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Diagnosis of pregnancy in moose using a bovine assay for pregnancy-specific protein B
Theriogenology
40:905-911,1993
DIAGNOSIS OF PREGNANCY IN MOOSE USING A BOVINE ASSAY FOR PREGNANCY-SPECIFICPROTEIN B J. C. Haigh! W. J. Dalton? C. A. Ruder3 and R. G. Sassera 1Departmentof Herd Medicine & Theriogenology, Western College of Veterinary Medicine University of Saskatchewan,Saskatoon, S7N OWO,Canada 2Ontario Ministry of Natural Resources, Thunder Bay, Ontario, WC 5G6, Canada 3Departmentof Animal Science, University of Idaho, Moscow, ID 83843 Received for publication: March 9, 1993 Accepted; August 3, 1993 ABSTRACT Blood samples were collected from 26 moose (Aloes ale@ and evaluated for the presence of an antigen that cross-reacted with antisera to bovine pregnancy-specific protein B (P-SPB). The objective of this study was to &tern-&e if the P-SPB radioimmunoassay(RIA) was a reliable indicator of pregnancy in these animals. In the first year of the study calf production the following summer was used as the index of previous pregnancy. In the second year all females were subjected to palpation per rectum after chemical immobilization.Seven of the 10 cows sampledin the first year were also sampled in the second year. All animals determined pregnant by rectal palpation were positive for P-SPB; however, P-SPB was not detected in males. Key
words: Moose, pregnancy diagnosis,pregnancy-specific protein B INTRODUCTION
Analysis of ungulate serum for chemical indicators of pregnancy provides a means of detecting pregnancy without having to perform palpation per rectum, infer pregnancy rates from ovarianexamina tion of hunter killed animals (22) or to sacrifice animals. This approach is particularly suited to population studies on endangered species or otherwise vahtable wild stock where it allows determmationof pregnancy rates in demographic studies without affecting animal survivorship. Bovine pregnancy-specific protein B (P-SPB) is produced by the bi-nucleate cells of the trophoblastof cattle (4,13), and its detection is used as a diagnostic technique in this species (16). The use of P-SPB as a means of pregnancy testing has been demonstrated m a number of ungulates (17). Cross-reactivity with bovine P-SPB in radioimmunoassayhas been demonstrated in other mammals with cotyledonary placentation(6,7,10,14). Here we report the result of a study, limited for practical reasons to a small number of free-ranging animals,to determine whether such crossreactivity would be useful as an aid to detect pregnancy diagnosis in moose. MATERIALS AND METHODS Moose in north-western Ontario area were captured by darting from a helicopter using carfentanil and xylazme (8,18). Capture was carried out in mid-Decemberor mid-Januaryin the first year, and in late February and March in the second year. An incisiform canine tooth was removed from each moose during the handling procedure. Cementurnannuli were counted in order
Copyright
0 1993 Butterworth-Heinemann
T~erjog~nu/ogy
906
to provide an estimateof the age of the animal. All the female moose captured were fitted with radio collars. Over the course of the 2 study seasonsblood samples from 26 free-ranging moose were collected (10 cows and 7 bulls in the first year, 13 cows and 4 bulls in the second). Of these, 8 cows and 1 bull were tested in both years; and, of these, sera from 2 cows collected in the first year were resubmitted with the samples taken in the second year. Two cows in the second year sample were aged 1.5 yr. All the other cows were between 2.5 and 12.5 years of age. The sampleswere allowed to clot, and the harvested serum was split into 3 sub-samplesbefore being frozen at -200C. Each sub-samplewas assigned a unique number selected from a random numbers table. The serum was shipped to the laboramry withont any key to indicate animal identity or sex. In the first study season no palpation per rectum was performed. In the following late Ma and mid June we ground-trackedall radiocollared cow moose for observation, particularly to see r*r there was a calf with the cow. During the course of handling procedures on captured moose in the second study season, palpation per rectum for determinationofpregnancy was carried out. The sub-sampleswere assayed in duplicate for P-SPB i~~~ti~~ by a double radioimmunoassay using rabbit antiserum to bovine P-SPB (16). The P-SPB was isolated t?om bovine cotyledonary tissue (preparation R-37) and was used for standardand for radioiodination with r z51. 200 microliters of plasma were added to duplicate assay tubes, and depressionof binding of ~oi~~~ bovine P-WE to the antiserum s 95% of controi values was considered indicative of pregnancy. Samplesin which binding was > 95% were considered to have come from animals that were nonpregnant. Intraassayprecision was measured by the median variance ratio of duplicate assays of samples. Interassay precision was not measured due to changes in laboratory serum pools over the 2 yr course of the study. Laboratory results related to the randomly numbered tubes were matched to the identification numbers of the animals. RESULTS Median variance ratios from assay of replicate samplesaveraged 0.025 and 0.01 in each assay, respectively. The cutoff for stating that an animal was pregnant [( 95% binding of label) was less than 0.043 and 0.042 ng of R-37 P-SPB\assay tube in the respective assays. No inhibition curve was generated by assaying different volumes of sera from the nonpregnantanimals. The rtage of binding in the various samples, sub-samplesand replicates are shown in Tables 1 and The P-SPB was detected in all 10 cows tested in the first year; 7 females were seen with calves during the May and June searches. Of the 3 not seen with calves 2 were not observed in May_ Of the 13 cows palpated in 1988 only the 2 yearlings (1.5 yr) were not pregnant. Neither of them had detectable P-SPB in their sera. All of the animals de&mined to be pregnant by palpation per rectum were positive for P-SPR. No P-SPB was detected in the serum of the 3 captive calves. None of the males had detectable P-SPB in their serum. One replication from each sub~ple of three in~vid~ males had a measured binding 9 95% P-SPB ~92~%-95%) (Table 2). DISCWSSION The dentin of a ~y~~ific protein, derived Erombovine biiucleate whilst cells (4,13), has allowed for the early detection of pregnancy in cattle (16). The same test has been
a b
P P P 0 P P 0 P
30-Mar 24-Feb 24-Feb 24-Feb 25-Feb 26-Feb
P P
25-Feb 25-Feb
‘I-Mar 26-Feb
P
25-Feb
Pb P
55 57 60 67 63 61 49 57 64 51 52 55 54 97 51 48 103 50
54 58 60 70 62 64 49 59 63 52 so 55 54 102 55 50 103 51
Ground check for calves was not conducted until mid-June. Parturition was in mid-May. Cow was eutbanatized and autopsied, twin fetuses were present.
1 1 Oa 2
1
P P P P P P
14&n
resubmit 16&n 16-Jan 14-Jan 16&l
2 Oa Oa
P P P
19-Dee 19-Dee 19-Dee
23-Feb 23-Feb
56 59 56 71 62 62 47 59 64 55 SO 55 55 103 56 41 101 53
5s 49 59 70 63 62 46 58 63 53 51 53 52 101 53 48 99 53
53 58 56 54 62 64 47 58 64 54 48 59 52 102 53 SO 101 51
53 58 60 67 62 63 48 58 68 52 51 57 55 98 60 49 101 52
9.5 8.5 8.5 9.5 3.5 7.5 5.5 3.5 3.5 11.5 6.5 4.5 3.5 1.5 8.5 2.5 1.5 9.5
2 3 3 6 7 9 11 13 13 14 IS 16 20 22 27 28 29 30
2 2
Year1 Year 1 No. of Year2 Palpation % Binding Sampling P-SPB calves Sampling ezam Sub-sample 3 date status seen date Sub-sample 1 Sub-sample 2 Replicate 1 Replicate 2 Replicate 1 Replicate 2 Replicate 1 Replicate 2 (day-month) (day-month)
Age (years) at 1st sample
ID #
P P
Serum assay of female moose for binding to antibody for bovine P-SPB. Pregnant animals are designated by a P in the table, nonpregnant animals are designated by an 0.
Table 1.
54.0 f 1.20 56.5 f 1.54 58.~5f 0.8i 66.5 f 2.59 62.3 -f 0.52 62.7 f 1.21 47.7 f 1.21 58.2 M.75 64.3 f 1.85 52.8 f 1.47 SO.3f 1.37 55.7 f 2.07 53.7 f 1.37 100.5f 2.43 54.7 i3.14 48.7 i 1.21 101.3 * 1.51 51.1 f 1.21
MeanfSD
4 5 8 8 10 12 17 18 19 26
1
ID #
date
SampIing
8.5 3.5 5.5 5.5 6.5 8.5 5.5 2.5 8.5 4.5 6.5
sample
19-Dee 15&n IS-Jm 14-Jan 25-Feb 26-Feb 15-h 14-h 23-Feb 14Jan 24-Feb
Sub-mqle
1 Sub-sample2
% Binding Sllbsample 3
101 100 96 98 99 101 96 100 100 102 99
101 102 93 97 98 99 101 100 101 100 93 105 97 96 91 102 103 82 96 98 102 99 98 97 98 103 98 95 98 loo
102
98 91 103 99 103 100 94 101
100 100 93 96 101 103 102 103 102 104
103 95
Replicate 1 Replicate 2 Replicate 1 Replicate 2 Repkate 1 Rqdicate2
at 1st (day-month)
Age (Ye=)
102.0 f 99.3 f 96.3 f 96.7 f 97.3 f loo.7 f 100.8 f 97.5 f 99.2 f 99.0 f 99.8 f
1.79 2.99 2.50 1.97 1.03 1.86 2.86 7.79 3.06 3.03 3.76
Mean&SD
Table 2Semm assay of malemoose. For 7 bulls 1 replicate showed 5 95% biadiq whichcan indicatapregnancy if the s 95% level is taken as the division point. Repeat measurementsand mean values were > 95%.
Theriogenology
909
found to have a high degree of cross-reactivity in bison (7), sheep (ovis spp; 15), mule and whitetailed deer (Gdocow spp;_23),mountain goats (Greamn amencanus; lo), red deer (m elanhus; 6), musk-oxen (ovlbos moschatus; 14), and a zty of other animals with cotyledonary placentation (17). Nothing is known of the timing of appearance of pregancy-specific protein in moose relative to conception, nor of its disappearanceafter parturition or abortion. In red deer, pregnancy-specific protein is present by 30 days post-conception (6), and in most cattle P-SPB is present by 24 days of gestation (16). In cattle, P-SPB has a half-life of 8.4 days (11). A&r parturition or abortion pregancy-specific protein has disappeared by 90 days in cattle, and by 25 days in sheep (Sasser RG, personal communication). Moose are seasonal breeders, the rut occurring in late September and early October. For practical pmposes sampling for pregancyspecific protein should not be carried out until mid-November at the earliest Samplingof &eeranging moose late in gestation (past mid-March)is contraindicatedbecause of the potential for capture-related mortality in heavily pregnant animals. The cut-off of 95% binding was chosen because it had been the established figure used in cattle In this study, with the exception of one outlier of 82%, all bulls had binding 2 93%) which is the figure currently used for cutoff in cattle (Sasser RG, personal communication). The d~scmpancy between non-pregnant animals and pregnant or presumed pregnant ones (571%) would have permitted a lower cut-off point without altering the results. If a cow was accompanied by a calf in the May/June following the first capture season this was taken to indicate that she had been pregnant at the time of capture. The 3 animalsthat were not observed with calves in the first year of the study all had diagnostic concentrations of P-SPB in December&nmary. It is possible that they were not pregnant, or that they had recently aborted. However, it is more likely that by the time they were seen in mid-June they had already lost their calves due to predation. Black bears (Urs m and wolves (Canis lunus) are known pmdators of moose calves in the first few weeks of life (5,21) and share range with moose in the study area. Both yearling females sampled in this study were nonpregnant. Yearling moose may conceive and carry calves, but the proportion of them that do so in any given year is primarily dependent upon the quahty of their range in the summer preceding the breeding season and pregnancy rates may range from 0 to 93% (1). Culling carried out during the gestation period obviously provides the most accurate indicator of pregnancy rates, but other less invasive methods are useful and desirable. Palpation per rectum is routinely used to detect pregnancy in cattle, and has been similarly used in immobilized bison and moose (7,s). Portable ultrasound equipment has also been used in free ranging moose (Glover GG, personal communication), mule deer (19), and bighorn sheep @&iscanadensis; 9). Serum progesterone assay has been reported as a means of determining pregnancy in ungulates and has been used in bighorn sheep (2) and moose (8,20). Progesterone as an indicator of pregnancy has the disadvantagethat it is also present during the luteal phase of the estrous cycle. Concentrations of progesterone measured during pregnancy or the cycle may overlap, so that high levels can only be taken as indicatingpregnancy if they are either obtained after the end of seasonalcyclic&y,or repeated iiequently during a period longer than 1 cycle in ordertoshowthattheyremainhigh. Immunoreactive fecal progestins and estrogen$F been used as indicators of cyclicity and pregnancy in cattle, muskoxen (3) and caribou @ana tarandus; 12). They have the advantage over other methods in that their collection is noninvasive,but in free-rangingpopulations they are only valuable as indicators of reproductive activity of populations rather than of individuals High
910
Theriogenology
total estrogen and progesterone concentrationsin feces have been shown to correlate well with late pregnancy in muskoxen (3) and caribou (12). The results in the study reported here show that a bovine P-SPB antibody is a useful indicator of well establishedpregnancy in moose. Determinationsof the time of its appearance in early pregnancy, and its disappearanceafter parturition, remain to be conducted. REFERENCES
1. Boer A. Fecundity of North American moose (Aloesalces): a review. Alces supplement. 1992; 1: l-10. 2. Brumge GC, Lane LJ, McCabe TR. Early pregnancy determination using serum progesterone concentration in bighorn sheep. J Wildl Manage 1988; 52: 610612 ). 3. Desaulniers DM, Goff AK, Be&ridge KJ, Rowell JE, Flood PF. Reproductive hormone concentrations in faeces during the oestrous cycle and pregnancy in cattle (Bos taurus) and muskoxen (Ovibos moschatus). Can J Zoo1 1987; 67: 1148-l 154. 4. Eckblad WP, Sasser RG, Ruder CA, PanlasuigiPM, Kuszynski TS. Locahzation of pregnancy-specific protein B (P-SPB) in bovine placenta cells using a glucose oxidase anti-glucose oxidase \ stain. J Anim Sci 1985; 61 (Suppl): 149-150. 5. Franzmann AW, Schwartz CC. Black bear predation on moose calves in highly productive versus marginal moose habitats on the Kenai peninsula, Alaska Alces 1986; 22: 139-154. 6. Haigh JC, Cranfield M, SassedRG. Estrus synchronization and pregnancy diagnosis in red deer. 3 Zoo Anim Med 1988; 19: 202-207. 7. Haigh JC, Gates C, Ruder A, Sasser R. Diagnosis of pregnancy in wood bison using a bovine assay for pregnancy-specific protein B. Theriogenomgy 1991; 36: 749-754. 8. Haigh JC, Kowal EH, Runge W, Wobeser G. Pregnancy diagnosis as a management tool for moose. Aloes. 1982; 18: 45-53. 9. Harper WL, Cohen RD. Accuracy of Doppler ultrasound in diagnosingpregnancy in bighorn sheep. J Wildl Manage 1985; 49: 793-796. 10. Houston DB, Robbii CT, Ruder CA, Sasser RG. Pregnancy detection in mountain goats by assay for pregnancy-specific protein B. J Wildl Manage 1986; 50: 740-742. 11. Kiicofe GH, Wright JM, Schalles RR, Ruder CA, Prish S, Sasser RG. Pregnancyspecific protein B in serum of postpartum beef cows. J Anim Sci 1993; 71: In press. 12. Messier F, Desaulniers DM, Goff AK, Nault R, Patenaudc R, Crete M. Caribou pregnancy diagnosis from immunoreactive progestins and estrogens excreted in feces. J Wiidl Manage 1990; 54: 279-283. 13. Reimers TJ, Sasser RG, Ruder CA. Production of pregnancy-specific protein by binucleate trophoblastic cells. Biol Reprod Suppll985; 132: 65. (Abstr.). 14. Rowell JE, Flood PF, Ruder CA, Sasser RG. Pregnancy-specificprotein in the plasma of captive muskoxen. J Wildl Manage 1989; 53: 899901. 15. Ruder CA, StelIfug JN, Dahmen JJ, Sasser RG. Deteceon of pregnancy in sheep rad&n;;“““oa”say of sera for pregnancy-specific protem B. Thermgenology 1988 29: 16. Sasser RG, Ruder CA, Ivani KA, Butler JE, Hamilton WC. Detection of pregnancy by radioimmunoassayof a novel pregnancy-specific protein in serum of cows and a profile of serum concentrations during gestation. Biol Reprod 1986; 35: 936-942. 17. Sasser RG, Ruder CA, Ivani KA, Short RE, Wood AK. Pregnancy-specific protein B in serum of various species. In: Ellemiorf, F., and Koch, E. (eds.), Reproductiveand PeriNatal Medicine. 1. Early Pregnancy Factors. Perinatology Press, Ithaca. 1985; 161. 18. Schmitt SM, Dalton WJ. Immobilization of moose by carfentanil and x lazine and reversal by nahrexone, a long acting antagonist. Aloes 1987;23: 195-213. 19. Smith RB, Lindzey FG. Use of ultrasound for detecting pregnancy in mule deer. J Wildl Manage 1982; 46: 1089-1092.
Theriogenology
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Comishen-StewartLM, Haigh JC. Levels of some reproductive hormones in relation to pregnancy in moose: a preliminary report. Alces 1985; 21: 393402. 21. Stewart RR. Kowal EXT.BeaulieuR. Rock TW. The imoact of black bear removal on moose calf survival in &st central S&atchewan. Alces 1985; 21: 403-418. 22. Verme, L.J. Problems in appraisingreproduction in Cervidae. Proc. N. Amer. Moose. Conf. Workshop. 1974; 10: 22-36. 23. Wood AK, Short RI$ Darling A, Dusek GL, Sasser RG, Ruder CA. Serum assays for detecting pregnancy m mule and white-tailed deer. J Wildl Manage,l986; 50: 684-687. 20. Stewart RR,
40:905-911,1993
DIAGNOSIS OF PREGNANCY IN MOOSE USING A BOVINE ASSAY FOR PREGNANCY-SPECIFICPROTEIN B J. C. Haigh! W. J. Dalton? C. A. Ruder3 and R. G. Sassera 1Departmentof Herd Medicine & Theriogenology, Western College of Veterinary Medicine University of Saskatchewan,Saskatoon, S7N OWO,Canada 2Ontario Ministry of Natural Resources, Thunder Bay, Ontario, WC 5G6, Canada 3Departmentof Animal Science, University of Idaho, Moscow, ID 83843 Received for publication: March 9, 1993 Accepted; August 3, 1993 ABSTRACT Blood samples were collected from 26 moose (Aloes ale@ and evaluated for the presence of an antigen that cross-reacted with antisera to bovine pregnancy-specific protein B (P-SPB). The objective of this study was to &tern-&e if the P-SPB radioimmunoassay(RIA) was a reliable indicator of pregnancy in these animals. In the first year of the study calf production the following summer was used as the index of previous pregnancy. In the second year all females were subjected to palpation per rectum after chemical immobilization.Seven of the 10 cows sampledin the first year were also sampled in the second year. All animals determined pregnant by rectal palpation were positive for P-SPB; however, P-SPB was not detected in males. Key
words: Moose, pregnancy diagnosis,pregnancy-specific protein B INTRODUCTION
Analysis of ungulate serum for chemical indicators of pregnancy provides a means of detecting pregnancy without having to perform palpation per rectum, infer pregnancy rates from ovarianexamina tion of hunter killed animals (22) or to sacrifice animals. This approach is particularly suited to population studies on endangered species or otherwise vahtable wild stock where it allows determmationof pregnancy rates in demographic studies without affecting animal survivorship. Bovine pregnancy-specific protein B (P-SPB) is produced by the bi-nucleate cells of the trophoblastof cattle (4,13), and its detection is used as a diagnostic technique in this species (16). The use of P-SPB as a means of pregnancy testing has been demonstrated m a number of ungulates (17). Cross-reactivity with bovine P-SPB in radioimmunoassayhas been demonstrated in other mammals with cotyledonary placentation(6,7,10,14). Here we report the result of a study, limited for practical reasons to a small number of free-ranging animals,to determine whether such crossreactivity would be useful as an aid to detect pregnancy diagnosis in moose. MATERIALS AND METHODS Moose in north-western Ontario area were captured by darting from a helicopter using carfentanil and xylazme (8,18). Capture was carried out in mid-Decemberor mid-Januaryin the first year, and in late February and March in the second year. An incisiform canine tooth was removed from each moose during the handling procedure. Cementurnannuli were counted in order
Copyright
0 1993 Butterworth-Heinemann
T~erjog~nu/ogy
906
to provide an estimateof the age of the animal. All the female moose captured were fitted with radio collars. Over the course of the 2 study seasonsblood samples from 26 free-ranging moose were collected (10 cows and 7 bulls in the first year, 13 cows and 4 bulls in the second). Of these, 8 cows and 1 bull were tested in both years; and, of these, sera from 2 cows collected in the first year were resubmitted with the samples taken in the second year. Two cows in the second year sample were aged 1.5 yr. All the other cows were between 2.5 and 12.5 years of age. The sampleswere allowed to clot, and the harvested serum was split into 3 sub-samplesbefore being frozen at -200C. Each sub-samplewas assigned a unique number selected from a random numbers table. The serum was shipped to the laboramry withont any key to indicate animal identity or sex. In the first study season no palpation per rectum was performed. In the following late Ma and mid June we ground-trackedall radiocollared cow moose for observation, particularly to see r*r there was a calf with the cow. During the course of handling procedures on captured moose in the second study season, palpation per rectum for determinationofpregnancy was carried out. The sub-sampleswere assayed in duplicate for P-SPB i~~~ti~~ by a double radioimmunoassay using rabbit antiserum to bovine P-SPB (16). The P-SPB was isolated t?om bovine cotyledonary tissue (preparation R-37) and was used for standardand for radioiodination with r z51. 200 microliters of plasma were added to duplicate assay tubes, and depressionof binding of ~oi~~~ bovine P-WE to the antiserum s 95% of controi values was considered indicative of pregnancy. Samplesin which binding was > 95% were considered to have come from animals that were nonpregnant. Intraassayprecision was measured by the median variance ratio of duplicate assays of samples. Interassay precision was not measured due to changes in laboratory serum pools over the 2 yr course of the study. Laboratory results related to the randomly numbered tubes were matched to the identification numbers of the animals. RESULTS Median variance ratios from assay of replicate samplesaveraged 0.025 and 0.01 in each assay, respectively. The cutoff for stating that an animal was pregnant [( 95% binding of label) was less than 0.043 and 0.042 ng of R-37 P-SPB\assay tube in the respective assays. No inhibition curve was generated by assaying different volumes of sera from the nonpregnantanimals. The rtage of binding in the various samples, sub-samplesand replicates are shown in Tables 1 and The P-SPB was detected in all 10 cows tested in the first year; 7 females were seen with calves during the May and June searches. Of the 3 not seen with calves 2 were not observed in May_ Of the 13 cows palpated in 1988 only the 2 yearlings (1.5 yr) were not pregnant. Neither of them had detectable P-SPB in their sera. All of the animals de&mined to be pregnant by palpation per rectum were positive for P-SPR. No P-SPB was detected in the serum of the 3 captive calves. None of the males had detectable P-SPB in their serum. One replication from each sub~ple of three in~vid~ males had a measured binding 9 95% P-SPB ~92~%-95%) (Table 2). DISCWSSION The dentin of a ~y~~ific protein, derived Erombovine biiucleate whilst cells (4,13), has allowed for the early detection of pregnancy in cattle (16). The same test has been
a b
P P P 0 P P 0 P
30-Mar 24-Feb 24-Feb 24-Feb 25-Feb 26-Feb
P P
25-Feb 25-Feb
‘I-Mar 26-Feb
P
25-Feb
Pb P
55 57 60 67 63 61 49 57 64 51 52 55 54 97 51 48 103 50
54 58 60 70 62 64 49 59 63 52 so 55 54 102 55 50 103 51
Ground check for calves was not conducted until mid-June. Parturition was in mid-May. Cow was eutbanatized and autopsied, twin fetuses were present.
1 1 Oa 2
1
P P P P P P
14&n
resubmit 16&n 16-Jan 14-Jan 16&l
2 Oa Oa
P P P
19-Dee 19-Dee 19-Dee
23-Feb 23-Feb
56 59 56 71 62 62 47 59 64 55 SO 55 55 103 56 41 101 53
5s 49 59 70 63 62 46 58 63 53 51 53 52 101 53 48 99 53
53 58 56 54 62 64 47 58 64 54 48 59 52 102 53 SO 101 51
53 58 60 67 62 63 48 58 68 52 51 57 55 98 60 49 101 52
9.5 8.5 8.5 9.5 3.5 7.5 5.5 3.5 3.5 11.5 6.5 4.5 3.5 1.5 8.5 2.5 1.5 9.5
2 3 3 6 7 9 11 13 13 14 IS 16 20 22 27 28 29 30
2 2
Year1 Year 1 No. of Year2 Palpation % Binding Sampling P-SPB calves Sampling ezam Sub-sample 3 date status seen date Sub-sample 1 Sub-sample 2 Replicate 1 Replicate 2 Replicate 1 Replicate 2 Replicate 1 Replicate 2 (day-month) (day-month)
Age (years) at 1st sample
ID #
P P
Serum assay of female moose for binding to antibody for bovine P-SPB. Pregnant animals are designated by a P in the table, nonpregnant animals are designated by an 0.
Table 1.
54.0 f 1.20 56.5 f 1.54 58.~5f 0.8i 66.5 f 2.59 62.3 -f 0.52 62.7 f 1.21 47.7 f 1.21 58.2 M.75 64.3 f 1.85 52.8 f 1.47 SO.3f 1.37 55.7 f 2.07 53.7 f 1.37 100.5f 2.43 54.7 i3.14 48.7 i 1.21 101.3 * 1.51 51.1 f 1.21
MeanfSD
4 5 8 8 10 12 17 18 19 26
1
ID #
date
SampIing
8.5 3.5 5.5 5.5 6.5 8.5 5.5 2.5 8.5 4.5 6.5
sample
19-Dee 15&n IS-Jm 14-Jan 25-Feb 26-Feb 15-h 14-h 23-Feb 14Jan 24-Feb
Sub-mqle
1 Sub-sample2
% Binding Sllbsample 3
101 100 96 98 99 101 96 100 100 102 99
101 102 93 97 98 99 101 100 101 100 93 105 97 96 91 102 103 82 96 98 102 99 98 97 98 103 98 95 98 loo
102
98 91 103 99 103 100 94 101
100 100 93 96 101 103 102 103 102 104
103 95
Replicate 1 Replicate 2 Replicate 1 Replicate 2 Repkate 1 Rqdicate2
at 1st (day-month)
Age (Ye=)
102.0 f 99.3 f 96.3 f 96.7 f 97.3 f loo.7 f 100.8 f 97.5 f 99.2 f 99.0 f 99.8 f
1.79 2.99 2.50 1.97 1.03 1.86 2.86 7.79 3.06 3.03 3.76
Mean&SD
Table 2Semm assay of malemoose. For 7 bulls 1 replicate showed 5 95% biadiq whichcan indicatapregnancy if the s 95% level is taken as the division point. Repeat measurementsand mean values were > 95%.
Theriogenology
909
found to have a high degree of cross-reactivity in bison (7), sheep (ovis spp; 15), mule and whitetailed deer (Gdocow spp;_23),mountain goats (Greamn amencanus; lo), red deer (m elanhus; 6), musk-oxen (ovlbos moschatus; 14), and a zty of other animals with cotyledonary placentation (17). Nothing is known of the timing of appearance of pregancy-specific protein in moose relative to conception, nor of its disappearanceafter parturition or abortion. In red deer, pregnancy-specific protein is present by 30 days post-conception (6), and in most cattle P-SPB is present by 24 days of gestation (16). In cattle, P-SPB has a half-life of 8.4 days (11). A&r parturition or abortion pregancy-specific protein has disappeared by 90 days in cattle, and by 25 days in sheep (Sasser RG, personal communication). Moose are seasonal breeders, the rut occurring in late September and early October. For practical pmposes sampling for pregancyspecific protein should not be carried out until mid-November at the earliest Samplingof &eeranging moose late in gestation (past mid-March)is contraindicatedbecause of the potential for capture-related mortality in heavily pregnant animals. The cut-off of 95% binding was chosen because it had been the established figure used in cattle In this study, with the exception of one outlier of 82%, all bulls had binding 2 93%) which is the figure currently used for cutoff in cattle (Sasser RG, personal communication). The d~scmpancy between non-pregnant animals and pregnant or presumed pregnant ones (571%) would have permitted a lower cut-off point without altering the results. If a cow was accompanied by a calf in the May/June following the first capture season this was taken to indicate that she had been pregnant at the time of capture. The 3 animalsthat were not observed with calves in the first year of the study all had diagnostic concentrations of P-SPB in December&nmary. It is possible that they were not pregnant, or that they had recently aborted. However, it is more likely that by the time they were seen in mid-June they had already lost their calves due to predation. Black bears (Urs m and wolves (Canis lunus) are known pmdators of moose calves in the first few weeks of life (5,21) and share range with moose in the study area. Both yearling females sampled in this study were nonpregnant. Yearling moose may conceive and carry calves, but the proportion of them that do so in any given year is primarily dependent upon the quahty of their range in the summer preceding the breeding season and pregnancy rates may range from 0 to 93% (1). Culling carried out during the gestation period obviously provides the most accurate indicator of pregnancy rates, but other less invasive methods are useful and desirable. Palpation per rectum is routinely used to detect pregnancy in cattle, and has been similarly used in immobilized bison and moose (7,s). Portable ultrasound equipment has also been used in free ranging moose (Glover GG, personal communication), mule deer (19), and bighorn sheep @&iscanadensis; 9). Serum progesterone assay has been reported as a means of determining pregnancy in ungulates and has been used in bighorn sheep (2) and moose (8,20). Progesterone as an indicator of pregnancy has the disadvantagethat it is also present during the luteal phase of the estrous cycle. Concentrations of progesterone measured during pregnancy or the cycle may overlap, so that high levels can only be taken as indicatingpregnancy if they are either obtained after the end of seasonalcyclic&y,or repeated iiequently during a period longer than 1 cycle in ordertoshowthattheyremainhigh. Immunoreactive fecal progestins and estrogen$F been used as indicators of cyclicity and pregnancy in cattle, muskoxen (3) and caribou @ana tarandus; 12). They have the advantage over other methods in that their collection is noninvasive,but in free-rangingpopulations they are only valuable as indicators of reproductive activity of populations rather than of individuals High
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total estrogen and progesterone concentrationsin feces have been shown to correlate well with late pregnancy in muskoxen (3) and caribou (12). The results in the study reported here show that a bovine P-SPB antibody is a useful indicator of well establishedpregnancy in moose. Determinationsof the time of its appearance in early pregnancy, and its disappearanceafter parturition, remain to be conducted. REFERENCES
1. Boer A. Fecundity of North American moose (Aloesalces): a review. Alces supplement. 1992; 1: l-10. 2. Brumge GC, Lane LJ, McCabe TR. Early pregnancy determination using serum progesterone concentration in bighorn sheep. J Wildl Manage 1988; 52: 610612 ). 3. Desaulniers DM, Goff AK, Be&ridge KJ, Rowell JE, Flood PF. Reproductive hormone concentrations in faeces during the oestrous cycle and pregnancy in cattle (Bos taurus) and muskoxen (Ovibos moschatus). Can J Zoo1 1987; 67: 1148-l 154. 4. Eckblad WP, Sasser RG, Ruder CA, PanlasuigiPM, Kuszynski TS. Locahzation of pregnancy-specific protein B (P-SPB) in bovine placenta cells using a glucose oxidase anti-glucose oxidase \ stain. J Anim Sci 1985; 61 (Suppl): 149-150. 5. Franzmann AW, Schwartz CC. Black bear predation on moose calves in highly productive versus marginal moose habitats on the Kenai peninsula, Alaska Alces 1986; 22: 139-154. 6. Haigh JC, Cranfield M, SassedRG. Estrus synchronization and pregnancy diagnosis in red deer. 3 Zoo Anim Med 1988; 19: 202-207. 7. Haigh JC, Gates C, Ruder A, Sasser R. Diagnosis of pregnancy in wood bison using a bovine assay for pregnancy-specific protein B. Theriogenomgy 1991; 36: 749-754. 8. Haigh JC, Kowal EH, Runge W, Wobeser G. Pregnancy diagnosis as a management tool for moose. Aloes. 1982; 18: 45-53. 9. Harper WL, Cohen RD. Accuracy of Doppler ultrasound in diagnosingpregnancy in bighorn sheep. J Wildl Manage 1985; 49: 793-796. 10. Houston DB, Robbii CT, Ruder CA, Sasser RG. Pregnancy detection in mountain goats by assay for pregnancy-specific protein B. J Wildl Manage 1986; 50: 740-742. 11. Kiicofe GH, Wright JM, Schalles RR, Ruder CA, Prish S, Sasser RG. Pregnancyspecific protein B in serum of postpartum beef cows. J Anim Sci 1993; 71: In press. 12. Messier F, Desaulniers DM, Goff AK, Nault R, Patenaudc R, Crete M. Caribou pregnancy diagnosis from immunoreactive progestins and estrogens excreted in feces. J Wiidl Manage 1990; 54: 279-283. 13. Reimers TJ, Sasser RG, Ruder CA. Production of pregnancy-specific protein by binucleate trophoblastic cells. Biol Reprod Suppll985; 132: 65. (Abstr.). 14. Rowell JE, Flood PF, Ruder CA, Sasser RG. Pregnancy-specificprotein in the plasma of captive muskoxen. J Wildl Manage 1989; 53: 899901. 15. Ruder CA, StelIfug JN, Dahmen JJ, Sasser RG. Deteceon of pregnancy in sheep rad&n;;“““oa”say of sera for pregnancy-specific protem B. Thermgenology 1988 29: 16. Sasser RG, Ruder CA, Ivani KA, Butler JE, Hamilton WC. Detection of pregnancy by radioimmunoassayof a novel pregnancy-specific protein in serum of cows and a profile of serum concentrations during gestation. Biol Reprod 1986; 35: 936-942. 17. Sasser RG, Ruder CA, Ivani KA, Short RE, Wood AK. Pregnancy-specific protein B in serum of various species. In: Ellemiorf, F., and Koch, E. (eds.), Reproductiveand PeriNatal Medicine. 1. Early Pregnancy Factors. Perinatology Press, Ithaca. 1985; 161. 18. Schmitt SM, Dalton WJ. Immobilization of moose by carfentanil and x lazine and reversal by nahrexone, a long acting antagonist. Aloes 1987;23: 195-213. 19. Smith RB, Lindzey FG. Use of ultrasound for detecting pregnancy in mule deer. J Wildl Manage 1982; 46: 1089-1092.
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Comishen-StewartLM, Haigh JC. Levels of some reproductive hormones in relation to pregnancy in moose: a preliminary report. Alces 1985; 21: 393402. 21. Stewart RR. Kowal EXT.BeaulieuR. Rock TW. The imoact of black bear removal on moose calf survival in &st central S&atchewan. Alces 1985; 21: 403-418. 22. Verme, L.J. Problems in appraisingreproduction in Cervidae. Proc. N. Amer. Moose. Conf. Workshop. 1974; 10: 22-36. 23. Wood AK, Short RI$ Darling A, Dusek GL, Sasser RG, Ruder CA. Serum assays for detecting pregnancy m mule and white-tailed deer. J Wildl Manage,l986; 50: 684-687. 20. Stewart RR,