYASHI and MOBERG (1987) reported that "acute restraint stress stimulates a transient release of LH" [luteinizing hormone; in male rhesus monkeys] and prevents the normal release of testosterone in response to GnRH administration.
The present study examines the information commonly provided for venipuncture of conscious macaques and discusses possible scientific implications.


Methods

A survey was conducted of all original research articles on macaques published in LABORATORY ANIMAL SCIENCE, the AMERICAN JOURNAL OF PRIMATOLOGY and the JOURNAL OF MEDICAL PRIMATOLOGY between 1981 and 1990. From each article the following information was compiled:

1. Were research data collected by means of venipuncture?
2. Is it possible that research data were influenced by stress reactions associated with the venipuncture procedure? It was assumed that research data may be influenced by venipuncture when blood samples served to evaluate the following physiological parameters:
a) White Blood Cell Count (IVES and DACK 1956),
b) SGO-T (ROBINSON et al. 1964),
c) Cortisol (MICHAEL et al. 1974),
d) Testosterone (PURI et al. 1981),
e) LH (HAYASHI and MOBERG 1987). 

3. How were the experimental animals caught? 
4. Where was venipuncture performed? 
5. How were the experimental animals restrained?


Results

Of 397 publications surveyed, 136 provided research data of animals that were handled during data collection. Handling involved venipuncture in 88 % (119/136) of cases. Blood samples collected from conscious animals were used to analyze possibly stress- sensitive physiological parameters in 58 articles. Hemograms (including WBC) and serum chemistry (including SGO-T) were analyzed in 20 and 13 studies, respectively. Cortisol and luteinizing hormone were analyzed in each of 9, testosterone in 7 studies.

Table 1. Number of studies (total 58) analyzing handling stress-sensitive parameters in 
venipunctured research macaques.
Procedure
Stress-sensitive parameters analyzed
 WBCC
SGO-T
Cortisol
Testost.
LH
 CATCHING  
 not necessary
 1
2
 -
2
 trained
-
1
-
-
manual
-
-
 1
-
-
with squeeze-back
-
1
-
-
not described 
 19
12
 4
7
7
VENIPUNCTURE  
in homecage
-
-
-
-
  trained
-
1
-
-
  squeeze-back
1
1
1
2
away from homecage
-
-
-
-
  manual immobilization
 1
1
3
-
  mechan. immobilization
-
-
-
-
not described
18
11
4
7
7


Nine of these 58 reports indicated if and how the research subjects were caught for venipuncture. No catching was required in 6 cases; in the other 3 cases, the research animals were either manually caught, trained to voluntarily enter a transport box or coaxed to do so using a squeeze-back mechanism (Table 1).

Eleven of the 58 reports described the handling procedure that was used to draw the blood samples. Venipuncture took place in the research animals' home cages in 6 studies; the subjects were trained to cooperate without use of restraint in 1 study, they were mechanically immobilized with a squeeze-back mechanism in the other 5 studies (Table 1). Venipuncture took place outside of the research animals' home cages in 5 studies and was accomplished while the subjects were immobilized manually (Table 1).

Forty-seven of the 58 investigations provided no information on the blood drawing procedure nor on the possibly preceding catching procedure.


Discussion

Venipuncture was the most common handling procedure to which research macaques were subjected in the present survey of 397 research publications. In 49 % of cases venipuncture served to collect data of stress -sensitive physiological parameters. The catching and restraining associated with venipuncture is a potentially stressful event for the research animal and hence a variable that may bias research data (HEMM and JOHNSON 1978; WICKINGS and NIESCHLAG 1980). It is surprising that the majority (81 %) of the 58 studies did not account for this circumstance by omitting information of how the research subjects were caught and how and where they were restrained for venipuncture. According to a 1989 census conducted at the National Institutes of Health "only 9 % of [56] scientists interviewed currently train their animals to enter transport cages" (BAYNE, 1989). This prompts the assumption that animals were forced with fear-inducing techniques (e. g., MASON et al. 1957) to leave their home cages and enter a transport cage in many of the above studies. It is therefore very likely that the research data collected from these subjects were influenced by stress reactions immediately prior to blood collection. The same is true for those animals that were not removed from their cages but were physically restrained with squeeze-backs. The research data were probably further biased by the animals' additional stress reactions triggered by manual or mechanical immobilization during actual venipuncture.

Only a minority (19 %) of the 58 studies provided information regarding the actual venipuncture procedure. Venipuncture took place in the subject's home cage in 6 studies, outside of the subject's cage in 5 studies. Adult rhesus macaques show a significant increase in serum cortisol concentrations as a response to venipuncture away from their familiar home cage; this response is absent when they are venipunctured in their own cages (REINHARDT et al. 1990, 1991). This suggests that research data are less confounded by stress when the subject is handled in its familiar cage than when it is handled away from it in a relatively unpredictable environment (cf. LINE et al. 1989).

Despite the fact that macaques can readily be conditioned to voluntarily leave their home area and enter a transport cage thereby avoiding the stress of being forcefully removed (MICHAEL et al. 1974; ELVIDGE et al. 1976; SMITH 1981; BUNYAK et al. 1982; SAINSBURY et al. 1990; WALKER et al. 1982; HEATH 1989; REINHARDT, 1990), only one study mentioned that subjects were "trained to enter a transport cage" (CLARKE et al. 1988). It is also surprising that only one study mentioned that "monkeys" were trained to present their arms through the bars of the cage to the technician who then draws blood from a vein without difficulty" (HEIN et al. 1989).. It has been reported for stump-tailed, cynomolgus and for rhesus macaques that animals can be conditioned to cooperate during venipuncture in their familiar surrounding, thus avoiding possible stress associated with catching and with use of restraint (Macaca artoides: BUNYAK et al. 1982; Macaca fascicularis: HEIN et al. 1989; Macaca mulatta: ROSENBLUM and CLOUSTON 1981; WALKER et al. 1982; SCALLET et al. 1989; VERTEIN and REINHARDT 1989). Even adult male rhesus monkeys, who are "aggressive animals and very difficult to handle" (WICKING and NIESCHLAG 1980) can be trained easily, quickly and safely to actively cooperate during in-homecage venipuncture (REINHARDT 1991). Such individuals do not show the significant corticosteroid response that typically occurs in animals venipunctured away from their home cage (REINHARDT et al.1991).

The present study leads to the conclusion that the description of the experimental animal's handling prior to and during venipuncture is a methodological issue which - like sex, age, health status, feeding regime, room temperature, lighting condition and housing condition - needs to be specified in order to account for a possibly data-biasing variable. Such information is a basic requirement to interpret research data and to compare them with those of different studies. Training techniques ensuring the experimental animals's cooperation during venipuncture in its familiar home environment should be applied whenever stress -sensitive blood parameters are examined.


Acknowledgements

I am very thankful to KLARI FAJZI and my wife ANNIE for critically reading the manuscript and providing constructive comments. Thanks are also due to JACKIE KINNEY for proofreading. This project was supported by NIH grant RR00167 to the Wisconsin Regional Primate Research Center.


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This article originally appeared in the Journal of Experimental Animal Science 34, 211-217, 1991. 
Reprinted with permission of the publisher.