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Blood Sampling from the Venous Occipital Sinus of Birds1,2
Blood Sampling from the Venous Occipital Sinus of Birds1,2 N. G. ZIMMERMANN3 and A. S. DHILLON4 Washington State University, Western Washington Research and Extension Center, Puyallup, Washington 98371-4998 (Received for publication January 21, 1985)
INTRODUCTION
avian species and to determine the validity of substituting this method for other routine sampling methods.
Nonlethal blood sampling is necessary for a wide variety of avian diagnostic and research purposes. Venipuncture, usually from the brachial vein, but also from the jugular and metatarsal veins, is most commonly used. This nonlife-threatening method frequently causes a hematoma, making multiple samplings difficult or impossible. Relatively large multiple blood samples are readily obtained by cardiac puncture but at greater risk to the bird. Recently, Vuillaume (1983) described a method for obtaining large multiple blood samples from the venous occipital sinus of ducks and geese. Richards' (1968) studies of cephalic vein anatomy suggested that occipital sinus blood was primarily of endocranial origin. Thus, it is possible that venous occipital sinus blood sample constituents vary from those of samples taken from other sources. The purpose of the study described below was to test the utility of venous occipital sinus blood sampling in several
Vuillaume's (1983) blood sampling procedure requires positioning of the head and neck of the bird at a right angle to increase the dorsal angle of articulation between the skull and the first cervical vertebra. This positioning creates a dorsal concavity which can be palpated. The skin is pierced in the center of this area on the median plane using a 21 to 25 gauge needle. The needle is slowly inserted at approximately a 45 angle to the neck in order to position the point in the dura mater under the posterior edge of the supraoccipital bone. This is an area rich in venous blood flow, where several vessels join to form the occipital and vertebral sinuses (Fig. 1). The blood-drawing efficacy of this technique was tested in several avian species.
1 A portion of these data was presented at the 73rd Annual Meeting of the Poultry Science Association, Inc., August 1984, Guelph, Ontario. 2 Scientific Paper No. 7039, College of Agriculture Research Center, Washington State University. Projects 0643 and 0576. 3 Department Animal Science. 4 Veterinary Microbiology and Pathology.
The maximum blood volume obtained by venous occipital sinus sampling was compared to maximum volumes obtained by cardiac puncture and decapitation. Seven 1-day-old broiler chicks were bled by each method. Data were compared by analysis of variance with Duncan's multiple range test. The utility of venous occipital sinus sampling for serial bleeding was tested by sequentially bleeding five, 10-week-old Leghorn chick-
MATERIALS AND METHODS
1859
Downloaded from http://ps.oxfordjournals.org/ at Michigan State University on February 23, 2015
ABSTRACT Venous occipital sinus blood sampling was demonstrated to be an effective nonlethal method in all species tested including ducks, geese, Chukar partridge, Japanese quail, peafowl, pheasants, parakeets, turkeys, and chickens. Maximum blood volume obtained from the occipital sinus of 1-day-old chicks equalled the volume obtained by cardiac puncture and exceeded the volume collected by decapitation. Venous occipital sinus puncture was successfully used to rapidly collect several successive blood samples from individual birds, thus indicating the method's utility for serial blood sampling. Hematocrit values, as well as infectious bronchitis and Newcastle disease virus antibody titers measured in blood samples drawn from the occipital sinus and the brachial vein, were compared. Significant correlation coefficients between the two blood sources for each of the variables measured suggest that occipital sinus blood sampling may be substituted for venipuncture for many routine measurements. (Key words: occipital sinus, blood sampling) 1985 Poultry Science 64:1859-1862
1860
ZIMMERMANN AND DHILLON RESULTS AND DISCUSSION
FIG. 1. Diagramic representation of the cephalic venous vasculature and approximate angle of sampling needle insertion (adapted from Richards, 1968).'
ens weighing approximately 650 g. Eight 1.0-ml blood samples were taken from each bird and the time of each bleeding recorded. A ninth sample for maximum volume was also taken and the volume recorded. The homology of blood hematocrit, infectious bronchitis, and Newcastle disease serum hemagglutination inhibition (HI) titer was determined between samples from the brachial vein and the venous occipital sinus. Hematocrit and serum antibody titer were determined in each of the 3.0-ml blood samples taken from the venous occipital sinus and the brachial vein of ten, 23-week-old Leghorn pullets. Infectious bronchitis (Arkansas 99 and Massachusetts 41 strains) and Newcastle disease serum HI titers were determined by the Poultry Diagnostic Laboratory, Western Washington Research and Extension Center, Puyallup, using procedures described by King and Hopkins (1983) and Beard and Wilkes (1973), respectively. All samples were assayed in duplicate. Hematocrit and reciprocal titer values between the two bleeding sites were analyzed by paired t-test and correlation coefficient. All statistics were compiled using SAS Institute Inc. (1982) procedures.
Although occipital sinus sampling is a very effective method of obtaining blood samples from birds, it is also potentially dangerous to the health of the bird. The sampling site in the dura mater is in close approximation to easily damaged neural tissue. If the bird moves during sampling, or if the syringe needle inadvertently penetrates too far into the skull, severe neural damage or death can occur. The former malady is quite prevalent especially in larger, stronger birds even when an extra technician is employed to hold the bird. The sampling procedure does not appear to be painful but some birds object to immobilization of the head. The latter situation often occurs due to the use of excessive pressure when pushing the sampling needle into the tough fibrous connective tissue of the dura mater. Additional pressure seemed to be required to puncture the dura in older birds. Losses, attributed to these problems, can be largely eliminated by retaining a trimmed needle sheath on the needle which allows only
Downloaded from http://ps.oxfordjournals.org/ at Michigan State University on February 23, 2015
'Abbreviations are: ACb, anterior cerebral vein; C, carotid vein; Cm, cisterna magna; Cv, cervical sinus; DCO, dorsal cerebral ophthalmic vein; E, ethmoid vein; EF, external facial vein; EOc, external occipital vein; FCV, first cervical vertebra; IF, internal facial vein; IOc, internal occipital vein; J. jugular vein; M, maxillary vein; MC, middle cerebral vein; MD, mid-dorsal sinus; Oc, occipital sinus; Op, ophthalmic vein; PC, posterior cephalic vein; ROp, recurrent ophthalmic vein; SC, sinus cavernosus; SOB, supra occipital bone; TA, transverse anastomosis; TOp, temporal ophthalmic vein; TR, temporal rete; V, vertebral vein.
Blood samples were successfully drawn from the venous occipital sinus in each of the species tested. These included ducks, geese, Chukar partridge, Japanese quail, peafowl, pheasants, parakeets, turkeys, and chickens. The angle of needle entry on the median plane varied considerably between species and age of birds. Often an angle of entry that was satisfactory on young birds caused the needle to impact on the supraoccipital bone in older birds of the same species. Generally, angling the needle such that it passed just under the supraoccipital bone and into the dura mater was most successful. The length of needle insertion, required to puncture the occipital sinus, varied from 2 to 3 mm for a small parakeet to 10 to 12 mm for a large Muscovy drake. Usually the slight resistance to needle insertion felt when the needle tip contacted the dura mater could be used to perceive impending entry into the venous occipital sinus. These observations confirm the blood sampling effectiveness of this method in ducks and geese as reported by Vuillaume (1983) and also demonstrate the effectiveness of this technique in several other avian species. Because it is unlikely that the occipital sinus vasculature varies greatly between species, and the sampling method has been demonstrated to be effective in several species ranging in body size from small to large, it is reasonable to assume the method will be useful in all avian species.
OCCIPITAL SINUS BLOOD SAMPLING TABLE 1. Maximal blood volume obtained from l-day-old chicks utilizing different sampling methods
Decapitation Cardiac puncture Occipital sinus puncture
(ml) 1.36 ± .07a 1.70 .04b 1.66 : .09b
7 7 7
a' bMeans followed by a different superscript are significantly different (P<.01). 1 Means + SEM.
the desired length of needle to protrude. The needle sheath, trimmed to an appropriate length depending on the size of the bird, prevents excessive needle penetration, thus reducing the risk of neural damage. Maximum blood volumes obtained from l-day-old chicks using occipital sinus sampling, cardiac puncture, and decapitation are listed in Table 1. The volumes of blood obtained in this test always caused death by exsanguination. As expected, decapitation was the least efficient sampling method due to loss of blood in the head. Sampling blood from the occipital sinus provided volumes similar to those obtained by cardiac puncture. These observations indicate that venous occipital sinus sampling is an effective method for obtaining large volumes of blood. The utility of occipital sinus sampling for obtaining serial blood samples from birds is demonstrated in Table 2. Eight 1.0-ml blood samples were sequentially collected from each of five young chickens. At the end of this
TABLE 2. Timing of serial blood samples taken from the venous occipital sinus* Time 1.0-ml sample taken Sample number Bird
1
2
3
4
5 •
1 4 7 8 9
1 2 3 4 5 1
10 12 13 14 16
Refer to text for details.
17 18 18 19 20
26 29 30 32 33
6
7
8
Ninth sample volume (ml)
X
34 35 36 37 38
38 40 41 42 43
77 78 79 81 82
84 86 87 88 88
9.5 19.0 16.5 9.5 16.5
Downloaded from http://ps.oxfordjournals.org/ at Michigan State University on February 23, 2015
sampling period, the birds were alert, active, and did not appear to be greatly stressed. All 40 samples were obtained by one technician in 88 min., including a 30-min break between sample Numbers 6 and 7. The rapidity with which these samples were collected, as well as bird livability, indicates that occipital sinus sampling is an excellent method for serial blood sampling. In addition, a ninth blood sample for maximum volume yielded respectable quantities of blood. Examination of the skin surface, where the needle penetrated and sampling took place, showed redness and some swelling. Dissection of tissue in the sampling area revealed minor capillary hemorrhage but no hematoma. Table 3 lists the hematocrit and serum HI titer values measured in blood samples obtained from the brachial vein and the occipital sinus. Paired t-tests were unable to detect any significant (P*S.05) differences between the values obtained from the two sources. In addition, strong correlation coefficients between values obtained from the two bleeding sites were observed. This result indicates that occipital sinus sampling may be safely substituted for brachial vein sampling for these diagnostic purposes. There is some concern that occipital sinus blood samples could be inadvertently diluted with cerebrospinal fluid (CSF) because the blood sampling method described here is very similar to the CSF sampling method described by Anderson and Hazelwood (1969). Excessive needle penetration could result in CSF sampling from the subarachnoid space and the cisterna magna (Fig. 1). Significant dilution of blood with CSF could affect subsequent tests. The
Blood volume1
Sampling method
1861
1862
ZIMMERMANN AND DHILLON TABLE 3. Comparison of blood variables measured in samples taken from the brachial vein and the occipital sinus Serum titer -1 Infectious bronchitis
Bleeding site
n
Hematocrit
Arkansas 99
Massachusetts 41
Newcastle disease
Brachial vein1 Occipital sinus1 Correlation
10 10
35.3 ±1.4 34.6 ± .9 .69*
64 ± 6 66 ± 6 .82"
21 ± 7 22 ± 8 .92*"
15 ± 5 16 ± 5 .99*"
1
"(P<.05). **(P<.01). *"(P«.001).
CSF has lower protein and glucose content than plasma (Anderson and Hazelwood, 1969) and probably varies in other constituents as well. A sufficiently large blood sample should reduce this suspected dilution effect due to the comparatively small CSF reservoir volume. Although the hematacrit values in Table 3 show no significant dilution of blood taken from the occipital sinus, this sampling method probably should be used with caution for some tests. Venipuncture, as a method of obtaining large blood samples, was not included in the maximum volume test due to the difficulty of sampling from a vein in very small birds. This fact emphasizes the usefulness of the venous occipital sinus to obtain blood samples from very small birds. The only other large volume, nonlethal-sampling technique for use in small birds is cardiac puncture. Although it was not objectively measured, we believe occipital sinus blood sampling by an experienced technician is less of a life-threatening procedure than cardiac puncture. However, the risk of neural trauma during occipital sinus blood sampling relegates it to a method of choice only when venipuncture is impractical.
ACKNOWLEDGMENTS
The authors wish to express their appreciation to Wayne Kuenzel for his advice and to the poultry and veterinary staff at Western Washington Research and Extension Center for their technical assistance. REFERENCES Anderson, D. K., and R. L. Hazelwood, 1969. Chicken cerebrospinal fluid: Normal composition and response to insulin administration. J. Physiol. (Lond.) 202:83-95. Beard, C. W., and W. J. Wilkes, 1973. A simple and rapid micro-test procedure for determining newcastle hemagglutination-inhibition (HI) antibody titers. 596—600 in Proc. 77th Annu. Mtg. US Anim. Health Assoc. King, D. J., and S. R. Hopkins, 1983. Evaluation of the hemagglutination-inhibition test for measuring the response of chickens to avian infectious bronchitis virus vaccination. Avian Dis. 27:100-112. Richards, S. A., 1968. Anatomy of the veins of the head in the domestic fowl. J. Zool. (Lond.) 154:223-234. SAS Institute Inc. 1982. SAS User's Guide: Statistics, 1982 Edition. SAS Inst. Inc., Cary, NC. Vuillaume, A., 1983. A new technique for taking blood samples from ducks and geese. Avian Pathol. 12:389-391.
Downloaded from http://ps.oxfordjournals.org/ at Michigan State University on February 23, 2015
Means ± SEM.
INTRODUCTION
avian species and to determine the validity of substituting this method for other routine sampling methods.
Nonlethal blood sampling is necessary for a wide variety of avian diagnostic and research purposes. Venipuncture, usually from the brachial vein, but also from the jugular and metatarsal veins, is most commonly used. This nonlife-threatening method frequently causes a hematoma, making multiple samplings difficult or impossible. Relatively large multiple blood samples are readily obtained by cardiac puncture but at greater risk to the bird. Recently, Vuillaume (1983) described a method for obtaining large multiple blood samples from the venous occipital sinus of ducks and geese. Richards' (1968) studies of cephalic vein anatomy suggested that occipital sinus blood was primarily of endocranial origin. Thus, it is possible that venous occipital sinus blood sample constituents vary from those of samples taken from other sources. The purpose of the study described below was to test the utility of venous occipital sinus blood sampling in several
Vuillaume's (1983) blood sampling procedure requires positioning of the head and neck of the bird at a right angle to increase the dorsal angle of articulation between the skull and the first cervical vertebra. This positioning creates a dorsal concavity which can be palpated. The skin is pierced in the center of this area on the median plane using a 21 to 25 gauge needle. The needle is slowly inserted at approximately a 45 angle to the neck in order to position the point in the dura mater under the posterior edge of the supraoccipital bone. This is an area rich in venous blood flow, where several vessels join to form the occipital and vertebral sinuses (Fig. 1). The blood-drawing efficacy of this technique was tested in several avian species.
1 A portion of these data was presented at the 73rd Annual Meeting of the Poultry Science Association, Inc., August 1984, Guelph, Ontario. 2 Scientific Paper No. 7039, College of Agriculture Research Center, Washington State University. Projects 0643 and 0576. 3 Department Animal Science. 4 Veterinary Microbiology and Pathology.
The maximum blood volume obtained by venous occipital sinus sampling was compared to maximum volumes obtained by cardiac puncture and decapitation. Seven 1-day-old broiler chicks were bled by each method. Data were compared by analysis of variance with Duncan's multiple range test. The utility of venous occipital sinus sampling for serial bleeding was tested by sequentially bleeding five, 10-week-old Leghorn chick-
MATERIALS AND METHODS
1859
Downloaded from http://ps.oxfordjournals.org/ at Michigan State University on February 23, 2015
ABSTRACT Venous occipital sinus blood sampling was demonstrated to be an effective nonlethal method in all species tested including ducks, geese, Chukar partridge, Japanese quail, peafowl, pheasants, parakeets, turkeys, and chickens. Maximum blood volume obtained from the occipital sinus of 1-day-old chicks equalled the volume obtained by cardiac puncture and exceeded the volume collected by decapitation. Venous occipital sinus puncture was successfully used to rapidly collect several successive blood samples from individual birds, thus indicating the method's utility for serial blood sampling. Hematocrit values, as well as infectious bronchitis and Newcastle disease virus antibody titers measured in blood samples drawn from the occipital sinus and the brachial vein, were compared. Significant correlation coefficients between the two blood sources for each of the variables measured suggest that occipital sinus blood sampling may be substituted for venipuncture for many routine measurements. (Key words: occipital sinus, blood sampling) 1985 Poultry Science 64:1859-1862
1860
ZIMMERMANN AND DHILLON RESULTS AND DISCUSSION
FIG. 1. Diagramic representation of the cephalic venous vasculature and approximate angle of sampling needle insertion (adapted from Richards, 1968).'
ens weighing approximately 650 g. Eight 1.0-ml blood samples were taken from each bird and the time of each bleeding recorded. A ninth sample for maximum volume was also taken and the volume recorded. The homology of blood hematocrit, infectious bronchitis, and Newcastle disease serum hemagglutination inhibition (HI) titer was determined between samples from the brachial vein and the venous occipital sinus. Hematocrit and serum antibody titer were determined in each of the 3.0-ml blood samples taken from the venous occipital sinus and the brachial vein of ten, 23-week-old Leghorn pullets. Infectious bronchitis (Arkansas 99 and Massachusetts 41 strains) and Newcastle disease serum HI titers were determined by the Poultry Diagnostic Laboratory, Western Washington Research and Extension Center, Puyallup, using procedures described by King and Hopkins (1983) and Beard and Wilkes (1973), respectively. All samples were assayed in duplicate. Hematocrit and reciprocal titer values between the two bleeding sites were analyzed by paired t-test and correlation coefficient. All statistics were compiled using SAS Institute Inc. (1982) procedures.
Although occipital sinus sampling is a very effective method of obtaining blood samples from birds, it is also potentially dangerous to the health of the bird. The sampling site in the dura mater is in close approximation to easily damaged neural tissue. If the bird moves during sampling, or if the syringe needle inadvertently penetrates too far into the skull, severe neural damage or death can occur. The former malady is quite prevalent especially in larger, stronger birds even when an extra technician is employed to hold the bird. The sampling procedure does not appear to be painful but some birds object to immobilization of the head. The latter situation often occurs due to the use of excessive pressure when pushing the sampling needle into the tough fibrous connective tissue of the dura mater. Additional pressure seemed to be required to puncture the dura in older birds. Losses, attributed to these problems, can be largely eliminated by retaining a trimmed needle sheath on the needle which allows only
Downloaded from http://ps.oxfordjournals.org/ at Michigan State University on February 23, 2015
'Abbreviations are: ACb, anterior cerebral vein; C, carotid vein; Cm, cisterna magna; Cv, cervical sinus; DCO, dorsal cerebral ophthalmic vein; E, ethmoid vein; EF, external facial vein; EOc, external occipital vein; FCV, first cervical vertebra; IF, internal facial vein; IOc, internal occipital vein; J. jugular vein; M, maxillary vein; MC, middle cerebral vein; MD, mid-dorsal sinus; Oc, occipital sinus; Op, ophthalmic vein; PC, posterior cephalic vein; ROp, recurrent ophthalmic vein; SC, sinus cavernosus; SOB, supra occipital bone; TA, transverse anastomosis; TOp, temporal ophthalmic vein; TR, temporal rete; V, vertebral vein.
Blood samples were successfully drawn from the venous occipital sinus in each of the species tested. These included ducks, geese, Chukar partridge, Japanese quail, peafowl, pheasants, parakeets, turkeys, and chickens. The angle of needle entry on the median plane varied considerably between species and age of birds. Often an angle of entry that was satisfactory on young birds caused the needle to impact on the supraoccipital bone in older birds of the same species. Generally, angling the needle such that it passed just under the supraoccipital bone and into the dura mater was most successful. The length of needle insertion, required to puncture the occipital sinus, varied from 2 to 3 mm for a small parakeet to 10 to 12 mm for a large Muscovy drake. Usually the slight resistance to needle insertion felt when the needle tip contacted the dura mater could be used to perceive impending entry into the venous occipital sinus. These observations confirm the blood sampling effectiveness of this method in ducks and geese as reported by Vuillaume (1983) and also demonstrate the effectiveness of this technique in several other avian species. Because it is unlikely that the occipital sinus vasculature varies greatly between species, and the sampling method has been demonstrated to be effective in several species ranging in body size from small to large, it is reasonable to assume the method will be useful in all avian species.
OCCIPITAL SINUS BLOOD SAMPLING TABLE 1. Maximal blood volume obtained from l-day-old chicks utilizing different sampling methods
Decapitation Cardiac puncture Occipital sinus puncture
(ml) 1.36 ± .07a 1.70 .04b 1.66 : .09b
7 7 7
a' bMeans followed by a different superscript are significantly different (P<.01). 1 Means + SEM.
the desired length of needle to protrude. The needle sheath, trimmed to an appropriate length depending on the size of the bird, prevents excessive needle penetration, thus reducing the risk of neural damage. Maximum blood volumes obtained from l-day-old chicks using occipital sinus sampling, cardiac puncture, and decapitation are listed in Table 1. The volumes of blood obtained in this test always caused death by exsanguination. As expected, decapitation was the least efficient sampling method due to loss of blood in the head. Sampling blood from the occipital sinus provided volumes similar to those obtained by cardiac puncture. These observations indicate that venous occipital sinus sampling is an effective method for obtaining large volumes of blood. The utility of occipital sinus sampling for obtaining serial blood samples from birds is demonstrated in Table 2. Eight 1.0-ml blood samples were sequentially collected from each of five young chickens. At the end of this
TABLE 2. Timing of serial blood samples taken from the venous occipital sinus* Time 1.0-ml sample taken Sample number Bird
1
2
3
4
5 •
1 4 7 8 9
1 2 3 4 5 1
10 12 13 14 16
Refer to text for details.
17 18 18 19 20
26 29 30 32 33
6
7
8
Ninth sample volume (ml)
X
34 35 36 37 38
38 40 41 42 43
77 78 79 81 82
84 86 87 88 88
9.5 19.0 16.5 9.5 16.5
Downloaded from http://ps.oxfordjournals.org/ at Michigan State University on February 23, 2015
sampling period, the birds were alert, active, and did not appear to be greatly stressed. All 40 samples were obtained by one technician in 88 min., including a 30-min break between sample Numbers 6 and 7. The rapidity with which these samples were collected, as well as bird livability, indicates that occipital sinus sampling is an excellent method for serial blood sampling. In addition, a ninth blood sample for maximum volume yielded respectable quantities of blood. Examination of the skin surface, where the needle penetrated and sampling took place, showed redness and some swelling. Dissection of tissue in the sampling area revealed minor capillary hemorrhage but no hematoma. Table 3 lists the hematocrit and serum HI titer values measured in blood samples obtained from the brachial vein and the occipital sinus. Paired t-tests were unable to detect any significant (P*S.05) differences between the values obtained from the two sources. In addition, strong correlation coefficients between values obtained from the two bleeding sites were observed. This result indicates that occipital sinus sampling may be safely substituted for brachial vein sampling for these diagnostic purposes. There is some concern that occipital sinus blood samples could be inadvertently diluted with cerebrospinal fluid (CSF) because the blood sampling method described here is very similar to the CSF sampling method described by Anderson and Hazelwood (1969). Excessive needle penetration could result in CSF sampling from the subarachnoid space and the cisterna magna (Fig. 1). Significant dilution of blood with CSF could affect subsequent tests. The
Blood volume1
Sampling method
1861
1862
ZIMMERMANN AND DHILLON TABLE 3. Comparison of blood variables measured in samples taken from the brachial vein and the occipital sinus Serum titer -1 Infectious bronchitis
Bleeding site
n
Hematocrit
Arkansas 99
Massachusetts 41
Newcastle disease
Brachial vein1 Occipital sinus1 Correlation
10 10
35.3 ±1.4 34.6 ± .9 .69*
64 ± 6 66 ± 6 .82"
21 ± 7 22 ± 8 .92*"
15 ± 5 16 ± 5 .99*"
1
"(P<.05). **(P<.01). *"(P«.001).
CSF has lower protein and glucose content than plasma (Anderson and Hazelwood, 1969) and probably varies in other constituents as well. A sufficiently large blood sample should reduce this suspected dilution effect due to the comparatively small CSF reservoir volume. Although the hematacrit values in Table 3 show no significant dilution of blood taken from the occipital sinus, this sampling method probably should be used with caution for some tests. Venipuncture, as a method of obtaining large blood samples, was not included in the maximum volume test due to the difficulty of sampling from a vein in very small birds. This fact emphasizes the usefulness of the venous occipital sinus to obtain blood samples from very small birds. The only other large volume, nonlethal-sampling technique for use in small birds is cardiac puncture. Although it was not objectively measured, we believe occipital sinus blood sampling by an experienced technician is less of a life-threatening procedure than cardiac puncture. However, the risk of neural trauma during occipital sinus blood sampling relegates it to a method of choice only when venipuncture is impractical.
ACKNOWLEDGMENTS
The authors wish to express their appreciation to Wayne Kuenzel for his advice and to the poultry and veterinary staff at Western Washington Research and Extension Center for their technical assistance. REFERENCES Anderson, D. K., and R. L. Hazelwood, 1969. Chicken cerebrospinal fluid: Normal composition and response to insulin administration. J. Physiol. (Lond.) 202:83-95. Beard, C. W., and W. J. Wilkes, 1973. A simple and rapid micro-test procedure for determining newcastle hemagglutination-inhibition (HI) antibody titers. 596—600 in Proc. 77th Annu. Mtg. US Anim. Health Assoc. King, D. J., and S. R. Hopkins, 1983. Evaluation of the hemagglutination-inhibition test for measuring the response of chickens to avian infectious bronchitis virus vaccination. Avian Dis. 27:100-112. Richards, S. A., 1968. Anatomy of the veins of the head in the domestic fowl. J. Zool. (Lond.) 154:223-234. SAS Institute Inc. 1982. SAS User's Guide: Statistics, 1982 Edition. SAS Inst. Inc., Cary, NC. Vuillaume, A., 1983. A new technique for taking blood samples from ducks and geese. Avian Pathol. 12:389-391.
Downloaded from http://ps.oxfordjournals.org/ at Michigan State University on February 23, 2015
Means ± SEM.