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Reproductive performance of farmed red deer (Cervus elaphus) in New Zealand
Animal Reproduction Science 55 Ž1999. 239–254
Reproductive performance of farmed red deer žCerÕus elaphus / in New Zealand IV. Biological markers as risk factors for yearling and adult hind conception Laurent Audige´ 1, Peter R. Wilson ) , Roger S. Morris Institute of Veterinary, Animal and Biomedical Sciences, Massey UniÕersity, Palmerston North, New Zealand Accepted 25 February 1999
Abstract A 2-year observational study of 15 red deer Ž CerÕus elephus . farms was carried out in New Zealand from March 1992. In each year of study, approximately 1650 hinds were individually monitored for reproductive success. During farm visits in March 1992 and 1993, five yearling and five adult hinds per farm were randomly selected and blood sampled to define their haematological, biochemical and blood mineral profile. Faecal samples were taken for parasite egg and larvae count. Biological markers potentially affecting the probability of conception before May 1 or of conception that year were investigated separately using multivariable logistic regression analysis. Adult hinds with low serum phosphorus concentrations were more likely to conceive before May 1. Lower conception rates were observed in yearling hinds when blood glutathione peroxidase, serum vitamin B12, and serum albumin concentrations were low, and when faecal lungworm larval counts were high. While these associations have yet to be proven as causal, data suggests that monitoring and maintaining adequate blood elements, and controlling internal parasites in yearling hinds, may assist farmers to achieve optimum reproductive performance in farmed red deer herds. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Red deer; Reproductive performance; Biological markers; New Zealand
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Corresponding author. Tel.: q64-6-350-5329; fax: q64-6-350-5616; e-mail: [email protected] Current address: Institute of Virology and Immunoprophylaxis, PO Box, CH-3147 Mittelhausern, ¨ Switzerland. Tel.: q41-31-848-92-52; fax: q41-31-848-92-22; e-mail: [email protected]. 1
0378-4320r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 4 3 2 0 Ž 9 9 . 0 0 0 1 8 - 4
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1. Introduction Reproductive success varies considerably between herds and between yearling Žprimiparous. and adult deer on commercial New Zealand deer farms ŽAudige, ´ 1995; Audige´ et al., 1999a.. A wide range of individual animal, management and grazing factors contribute to that variation and have been discussed elsewhere ŽAudige´ et al., 1999b,c.. In addition, there is variation both between animals and between farms in a number of biochemical measurements ŽAudige, ´ 1995.. That variation may be associated with variation in reproductive performance. However, there are no reports of relationships between biological markers and reproductive outcomes on commercial deer farms. Furthermore, there is a paucity on information on the influence of environmental factors on farmed deer reproductive performance as reviewed by Asher et al. Ž1996., although those authors proposed that feed supply, confinement and mating and calving management practices are key factors. There are a few reports of causal relationships between individual animal biological markers and reproductive outcomes in other species farmed in a commercial pastoral environment. Low selenium concentrations have been associated with poor reproductive performance in sheep ŽScales, 1974.. That relationship has been suspected but not proven in cattle ŽSpears et al., 1986; Ropstad, 1990; Corah and Ives, 1991.. Wilson et al. Ž1985. refer to variation in serum protein as a marker for reproductive success in cattle. Copper deficiency is widespread in farmed deer in New Zealand ŽEllison, 1995. and may contribute to poor reproductive performance, although there is no published evidence in this species. In cattle, low copper levels have been associated with changes in oestrous cycle length, anoestrus, increased number of services per conception and suspected embryonic loss ŽCorah and Ives, 1991.. There is little evidence of a direct association between vitamin B12 levels and reproduction. A 2-year observational study of 15 red deer farms was conducted to provide reference data for health and productivity outcomes, and to generate hypotheses on the relationship between various factors and productivity ŽAudige´ et al., 1993, 1994, 1999a,b,c; Audige, ´ 1995.. Blood and faeces were analysed for parameters from sentinel animals within those herds. While there may be no currently known biological relationship between some of the variables measured or recorded in this study, they were included to explore whether factors not studied to date may be associated with reproductive outcomes. Such factors, if identified, can subsequently be evaluated for causality. This paper presents biological markers associated with the likelihood of conceiving that year ŽCONC. or conceiving before May 1 ŽADVC. for yearling and adult hinds.
2. Materials and methods Fifteen commercial red deer herds were chosen for a 2-year longitudinal observational study from March 1992 as described elsewhere ŽAudige´ et al., 1993, 1994, 1999a,b,c; Audige, ´ 1995.. In each of the 2 years, hinds were individually monitored for reproductive success ŽAudige´ et al., 1999a. and were classified as conceiving before
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May 1, conceiving at all that year, even if after May 1, or being diagnosed non-pregnant, using rectal ultrasound pregnancy diagnostic techniques as described by Revol and Wilson Ž1991.. From individual animal data, early conception rates ŽADVCR. or conception rates that year ŽCONCR. were calculated within each farm and each year. During farm visits in March 1992 and 1993, 10 randomly selected sentinel hinds including five yearlings Ž15 months of age. and five adults Ž3 years of age or more. were physically restrained and blood sampled by jugular venipuncture into evacuated glass tubes containing either no anticoagulant or EDTA, for haematological, biochemical Table 1 Descriptive statistics of dichotomous biological markers screened for association with the ability of yearling and adult hinds to conceive before May 1 ŽADVC. and of conceiving that year ŽCONC., respectively Risk factorsa
Yearling hinds CU8 Yes No B12185 Yes No MGSHPX3 Yes No FEC ) 0 Yes No FLC ) 0 Yes No Adult hinds CU8 Yes No B12185 Yes No MGSHPX3 Yes No FEC ) 0 Yes No FLC ) 0 Yes No
Conception before May 1
Conception that year
Yes
No
OR
95% CI
p-value
Yes
No
OR
95% CI
p-value
64 10
33 1
0.2
0, 1.2
0.17
84 11
13 0
24 50
5 29
2.8
1, 7.8
0.06
28 67
1 12
5.0
0.6, 40.4
0.18 b
72 2
26 8
1.1
1, 1.3
0.001b
88 7
10 3
3.8
0.9, 15.9
0.10
15 59
5 29
1.5
0.5, 4.3
ns
16 79
4 9
0.5
0.1, 1.6
ns
44 30
19 15
1.2
0.5, 2.6
ns
51 44
12 1
0.1
0, 0.6
0.01
104 12
10 2
1.7
0, 8
ns
110 13
4 1
2.1
0, 15.6
ns
29 87
1 11
3.7
0.5, 29.6
ns b
29 94
1 4
1.2
0.1, 11.5
ns b
98 18
10 2
1.1
0, 4.9
ns
103 20
5 0
18 98
1 11
2.0
0.2, 16.6
ns b
19 104
0 5
63 53
7 5
0.8
0.3, 2.7
ns
67 56
3 2
0.8
0, 4.2
ns
OR: Odds ratio; 95% CI: 95% confidence interval. a Risk factor codes are defined in the text. b OR and 95% CI obtained using logistic regression.
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and blood mineral profile evaluation. Haematological measurements were carried out on fresh EDTA blood within 24 h of collection. White cell counts ŽWCC., and haemoglobin concentration ŽHB. were performed using a haematology analyser ŽCELL-DYN 900 Haematology Analyser, Sequoia-Turner, CA.. Packed cell volumes ŽPCV. were determined after centrifugation of capillary tube. Blood smears were stained, covered and stored to be read later. Differential leucocyte counts were performed by counting 100 cells under magnification Ž=40.. Leukocytes were classified as lymphocytes, polymorphonuclear neutrophils, eosinophils, basophils, band cells, monocytes and ‘unusual lymphocytes’ which were cells not conforming to normal morphology of lymphocytes because nuclei were severely indented. Variables lymphocyte percentage ŽLP., neutrophil percentage ŽNP. and eosinophil percentage ŽEOP. were calculated.
Table 2 Descriptive statistics of biological markers screened for association with the ability of adult hinds to conceive early Žbefore May 1. ŽADVC., in a preliminary univariable analysis and Rank sum test p-values Risk factor code a
Unit
Rank sum Range of all test values
ADVC s1 early conception
p-value
No. of Mean deer
Haematological markers WCC 10 9 rl 0.49 HB grdl 0.16 PCV % 0.12 MCHC grdl 1.00 NP % 0.02 EOP % 0.25 LP % 0.01 Haematological markers TP grl 0.31 ALB grl 0.76 GLOB grl 0.45 P mmolrl 0.01 GGT IU 0.71 BUN mmolrl 0.82 CU mmolrl 0.65 B12 pmolrl 0.40 GSHPx MGSHPxU kIUrl 0.37 PEPS mU 0.95 tyrosinerl Parasitological marker FLC larvaerg 0.86
Min
Max
SD
No. of Mean deer
1.7 12 3.3 12 0.05 12 7.5 12 15 12 5 12 15 12
5.5 18.6 0.41 46.0 40 8 49
SD
2.5 8.8 0.23 29.2 16 0 18
13.6 28.1 0.48 77.4 75 25 81
116 116 116 116 115 115 115
55.7 28.5 24.8 1.14 17 6.7 2.7 65 0.9 1.4 18
86.2 41.5 50.7 3.84 211 16.0 23 530 29.7 24.1 2719
116 116 116 116 116 116 116 116 62 116 116
70.7 35.1 35.7 2.4 42 11.1 13.2 161 6.9 7.3 585
5.5 2.9 5.1 0.5 25 1.7 4.2 77 6.7 6.0 447
12 12 12 12 12 12 12 12 3 12 12
69.5 34.8 34.7 2.8 36 11.2 13.3 134 14.0 7.5 528
4.3 2.5 4.3 0.5 9 1.8 3.7 37 8.6 5.8 333
14.8
116
0.8
1.4
12
1.9
4.3
0
5.5 16.0 0.39 41.6 48 7 42
ADVC s 0 late or no conception
1.1 5.0 0.05 13.2 10 4 10
Mins minimum; Max s maximum; SDsstandard deviation. The mean of the three individual measures of GSHPx from each farm each year has been taken as individual values for multivariable analysis of individual data. a Risk factor codes are described in the text.
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Sera were analysed for biochemical and mineral characteristics after being stored frozen. Total protein ŽTP., albumin ŽALB., phosphorus ŽP. and blood urea nitrogen ŽBUN. concentrations, and gamma glutamyl transferase ŽGGT. activity were measured from sera using an automatic analyser ŽCOBAS MIRA, F. Hoffmann-La Roche, Switzerland.. Whole blood glutathione peroxidase ŽGSHPx., from three hinds per farm, was measured by the MAF Bachelar Animal Health Laboratory, Palmerston North, New Zealand. Serum copper ŽCU. and vitamin B12 ŽB12. concentrations were analysed from frozen sera at the MAF Ruakura Animal Health Laboratory, Hamilton, New Zealand. Serum pepsinogen levels ŽPEPS. were assayed using the method described by Pomroy and Charleston Ž1989.. Faecal samples were taken per-rectum for gastrointestinal nematode egg ŽFEC. and lungworm larvae counts ŽFLC. which were performed the day after collection. Faecal egg counts were performed by mixing 2 g of faeces in 28 ml of Table 3 Descriptive statistics of biological markers screened for association with the ability of yearling hinds to conceive early Žbefore May 1. ŽADVC., in a preliminary univariable analysis and Rank sum test p-values Risk factor code a
Unit
Rank sum Range of all test values
ADVC s1 early conception
p-value
No. of Mean deer
Haematological markers WCC 10 9 rl 0.57 HB grdl 0.53 PCV % 0.93 MCHC grdl 0.59 NP % 0.24 EOP % 0.55 LP % 0.08 Haematological markers TP grl 0.05 ALB grl 0.22 GLOB grl 0.15 P mmolrl 0.45 GGT IU 0.97 BUN mmolrl 0.29 CU mmolrl 0.18 B12 pmolrl 0.03 GSHPx kIUrl MGSHPxU kIUrl 0.01 PEPS mU 0.91 tyrosinerl Parasitological marker FLC larvaerg 0.353
Min
2.8 12.1 0.33 32.2 11 0 23
56.2 30 22.4 1.5 16 6.5 1 65 2 1.4 18
0
Max
22 26.6 0.52 73.9 67 17 82
74 74 74 74 74 74 74
5.5 16.5 0.42 39.5 36 5 57
ADVC s 0 late or no conception SD
1.5 1.6 0.04 3.3 13 4 13
No. of Mean deer 34 34 34 34 34 34 34
6.3 17.5 0.42 42.0 39 5 52
SD
3.4 3.4 0.04 8.3 13 4 13
83.2 42.9 44.7 3.5 139 13.5 24 1500 29.2 24.1 1522
74 74 74 74 74 74 74 74 10 74 72
69.0 36.5 32.5 2.6 31 10.0 13.1 170.3 8.4 9.1 450.7
4.5 2.8 4.4 0.4 11 1.1 4.7 81.3 8.2 6.8 257.3
34 34 34 34 34 34 34 34 3 34 33
67.2 35.6 31.5 2.6 33 10.1 14.3 136.2 9.6 5.7 457.8
4.3 3.1 4.1 0.4 21 1.8 3.1 53.1 7.9 3.6 251.1
21.8
74
1.6
3.4
34
0.7
0.9
Mins minimum; Max s maximum; SDsstandard deviation. The mean of the three individual measures of GSHPx from each farm each year has been taken as individual values for multivariable analysis of individual data. a Risk factor codes are described in the text.
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saturated salt water, the count of one egg was equivalent to 50 eggs per gram of faeces Žmodified Master technique.. Faecal lungworm larvae counts were performed using a modified Baerman technique ŽHenricksen, 1965.. Faecal egg and larvae count data were categorised as positive faecal egg count ŽFEC ) 0. and positive faecal larval count ŽFLC ) 0., respectively. For statistical analyses, the mean of the three values of GSHPx within farms ŽMGSHPx. was used as the value for individual sentinel hinds since variability of GSHPx was mainly due to farm differences ŽAudige, ´ 1995.. Serum copper, ŽB12. and MGSHPx were categorised as adequate or deficient based on available data from deer and other domestic species. The categorical variables were: serum copper concentration G 8 mmolrl ŽCU8.; serum vitamin B12 concentration G 185 pmolrl ŽB12185.; and mean glutathione peroxidase activity from three hinds G 3 kIUrl ŽMGSHPx3.. These Table 4 Descriptive statistics of biological markers screened for association with the ability of adult hinds to conceive that year ŽCONC., in a preliminary univariable analysis and Rank sum test p-values Risk factor code a
Unit
Rank sum Range of all test values
CONC s1 coneption
p-value
No. of Mean deer
Haematological markers WCC 10 9 rl 0.43 HB grdl 0.57 PCV % 0.27 MCHC grdl 0.63 NP % 0.05 EOP % 0.73 LP % 0.03 Haematological markers TP grl 0.04 ALB grl 0.82 GLOB grl 0.04 P mmolrl 0.00 GGT IU 0.62 BUN mmolrl 0.11 CU mmolrl 0.66 B12 pmolrl 0.41 GSHPx MGSHPxU kIUrl 0.06 PEPS mU 0.99 tyrosinerl Parasitological markers FLC larvaerg 0.98
Min
2.5 8.8 0.23 29.2 16 0 18
55.7 28.5 24.8 1.1 17 6.7 2.7 65 0.9 1.4 18
0
Max
13.6 28.1 0.48 77.4 75 25 81
123 123 123 123 122 122 122
CONC s 0 no conception
5.4 16.2 0.39 42.1 48 7 42
SD
1.7 3.5 0.05 8.3 15 5 14
No. of Mean deer 5 5 5 5 5 5 5
5.5 17.6 0.42 41.6 36 6 54
SD
0.9 4.8 0.05 8.1 11 3 10
86.2 41.5 50.7 3.8 211 16.0 23 530 29.7 24.1 2719
123 123 123 123 123 123 123 123 64 123 123
70.8 35.1 35.7 2.4 41 11.1 13.2 159.5 7.1 7.2 581
5.5 2.9 5.0 0.5 24 1.7 4.1 74.9 6.9 6.0 440
5 5 5 5 5 5 5 5 1 5 5
66.9 34.7 32.2 3.1 35 10.6 13.7 126.0 13.3 8.4 546
0.7 2.5 2.3 0.2 13 0.6 3.9 46.2 1.8 416
14.7
123
0.8
1.4
5
3.1
6.5
Mins minimum; Max s maximum; SDsstandard deviation. The mean of the three individual measures of GSHPx from each farm each year has been taken as individual values for multivariable analysis of individual data. a Risk factor codes are described in the text.
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markers were also analysed as continuous variables, although both forms of the same variables were not included in the same multivariable analysis. Data from both years of study were combined for statistical analyses. Analyses were carried out separately for yearling and adult hinds. All analyses were carried out using the statistical software Stata version 5.0 ŽStata, TX, USA.. A full description of statistical methods applied in this study are presented elsewhere ŽAudige´ et al., 1999b.. Data were initially screened for univariable statistical association with both ADVC and CONC outcomes. Continuous risk factors were analysed without transformation, by the rank sum test, while categorical risk factors were analysed by the Fisher’s exact test. Potential risk factors statistically significantly associated with the outcome at p - 0.20 were included together in a stepwise multivariablte logistic regression analysis with a forward selection procedure. Risk factors classified as significant were associated with respective outcomes at the 5% confidence level. Table 5 Descriptive statistics of biological markers screened for association with the ability of yearling hinds to conceive that year ŽCONC., in a preliminary univariable analysis and Rank sum test p-values Risk factor code a
Unit
Rank sum Range of all test values
CONC s1 conception
p-value
No. of Mean deer
Haematological markers WCC 10 9 rl 0.56 HB grdl 0.52 PCV % 0.51 MCHC grdl 0.10 NP % 0.64 EOP % 0.67 LP % 0.23 Haematological markers TP grl 0.08 ALB grl 0.01 GLOB grl 0.82 P mmolrl 0.89 GGT IU 0.10 BUN mmolrl 0.43 CU mmolrl 0.00 B12 pmolrl 0.02 GSHPx kIUrl MGSHPxU kIUrl 0.21 PEPS mU 0.81 tyrosinerl Parasitological markers FLC larvaerg 0.04
Min
Max
CONC s 0 no conception
1.7 2.0 0.04 5.0 13 4 12
No. of Mean deer 13 13 13 13 13 13 13
6.9 18.0 0.41 43.5 40 5 50
SD
2.8 12.1 0.33 32.2 11 0 23
22 26.6 0.52 73.9 67 17 82
95 95 95 95 95 95 95
56.2 30 22.4 1.53 16 6.51 1 65 2 1.37 18
83.2 42.9 44.7 3.49 139 13.5 24 1500 29.2 24.13 1522
95 95 95 95 95 95 95 95 11 95 93
68.7 36.5 32.2 2.6 32 10.0 13.1 165.6 9.4 8.4 453
4.3 2.9 4.4 0.4 16 1.3 4.4 76.2 8.4 6.4 256
13 13 13 13 13 13 13 13 2 13 12
66.3 34.2 32.1 2.6 32 10.3 16.1 115.2 5.1 5.5 455
5.1 2.3 4.1 0.4 7 1.8 2.5 48.4 0.9 2.7 255
21.8
95
1.4
3.1
13
1.1
1.0
0
5.6 16.7 0.42 39.9 37 5 56
SD
4.9 3.9 0.04 7.5 15 4 14
Mins minimum; Max s maximum; SDsstandard deviation. The mean of the three individual measures of GSHPx from each farm each year has been taken as individual values for multivariable analysis of individual data. a Risk factor codes are described in the text.
U
246
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In a first analysis, the potential effect of clustering due to farm or year of study on the significance of risk factors was not taken into account. In a second step, the final models were checked considering clustering due to farms within years, as most of the variability of biological markers were due to farms ŽAudige, ´ 1995.. In addition, individual hind characteristics that were identified as important factors affecting conception in earlier analyses were forced into final models Ždepending on the outcome as presented in result tables. as otherwise they may be confounding factors. These adult-hind individual characteristics were: adult hind ) 3 years old at mating ŽAGE ) 3.; pre-mating bodycondition score ) 2.5 ŽBCSM) 2.5. or ) 2 ŽBCSM) 2.; pure New Zealand-type hind ŽNZ100., hind lactating at weaning ŽWET. and body weight standardised to April 1 from body weights recorded in March and June Žbody weight change was considered linear in this time interval. ŽW4. ŽAudige´ et al., 1999b.. Important yearling-hind characteristics were: pre-mating body-condition score - 3 ŽBCSM- 3.; percentage of New Zealand blood line ŽNZ.; growth rate between April 1 and June 1 ŽGR46. and W4 ŽAudige´ et al., 1999c.. Final models of all selected factors, after adjustment for potential clustering effect due to farm and after inclusion of hind variables defined above are presented in tables. The non-significance of some factors presented in result tables was because of this inclusion of variables and adjustment. Biological markers were initially investigated at the individual-hind level. Farm mean blood markers, using the geometric or arithmetic mean where appropriate, were investi-
Table 6 Logistic regression coefficient estimates of biological markers statistically and significantly associated with adult hind conception status Žafter adjustment for farm effect. Risk factor code a
Regression coefficient
Standard error
Hind conception before May 1 (ADVC) Before the inclusion of individual hind characteristics; no. of hinds: 128 P y2.78 0.61 After the inclusion of individual hind characteristics; no. of hinds: 125 NZ100 0.14 1.18 WET 1.05 0.81 AGE) 3 4.10 2.24 BCSM) 2 1.19 0.91 P y5.50 1.76 Hind conception that year (CONC) Before the inclusion of individual hind characteristics; no. of hinds: 127 P y4.41 0.99 NP 0.12 0.04 After the inclusion of individual hind characteristics; no. of hinds: 125 WET 2.25 0.88 BCSM) 2.5 0.52 0.63 P y4.86 1.35 NP 0.14 0.06 a
Risk factor codes are described in the text. OR s odds ratio.
p-value
- 0.001 0.91 0.19 0.07 0.19 - 0.001
- 0.001 0.003 0.91 0.19 0.07 0.19
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gated in association with conception rates in follow-up investigations of the most important individual biological markers. Scatterplots of farm mean blood markers vs. ADVCR or CONCR were created with Statistica ŽStatsoft, Tulsa, OK, USA..
3. Results Results of Fisher exact tests to screen association between each dichotomous sentinel hind characteristic and biological markers and outcome variables ADVC and CONC are presented in Table 1. The ranges of values, means and standard deviations and results of statistical analyses of continuous biological markers screened for association with ADVC and CONC for both adult and yearling hinds are presented in Tables 2–5.
Table 7 Logistic regression coefficient estimates of biological markers statistically and significantly associated with yearling hind conception status Žafter adjustment for farm effect. Risk factor code a
Regression coefficient
Standard error
p-value
Hind conception before May 1 (ADVC) Before the inclusion of individual hind characteristics; no. of hinds: 108 With continuous risk factors CU y0.13 0.06 0.02 MGSHPx 0.16 0.05 0.001 With some risk factors categorised MGSHPx3 2.40 0.33 - 0.001 After the inclusion of individual hind characteristics; no. of hinds: 88 With continuous risk factors W4 y0.003 0.03 0.92 CU y0.09 0.07 0.17 MGSHPx 0.19 0.07 - 0.001 With some risk factors categorised W4 0.02 0.04 0.64 MGSHPx3 2.42 0.42 - 0.001 Hind conception that year (CONC) Before the inclusion of individual hind characteristics; no. of hinds: 108 With continuous risk factorsb,c B12 0.02 0.01 0.06 ALB 0.28 0.11 0.01 CU y0.22 0.05 - 0.001 FLCPOS y2.43 0.92 0.01
OR
95% CI
11.1
5.8, 21.1
11.3
4.9, 25.8
0.09
0.01, 0.54
OR s odds ratio. a Risk factor codes are described in the text. b When replacing B12 by B12185 in the analysis, the final model included the variables ALB, CU and FLCPOS. This final model was ignored, as it provided no additional information. c After inclusion of BCSM- 3, NZ, W4, GR46 into this model, the factors FLCPOS and BCSM- 3 were dropped from the model as, if equal to null, they predicted perfectly the outcome CONC, and the number of observations was restricted to 30 hinds. Consequently, the resulting model is not presented in this table.
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Fig. 1. Relationship between mean pre-mating phosphorus concentrations of five adult hinds and conception rates Žbefore May 1 and overall conception. of adult hinds within each survey farm. Data from 1992 and 1993 combined.
Fig. 2. Relationship between mean pre-mating glutathione peroxidase from three hinds and yearling hind early conception rates within each survey farm. Data from 1992 and 1993 combined. Note: The line is a logarithm fitted line.
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Fig. 3. Relationship between geometric mean pre-mating serum vitamin B12 from five sentinel yearling hinds and yearling hind conception rates within each survey farm. Data from 1992 and 1993 combined. Note: The line is a logarithm fitted line.
Variables which were associated with an outcome with a p - 0.20 were those subjected to stepwise multiple logistic regression analysis to produce the final results presented in Tables 6 and 7, which are discussed. There was an inverse relationship between blood phosphorus concentration ŽP., Žrange 1.1–3.8 mmolrl, Table 2. and early conception and conception that year. However, this latter association became non-significant when individual hind variables
Fig. 4. Relationship between mean pre-mating albumin concentrations of five yearling hinds and conception rates of yearling hinds within each survey farm. Data from 1992 and 1993 combined. Note: The fitted line is an inverse exponential smoothing curve.
250
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WET and BCSM) 2.5 were included in the final model ŽTable 6.. There was a positive relationship between blood neutrophil percentage ŽNP. which ranged from 16–75% ŽTable 2. and CONC, but it became non-significant after the inclusion of WET and BCSM) 2.5 in the final model ŽTable 6.. In yearling hinds ŽTable 7., the serum copper concentration, which ranged from 1 to 24 mmolrl ŽTable 3., was negatively associated with the probability of early conception and conception that year. However, the association between CU and ADVC became non-significant when individual hind variable W4 was included in the final model. Mean glutathione peroxidase Žrange 21.4–29.24.1 nmolrl, Table 3. was positively associated with the probability of early conception. Furthermore, yearling hinds on farms with a mean GSHPx of more than 3 nmolrl had 11 times higher odds of conceiving early. Serum albumin Žrange 30–42.9 grl, Table 3. and vitamin B12 concentration Žrange 65–1500 pmolrl, Table 3. were positively associated with the probability of conception that year. Yearling hinds with lungworm larvae detected in faeces had about 11 times lower odds of conceiving that year than other hinds. Scatterplots ŽFigs. 1–4. show herd blood phosphorus in adult hinds, and mean GSHPx, individual blood albumin, and geometric mean serum vitamin B12 in yearling hinds, and blood phosphorus in adult hinds, plotted against conception rates. 4. Discussion This presentation is part of an extensive investigation of a large number of characteristics and outcomes from commercial red deer farms ŽAudige, ´ 1995.. It is the first to attempt to relate a range of deer blood characteristics and faecal parasite larvae output to measures of reproductive success. Previous analyses highlighted the importance of a wide range of individual hind characteristics such as weight and body condition score in yearling and adult hinds ŽAudige´ et al., 1999a,b,c.. In those analyses, 2600 adult and 700 yearling hinds were monitored for a limited range of individual characteristics which were simple, time efficient and inexpensive to measure or record. Much data used in this study was time-consuming to collect on the farm, and the laboratory analyses were costly to perform. For these reasons, it was necessary to limit the sample size to five hinds of each age category, from the 15 farms studied over the 2-year period. That only a small number of biological markers were found to be associated with the likelihood of conception or early conception in this study may have been due to sample size. Alternatively, the variation in many measurements may not have been large enough, or they were all within a normal range for physiological function, e.g., haematological and biochemical measurements such as HB, WCC, GGT, BUN and PEPS. Yet another explanation may be that the principal influences on individual deer conception, particularly in primiparous hinds, may be due to endocrine or behavioural factors not measured in this study. These warrant further investigation. Serum copper, vitamin B12 and glutathione peroxidase were analysed both as continuous and as categorical variables. All three markers have a minimum threshold level under which health problems may occur and production levels may be reduced. The minimum required blood level for copper in deer is thought to be 8 mmolrl ŽClark
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and Hepburn, 1986; Mackintosh et al., 1986.. Minimum levels for GSHPx in sheep are reported as 3 kIUrl ŽMAF Optigrowth Animal Health profile.. There are no reference data available for GSHPx in deer. Critical values of serum vitamin B12 in cattle are 38–76 pmolrl ŽSuttle, 1991., but concentrations below 185 pmolrl are regarded as deficient in sheep ŽClark and Millar, 1983.. No reference range exists for deer vitamin B12 concentrations ŽClark et al., 1986.. The threshold of 185 pmolrl has been chosen because of the sensitivity of the assay and the distribution of the data. These blood markers were also investigated as continuous variables because extrapolation of thresholds from other species may not be appropriate for deer. This study showed a negative relationship between serum phosphorus concentrations and adult hind ADVC. Many other studies failed to identify a significant relationship between phosphorus levels and fertility in cattle ŽRopstad, 1990; Forshell et al., 1991; Glauber et al., 1993.. However, there have been reports that high phosphorus concentrations could limit optimal fertility in cattle ŽHewett, 1975.. The non-significance of phosphorus levels on the onset of puberty and conception in yearling hinds is consistent with observations in heifers ŽWilliams et al., 1992.. The scatterplot of phosphorus data ŽFig. 1. suggests it is not obvious that phosphorus can be used to predict conception rates in adult hinds, although the relationship with overall conception rates was significant. It appears that low selenium concentrations as measured by GSHPx may impair early conception in yearling hinds. This effect was not evident in adults. Individual serum GSHPx activity was not investigated because only three hind samples per farm were analysed. However, because the variability of GSHPx is essentially due to farm difference Ž66.8% of the total variance ŽAudige, ´ 1995.., the mean GSHPx ŽMGSHPx. was used as an individual value for every hind within each farm. When MGSHPx was over 3 kIUrl, which has been proposed as the minimum threshold value in sheep ŽMAF Optigrow Animal Health profile., yearling hinds had 11 times higher odds of conceiving early. Fig. 2 demonstrates the possible negative effect of low selenium levels on yearling hind early conception, and suggests a threshold value may stand around 5–7 kIUrl, instead of 3 kIUrl as identified in sheep. Why this relationship in yearlings was not significant in adult hinds is not known, and requires further investigation. Furthermore, this finding questions the relevance of sheep reference ranges in yearling deer. More work is required to establish appropriate selenium reference ranges in deer. Many studies have failed to identify a significant relationship between blood selenium levels and fertility, and Se supplementation trials have failed to influence reproductive performance in cattle ŽSpears et al., 1986; Ropstad, 1990; Braun et al., 1991.. In sheep, however, the percentage of barrenness was significantly reduced by dosing with selenium prior to mating ŽScales, 1974., possibly due to reduced embryonic mortality. The negative relationship between serum copper concentrations and yearling-hind early conception and conception that year is intriguing and is difficult to explain. However, this association with ADVC became non-significant when W4 was forced into the model. The inclusion of yearling-hind individual characteristics into the model explaining CONC was not possible because of limited number of observations used for the regression analysis. The negative effect of CU may be a spurious result since it is
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contrary to observations in cattle where changes in oestrous cycle length, anoestrus, and an increased number of services per conception have been proven to be associated with low copper status, and embryonic death is suspected ŽCorah and Ives, 1991.. More intensive study of reproductive performance in deer herds of low copper status is required. Serum vitamin B12 was positively associated with the probability of yearling hinds conceiving. Cobalt is needed for optimal function of rumen micro-organisms and thus optimal forage digestibility ŽParagon, 1993.. The categorical variable B12 above 185 pmolrl was not significant, which suggests that 185 pmolrl may not be the appropriate threshold level in deer. This is also suggested by data in Fig. 3, supporting the observation of Clark et al. Ž1986., who reported no growth response following vitamin B12 supplementation trials in deer with B12 levels below 185 pmolrl. In sheep, vitamin B12 deficiency was not associated with lower conception rate, but lower lamb survival ŽFisher and McPherson, 1991.. Farm differences ‘explained’ 53.9% of the variability of pre-mating B12 in yearling hinds ŽAudige, ´ 1995.. There was a negative relationship between faecal larval count ŽFLC ) 0. and conception in yearling hinds. Hinds with positive FLC had 11 times lower odds of conceiving. The mechanism by which lung parasitism would affect reproductive performance directly is not clear, but it is believed to be mediated through the nutritional status of the animal ŽMorris and Meek, 1980.. This would be consistent with the observation that yearling hinds gaining body weight during mating were more likely to conceive that year ŽAudige´ et al., 1999c.. High serum albumin concentrations were associated with conception in individual yearling hinds, consistent with previous observation of the positive relationship with high growth rates during mating ŽAudige´ et al., 1999c.. This suggests serum albumin may be a useful marker to assess the likelihood of conception in yearling hinds. Many publications support the potential benefit of measuring serum albumin concentrations in dairy cattle ŽWilson et al., 1985.. Other authors referred to a reduction in the albumin:globulin ratio in infertile cows ŽRowlands et al., 1980.. Farm differences ‘explained’ 32.7% of the total variance of pre-mating serum albumin concentrations in yearling hinds ŽAudige, ´ 1995.. There was a statistically significant positive linear relationship between mean serum albumin concentrations and conception rates in this age group. Fig. 4 suggests a threshold value may stand around 35 grl, under which reproductive performance may be impaired. This will need to be further investigated.
5. Conclusion This study suggests that whereas no biological markers appeared useful in evaluating the risk of infertility in adult hinds, there is potential for the use of blood markers in yearling hinds. Measurement of selenium, serum vitamin B12, albumin concentrations, and faecal lungworm larval counts may be useful to trigger management decisions related to feeding, supplementation or treatment, to improve reproductive performance in that age group.
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Acknowledgements We wish to thank participating farmers for their active co-operation during this health and production research project. We thank the personnel of the Veterinary Faculty at Massey University for help and assistance. Financial assistance was provided by the New Zealand Game Industry Board, the French Government Žfor scholarship., CIRADEMVT, Deer Branch of the NZVA, Massey University, Pitman Moore Žnow Schering Plough., Rhone Cyanamid, AgResearch, Allflex New Zealand and Te Pari ˆ Merieux, ´ Products. References Asher, G.W., Fisher, M.W., Fennessy, P.F., 1996. Environmental constraints on reproductive performance of farmed deer. Anim. Reprod. Sci. 42, 35–44. Audige, ´ L., 1995. Deer herd health and production profiling. PhD thesis, Massey University, New Zealand, 550 pp. Audige, ´ L., Wilson, P.R., Morris, R.S., 1993. Deer herd health and production profiling the method. In: Wilson, P.R. ŽEd.., Proceedings of a Deer Course for Veterinarians. Deer Branch of the New Zealand Veterinary Association, No. 10, pp. 78–100. Audige, ´ L., Wilson, P.R., Morris, R.S., 1994. Deer-herd health and production profiling in New Zealand: 1. Study design. Vet. Res. 25 Ž2–3., 126–129. Audige, ´ L., Wilson, P.R., Morris, R.S., 1999a. Reproductive performance of farmed red deer Ž CerÕus elaphus. in New Zealand: I. Descriptive analysis. Anim. Reprod. Sci. 55, 127–141. Audige, ´ L., Wilson, P.R., Morris, R.S., 1999b. Reproductive performance of farmed red deer Ž CerÕus elaphus. in New Zealand: II. Risk factors for adult hind conception. Prev. Vet. Med. 40, 33–51. Audige, ´ L., Wilson, P.R., Morris, R.S., 1999c. Reproductive performance of farmed red deer Ž CerÕus elaphus. in New Zealand: III. Risk factors for yearling hind conception. Prev. Vet. Med. 40, 53–65. Braun, U., Forrer, R., Furer, W., Lutz, H., 1991. Selenium and vitamin E in blood sera of cows from farms with increased incidence of disease. Vet. Rec. 128 Ž23., 543–547. Clark, R.G., Hepburn, J.D., 1986. Deer liver and serum copper levels. Surveillance 13 Ž1., 11–14. Clark, R.G., Millar, K.R., 1983. Cobalt ŽCo.. In: Grace, N.D. ŽEd.., The Mineral Requirements of Grazing Ruminants. New Zealand Society of Animal Production, Palmerston North, No Occasional Publication No. 9, pp. 27–37. Clark, R., Burbage, J., Marshall, P.M., Valler, T., Wallace, D., 1986. Absence of a vitamin B12 weight gain response in two trials with growing red deer Ž CerÕus elaphus .. N. Z. Vet. J. 34 Ž11., 199–201. Corah, L.R., Ives, S., 1991. The effects of essential trace elements on reproduction in beef cattle. Vet. Clin. North Am. 7 Ž1., 41–57. Ellison, R.S., 1995. Trace elements in deer. In: Wilson, P.R. ŽEd.., Proceedings of a Deer Course for Veterinarians. Deer Branch of the New Zealand Veterinary Association, No. 12, pp. 57–68. Fisher, G.E.J., McPherson, A., 1991. Effect of cobalt deficiency in the pregnant ewe on reproductive performance and lamb viability. Res. Vet. Sci. 50 Ž3., 319–327. Forshell, K.P., Andersson, L., Pehrson, B., 1991. The relationships between the fertility of dairy cows and clinical and biochemical measurements, with special reference to plasma glucose and milk acetone. J. Vet. Med. Ser. A 38 Ž8., 608–616. Glauber, C.E., Lagger, J., Recoulat, A., 1993. Effect of supplementation with injectable phosphorus on the reproductive performance in dairy cows. Phosphataemia evaluation. Veterinaria ŽArgentina. 10 Ž97., 462–465. Henricksen, S.A., 1965. En forbedret teknik ved undersogelse for lungeormelarver I faeces. Nord. Vet. Med. 17, 446–454. Hewett, C., 1975. Importance of variations in the blood profile in cows in dairy herds. Svensk Veterinartidning 27 Ž16., 663–670.
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Mackintosh, C.G., Wilson, P.R., Beatson, N.S., Turner, K., Johnstone, P., 1986. Preliminary report of the liver: serum copper relationship in red deer. In: Wilson, P.R. ŽEd.., Proceedings of a Deer Course for Veterinarians, Vol. 3. Deer Branch of the New Zealand Veterinary Association, Rotorua, pp. 156–164. Morris, R.S., Meek, A.H., 1980. Measurement and evaluation of the economic effects of parasitic disease. Vet. Parasitol. 6, 165–184. Paragon, B.M., 1993. Readjustment of the supply of cobalt in the ruminant—a critical approach. Recl. Med. Vet. 169 Ž10., 759–761. Pomroy, W.E., Charleston, W.A.G., 1989. Failure of young goats to acquire resistance to Haemonchus contortus. N. Z. Vet. J. 37, 26–26. Revol, B., Wilson, P.R., 1991. Foetal ageing in farmed red deer using real-time ultrasonography. Anim. Reprod. Sci. 25 Ž3., 241–253. Ropstad, E., 1990. Constituents of blood and milk in relation to fertility, nutrition and metabolic status in dairy cows. Diss. Abstr. Int. 51 Ž2., 223C. Rowlands, G.J., Manston, R., Stark, A.J., Russell, A.M., Collis, K.A., Collis, S.C., 1980. Changes in albumin, globulin, glucose and cholesterol concentrations in the blood of dairy cows in late pregnancy and early lactation and relationships with subsequent fertility. J. Agric. Sci. 94 Ž3., 517–527. Scales, G.H., 1974. Reproductive performance of Merino ewes dosed with selenium prior to mating. Proc. N. Z. Soc. Anim. Prod. 34, 103–113. Spears, J.W., Harvey, R.W., Segerson, E.C., 1986. Effects of marginal selenium deficiency and winter protein supplementation on growth, reproduction and selenium status of beef cattle. J. Anim. Sci. 63 Ž2., 586–594. Suttle, N.F., 1991. The interactions between copper, molybdenum, and sulphur in ruminant nutrition. Annu. Rev. Nutr. 11, 121–140. Williams, S.N., McDowell, L.R., Warnick, A.C., Lawrence, L.A., Wilkinson, N.S., 1992. Influence of dietary phosphorus level on growth and reproduction of growing beef heifers. Int. J. Anim. Sci. 7 Ž2., 137–142. Wilson, G.F., Mackenzie, D.D.S., Holmes, C.W., 1985. Blood metabolites and infertility in dairy cows. Proc. N. Z. Soc. Anim. Prod. 45, 17–20.
Reproductive performance of farmed red deer žCerÕus elaphus / in New Zealand IV. Biological markers as risk factors for yearling and adult hind conception Laurent Audige´ 1, Peter R. Wilson ) , Roger S. Morris Institute of Veterinary, Animal and Biomedical Sciences, Massey UniÕersity, Palmerston North, New Zealand Accepted 25 February 1999
Abstract A 2-year observational study of 15 red deer Ž CerÕus elephus . farms was carried out in New Zealand from March 1992. In each year of study, approximately 1650 hinds were individually monitored for reproductive success. During farm visits in March 1992 and 1993, five yearling and five adult hinds per farm were randomly selected and blood sampled to define their haematological, biochemical and blood mineral profile. Faecal samples were taken for parasite egg and larvae count. Biological markers potentially affecting the probability of conception before May 1 or of conception that year were investigated separately using multivariable logistic regression analysis. Adult hinds with low serum phosphorus concentrations were more likely to conceive before May 1. Lower conception rates were observed in yearling hinds when blood glutathione peroxidase, serum vitamin B12, and serum albumin concentrations were low, and when faecal lungworm larval counts were high. While these associations have yet to be proven as causal, data suggests that monitoring and maintaining adequate blood elements, and controlling internal parasites in yearling hinds, may assist farmers to achieve optimum reproductive performance in farmed red deer herds. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Red deer; Reproductive performance; Biological markers; New Zealand
)
Corresponding author. Tel.: q64-6-350-5329; fax: q64-6-350-5616; e-mail: [email protected] Current address: Institute of Virology and Immunoprophylaxis, PO Box, CH-3147 Mittelhausern, ¨ Switzerland. Tel.: q41-31-848-92-52; fax: q41-31-848-92-22; e-mail: [email protected]. 1
0378-4320r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 4 3 2 0 Ž 9 9 . 0 0 0 1 8 - 4
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1. Introduction Reproductive success varies considerably between herds and between yearling Žprimiparous. and adult deer on commercial New Zealand deer farms ŽAudige, ´ 1995; Audige´ et al., 1999a.. A wide range of individual animal, management and grazing factors contribute to that variation and have been discussed elsewhere ŽAudige´ et al., 1999b,c.. In addition, there is variation both between animals and between farms in a number of biochemical measurements ŽAudige, ´ 1995.. That variation may be associated with variation in reproductive performance. However, there are no reports of relationships between biological markers and reproductive outcomes on commercial deer farms. Furthermore, there is a paucity on information on the influence of environmental factors on farmed deer reproductive performance as reviewed by Asher et al. Ž1996., although those authors proposed that feed supply, confinement and mating and calving management practices are key factors. There are a few reports of causal relationships between individual animal biological markers and reproductive outcomes in other species farmed in a commercial pastoral environment. Low selenium concentrations have been associated with poor reproductive performance in sheep ŽScales, 1974.. That relationship has been suspected but not proven in cattle ŽSpears et al., 1986; Ropstad, 1990; Corah and Ives, 1991.. Wilson et al. Ž1985. refer to variation in serum protein as a marker for reproductive success in cattle. Copper deficiency is widespread in farmed deer in New Zealand ŽEllison, 1995. and may contribute to poor reproductive performance, although there is no published evidence in this species. In cattle, low copper levels have been associated with changes in oestrous cycle length, anoestrus, increased number of services per conception and suspected embryonic loss ŽCorah and Ives, 1991.. There is little evidence of a direct association between vitamin B12 levels and reproduction. A 2-year observational study of 15 red deer farms was conducted to provide reference data for health and productivity outcomes, and to generate hypotheses on the relationship between various factors and productivity ŽAudige´ et al., 1993, 1994, 1999a,b,c; Audige, ´ 1995.. Blood and faeces were analysed for parameters from sentinel animals within those herds. While there may be no currently known biological relationship between some of the variables measured or recorded in this study, they were included to explore whether factors not studied to date may be associated with reproductive outcomes. Such factors, if identified, can subsequently be evaluated for causality. This paper presents biological markers associated with the likelihood of conceiving that year ŽCONC. or conceiving before May 1 ŽADVC. for yearling and adult hinds.
2. Materials and methods Fifteen commercial red deer herds were chosen for a 2-year longitudinal observational study from March 1992 as described elsewhere ŽAudige´ et al., 1993, 1994, 1999a,b,c; Audige, ´ 1995.. In each of the 2 years, hinds were individually monitored for reproductive success ŽAudige´ et al., 1999a. and were classified as conceiving before
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May 1, conceiving at all that year, even if after May 1, or being diagnosed non-pregnant, using rectal ultrasound pregnancy diagnostic techniques as described by Revol and Wilson Ž1991.. From individual animal data, early conception rates ŽADVCR. or conception rates that year ŽCONCR. were calculated within each farm and each year. During farm visits in March 1992 and 1993, 10 randomly selected sentinel hinds including five yearlings Ž15 months of age. and five adults Ž3 years of age or more. were physically restrained and blood sampled by jugular venipuncture into evacuated glass tubes containing either no anticoagulant or EDTA, for haematological, biochemical Table 1 Descriptive statistics of dichotomous biological markers screened for association with the ability of yearling and adult hinds to conceive before May 1 ŽADVC. and of conceiving that year ŽCONC., respectively Risk factorsa
Yearling hinds CU8 Yes No B12185 Yes No MGSHPX3 Yes No FEC ) 0 Yes No FLC ) 0 Yes No Adult hinds CU8 Yes No B12185 Yes No MGSHPX3 Yes No FEC ) 0 Yes No FLC ) 0 Yes No
Conception before May 1
Conception that year
Yes
No
OR
95% CI
p-value
Yes
No
OR
95% CI
p-value
64 10
33 1
0.2
0, 1.2
0.17
84 11
13 0
24 50
5 29
2.8
1, 7.8
0.06
28 67
1 12
5.0
0.6, 40.4
0.18 b
72 2
26 8
1.1
1, 1.3
0.001b
88 7
10 3
3.8
0.9, 15.9
0.10
15 59
5 29
1.5
0.5, 4.3
ns
16 79
4 9
0.5
0.1, 1.6
ns
44 30
19 15
1.2
0.5, 2.6
ns
51 44
12 1
0.1
0, 0.6
0.01
104 12
10 2
1.7
0, 8
ns
110 13
4 1
2.1
0, 15.6
ns
29 87
1 11
3.7
0.5, 29.6
ns b
29 94
1 4
1.2
0.1, 11.5
ns b
98 18
10 2
1.1
0, 4.9
ns
103 20
5 0
18 98
1 11
2.0
0.2, 16.6
ns b
19 104
0 5
63 53
7 5
0.8
0.3, 2.7
ns
67 56
3 2
0.8
0, 4.2
ns
OR: Odds ratio; 95% CI: 95% confidence interval. a Risk factor codes are defined in the text. b OR and 95% CI obtained using logistic regression.
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and blood mineral profile evaluation. Haematological measurements were carried out on fresh EDTA blood within 24 h of collection. White cell counts ŽWCC., and haemoglobin concentration ŽHB. were performed using a haematology analyser ŽCELL-DYN 900 Haematology Analyser, Sequoia-Turner, CA.. Packed cell volumes ŽPCV. were determined after centrifugation of capillary tube. Blood smears were stained, covered and stored to be read later. Differential leucocyte counts were performed by counting 100 cells under magnification Ž=40.. Leukocytes were classified as lymphocytes, polymorphonuclear neutrophils, eosinophils, basophils, band cells, monocytes and ‘unusual lymphocytes’ which were cells not conforming to normal morphology of lymphocytes because nuclei were severely indented. Variables lymphocyte percentage ŽLP., neutrophil percentage ŽNP. and eosinophil percentage ŽEOP. were calculated.
Table 2 Descriptive statistics of biological markers screened for association with the ability of adult hinds to conceive early Žbefore May 1. ŽADVC., in a preliminary univariable analysis and Rank sum test p-values Risk factor code a
Unit
Rank sum Range of all test values
ADVC s1 early conception
p-value
No. of Mean deer
Haematological markers WCC 10 9 rl 0.49 HB grdl 0.16 PCV % 0.12 MCHC grdl 1.00 NP % 0.02 EOP % 0.25 LP % 0.01 Haematological markers TP grl 0.31 ALB grl 0.76 GLOB grl 0.45 P mmolrl 0.01 GGT IU 0.71 BUN mmolrl 0.82 CU mmolrl 0.65 B12 pmolrl 0.40 GSHPx MGSHPxU kIUrl 0.37 PEPS mU 0.95 tyrosinerl Parasitological marker FLC larvaerg 0.86
Min
Max
SD
No. of Mean deer
1.7 12 3.3 12 0.05 12 7.5 12 15 12 5 12 15 12
5.5 18.6 0.41 46.0 40 8 49
SD
2.5 8.8 0.23 29.2 16 0 18
13.6 28.1 0.48 77.4 75 25 81
116 116 116 116 115 115 115
55.7 28.5 24.8 1.14 17 6.7 2.7 65 0.9 1.4 18
86.2 41.5 50.7 3.84 211 16.0 23 530 29.7 24.1 2719
116 116 116 116 116 116 116 116 62 116 116
70.7 35.1 35.7 2.4 42 11.1 13.2 161 6.9 7.3 585
5.5 2.9 5.1 0.5 25 1.7 4.2 77 6.7 6.0 447
12 12 12 12 12 12 12 12 3 12 12
69.5 34.8 34.7 2.8 36 11.2 13.3 134 14.0 7.5 528
4.3 2.5 4.3 0.5 9 1.8 3.7 37 8.6 5.8 333
14.8
116
0.8
1.4
12
1.9
4.3
0
5.5 16.0 0.39 41.6 48 7 42
ADVC s 0 late or no conception
1.1 5.0 0.05 13.2 10 4 10
Mins minimum; Max s maximum; SDsstandard deviation. The mean of the three individual measures of GSHPx from each farm each year has been taken as individual values for multivariable analysis of individual data. a Risk factor codes are described in the text.
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Sera were analysed for biochemical and mineral characteristics after being stored frozen. Total protein ŽTP., albumin ŽALB., phosphorus ŽP. and blood urea nitrogen ŽBUN. concentrations, and gamma glutamyl transferase ŽGGT. activity were measured from sera using an automatic analyser ŽCOBAS MIRA, F. Hoffmann-La Roche, Switzerland.. Whole blood glutathione peroxidase ŽGSHPx., from three hinds per farm, was measured by the MAF Bachelar Animal Health Laboratory, Palmerston North, New Zealand. Serum copper ŽCU. and vitamin B12 ŽB12. concentrations were analysed from frozen sera at the MAF Ruakura Animal Health Laboratory, Hamilton, New Zealand. Serum pepsinogen levels ŽPEPS. were assayed using the method described by Pomroy and Charleston Ž1989.. Faecal samples were taken per-rectum for gastrointestinal nematode egg ŽFEC. and lungworm larvae counts ŽFLC. which were performed the day after collection. Faecal egg counts were performed by mixing 2 g of faeces in 28 ml of Table 3 Descriptive statistics of biological markers screened for association with the ability of yearling hinds to conceive early Žbefore May 1. ŽADVC., in a preliminary univariable analysis and Rank sum test p-values Risk factor code a
Unit
Rank sum Range of all test values
ADVC s1 early conception
p-value
No. of Mean deer
Haematological markers WCC 10 9 rl 0.57 HB grdl 0.53 PCV % 0.93 MCHC grdl 0.59 NP % 0.24 EOP % 0.55 LP % 0.08 Haematological markers TP grl 0.05 ALB grl 0.22 GLOB grl 0.15 P mmolrl 0.45 GGT IU 0.97 BUN mmolrl 0.29 CU mmolrl 0.18 B12 pmolrl 0.03 GSHPx kIUrl MGSHPxU kIUrl 0.01 PEPS mU 0.91 tyrosinerl Parasitological marker FLC larvaerg 0.353
Min
2.8 12.1 0.33 32.2 11 0 23
56.2 30 22.4 1.5 16 6.5 1 65 2 1.4 18
0
Max
22 26.6 0.52 73.9 67 17 82
74 74 74 74 74 74 74
5.5 16.5 0.42 39.5 36 5 57
ADVC s 0 late or no conception SD
1.5 1.6 0.04 3.3 13 4 13
No. of Mean deer 34 34 34 34 34 34 34
6.3 17.5 0.42 42.0 39 5 52
SD
3.4 3.4 0.04 8.3 13 4 13
83.2 42.9 44.7 3.5 139 13.5 24 1500 29.2 24.1 1522
74 74 74 74 74 74 74 74 10 74 72
69.0 36.5 32.5 2.6 31 10.0 13.1 170.3 8.4 9.1 450.7
4.5 2.8 4.4 0.4 11 1.1 4.7 81.3 8.2 6.8 257.3
34 34 34 34 34 34 34 34 3 34 33
67.2 35.6 31.5 2.6 33 10.1 14.3 136.2 9.6 5.7 457.8
4.3 3.1 4.1 0.4 21 1.8 3.1 53.1 7.9 3.6 251.1
21.8
74
1.6
3.4
34
0.7
0.9
Mins minimum; Max s maximum; SDsstandard deviation. The mean of the three individual measures of GSHPx from each farm each year has been taken as individual values for multivariable analysis of individual data. a Risk factor codes are described in the text.
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saturated salt water, the count of one egg was equivalent to 50 eggs per gram of faeces Žmodified Master technique.. Faecal lungworm larvae counts were performed using a modified Baerman technique ŽHenricksen, 1965.. Faecal egg and larvae count data were categorised as positive faecal egg count ŽFEC ) 0. and positive faecal larval count ŽFLC ) 0., respectively. For statistical analyses, the mean of the three values of GSHPx within farms ŽMGSHPx. was used as the value for individual sentinel hinds since variability of GSHPx was mainly due to farm differences ŽAudige, ´ 1995.. Serum copper, ŽB12. and MGSHPx were categorised as adequate or deficient based on available data from deer and other domestic species. The categorical variables were: serum copper concentration G 8 mmolrl ŽCU8.; serum vitamin B12 concentration G 185 pmolrl ŽB12185.; and mean glutathione peroxidase activity from three hinds G 3 kIUrl ŽMGSHPx3.. These Table 4 Descriptive statistics of biological markers screened for association with the ability of adult hinds to conceive that year ŽCONC., in a preliminary univariable analysis and Rank sum test p-values Risk factor code a
Unit
Rank sum Range of all test values
CONC s1 coneption
p-value
No. of Mean deer
Haematological markers WCC 10 9 rl 0.43 HB grdl 0.57 PCV % 0.27 MCHC grdl 0.63 NP % 0.05 EOP % 0.73 LP % 0.03 Haematological markers TP grl 0.04 ALB grl 0.82 GLOB grl 0.04 P mmolrl 0.00 GGT IU 0.62 BUN mmolrl 0.11 CU mmolrl 0.66 B12 pmolrl 0.41 GSHPx MGSHPxU kIUrl 0.06 PEPS mU 0.99 tyrosinerl Parasitological markers FLC larvaerg 0.98
Min
2.5 8.8 0.23 29.2 16 0 18
55.7 28.5 24.8 1.1 17 6.7 2.7 65 0.9 1.4 18
0
Max
13.6 28.1 0.48 77.4 75 25 81
123 123 123 123 122 122 122
CONC s 0 no conception
5.4 16.2 0.39 42.1 48 7 42
SD
1.7 3.5 0.05 8.3 15 5 14
No. of Mean deer 5 5 5 5 5 5 5
5.5 17.6 0.42 41.6 36 6 54
SD
0.9 4.8 0.05 8.1 11 3 10
86.2 41.5 50.7 3.8 211 16.0 23 530 29.7 24.1 2719
123 123 123 123 123 123 123 123 64 123 123
70.8 35.1 35.7 2.4 41 11.1 13.2 159.5 7.1 7.2 581
5.5 2.9 5.0 0.5 24 1.7 4.1 74.9 6.9 6.0 440
5 5 5 5 5 5 5 5 1 5 5
66.9 34.7 32.2 3.1 35 10.6 13.7 126.0 13.3 8.4 546
0.7 2.5 2.3 0.2 13 0.6 3.9 46.2 1.8 416
14.7
123
0.8
1.4
5
3.1
6.5
Mins minimum; Max s maximum; SDsstandard deviation. The mean of the three individual measures of GSHPx from each farm each year has been taken as individual values for multivariable analysis of individual data. a Risk factor codes are described in the text.
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markers were also analysed as continuous variables, although both forms of the same variables were not included in the same multivariable analysis. Data from both years of study were combined for statistical analyses. Analyses were carried out separately for yearling and adult hinds. All analyses were carried out using the statistical software Stata version 5.0 ŽStata, TX, USA.. A full description of statistical methods applied in this study are presented elsewhere ŽAudige´ et al., 1999b.. Data were initially screened for univariable statistical association with both ADVC and CONC outcomes. Continuous risk factors were analysed without transformation, by the rank sum test, while categorical risk factors were analysed by the Fisher’s exact test. Potential risk factors statistically significantly associated with the outcome at p - 0.20 were included together in a stepwise multivariablte logistic regression analysis with a forward selection procedure. Risk factors classified as significant were associated with respective outcomes at the 5% confidence level. Table 5 Descriptive statistics of biological markers screened for association with the ability of yearling hinds to conceive that year ŽCONC., in a preliminary univariable analysis and Rank sum test p-values Risk factor code a
Unit
Rank sum Range of all test values
CONC s1 conception
p-value
No. of Mean deer
Haematological markers WCC 10 9 rl 0.56 HB grdl 0.52 PCV % 0.51 MCHC grdl 0.10 NP % 0.64 EOP % 0.67 LP % 0.23 Haematological markers TP grl 0.08 ALB grl 0.01 GLOB grl 0.82 P mmolrl 0.89 GGT IU 0.10 BUN mmolrl 0.43 CU mmolrl 0.00 B12 pmolrl 0.02 GSHPx kIUrl MGSHPxU kIUrl 0.21 PEPS mU 0.81 tyrosinerl Parasitological markers FLC larvaerg 0.04
Min
Max
CONC s 0 no conception
1.7 2.0 0.04 5.0 13 4 12
No. of Mean deer 13 13 13 13 13 13 13
6.9 18.0 0.41 43.5 40 5 50
SD
2.8 12.1 0.33 32.2 11 0 23
22 26.6 0.52 73.9 67 17 82
95 95 95 95 95 95 95
56.2 30 22.4 1.53 16 6.51 1 65 2 1.37 18
83.2 42.9 44.7 3.49 139 13.5 24 1500 29.2 24.13 1522
95 95 95 95 95 95 95 95 11 95 93
68.7 36.5 32.2 2.6 32 10.0 13.1 165.6 9.4 8.4 453
4.3 2.9 4.4 0.4 16 1.3 4.4 76.2 8.4 6.4 256
13 13 13 13 13 13 13 13 2 13 12
66.3 34.2 32.1 2.6 32 10.3 16.1 115.2 5.1 5.5 455
5.1 2.3 4.1 0.4 7 1.8 2.5 48.4 0.9 2.7 255
21.8
95
1.4
3.1
13
1.1
1.0
0
5.6 16.7 0.42 39.9 37 5 56
SD
4.9 3.9 0.04 7.5 15 4 14
Mins minimum; Max s maximum; SDsstandard deviation. The mean of the three individual measures of GSHPx from each farm each year has been taken as individual values for multivariable analysis of individual data. a Risk factor codes are described in the text.
U
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In a first analysis, the potential effect of clustering due to farm or year of study on the significance of risk factors was not taken into account. In a second step, the final models were checked considering clustering due to farms within years, as most of the variability of biological markers were due to farms ŽAudige, ´ 1995.. In addition, individual hind characteristics that were identified as important factors affecting conception in earlier analyses were forced into final models Ždepending on the outcome as presented in result tables. as otherwise they may be confounding factors. These adult-hind individual characteristics were: adult hind ) 3 years old at mating ŽAGE ) 3.; pre-mating bodycondition score ) 2.5 ŽBCSM) 2.5. or ) 2 ŽBCSM) 2.; pure New Zealand-type hind ŽNZ100., hind lactating at weaning ŽWET. and body weight standardised to April 1 from body weights recorded in March and June Žbody weight change was considered linear in this time interval. ŽW4. ŽAudige´ et al., 1999b.. Important yearling-hind characteristics were: pre-mating body-condition score - 3 ŽBCSM- 3.; percentage of New Zealand blood line ŽNZ.; growth rate between April 1 and June 1 ŽGR46. and W4 ŽAudige´ et al., 1999c.. Final models of all selected factors, after adjustment for potential clustering effect due to farm and after inclusion of hind variables defined above are presented in tables. The non-significance of some factors presented in result tables was because of this inclusion of variables and adjustment. Biological markers were initially investigated at the individual-hind level. Farm mean blood markers, using the geometric or arithmetic mean where appropriate, were investi-
Table 6 Logistic regression coefficient estimates of biological markers statistically and significantly associated with adult hind conception status Žafter adjustment for farm effect. Risk factor code a
Regression coefficient
Standard error
Hind conception before May 1 (ADVC) Before the inclusion of individual hind characteristics; no. of hinds: 128 P y2.78 0.61 After the inclusion of individual hind characteristics; no. of hinds: 125 NZ100 0.14 1.18 WET 1.05 0.81 AGE) 3 4.10 2.24 BCSM) 2 1.19 0.91 P y5.50 1.76 Hind conception that year (CONC) Before the inclusion of individual hind characteristics; no. of hinds: 127 P y4.41 0.99 NP 0.12 0.04 After the inclusion of individual hind characteristics; no. of hinds: 125 WET 2.25 0.88 BCSM) 2.5 0.52 0.63 P y4.86 1.35 NP 0.14 0.06 a
Risk factor codes are described in the text. OR s odds ratio.
p-value
- 0.001 0.91 0.19 0.07 0.19 - 0.001
- 0.001 0.003 0.91 0.19 0.07 0.19
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gated in association with conception rates in follow-up investigations of the most important individual biological markers. Scatterplots of farm mean blood markers vs. ADVCR or CONCR were created with Statistica ŽStatsoft, Tulsa, OK, USA..
3. Results Results of Fisher exact tests to screen association between each dichotomous sentinel hind characteristic and biological markers and outcome variables ADVC and CONC are presented in Table 1. The ranges of values, means and standard deviations and results of statistical analyses of continuous biological markers screened for association with ADVC and CONC for both adult and yearling hinds are presented in Tables 2–5.
Table 7 Logistic regression coefficient estimates of biological markers statistically and significantly associated with yearling hind conception status Žafter adjustment for farm effect. Risk factor code a
Regression coefficient
Standard error
p-value
Hind conception before May 1 (ADVC) Before the inclusion of individual hind characteristics; no. of hinds: 108 With continuous risk factors CU y0.13 0.06 0.02 MGSHPx 0.16 0.05 0.001 With some risk factors categorised MGSHPx3 2.40 0.33 - 0.001 After the inclusion of individual hind characteristics; no. of hinds: 88 With continuous risk factors W4 y0.003 0.03 0.92 CU y0.09 0.07 0.17 MGSHPx 0.19 0.07 - 0.001 With some risk factors categorised W4 0.02 0.04 0.64 MGSHPx3 2.42 0.42 - 0.001 Hind conception that year (CONC) Before the inclusion of individual hind characteristics; no. of hinds: 108 With continuous risk factorsb,c B12 0.02 0.01 0.06 ALB 0.28 0.11 0.01 CU y0.22 0.05 - 0.001 FLCPOS y2.43 0.92 0.01
OR
95% CI
11.1
5.8, 21.1
11.3
4.9, 25.8
0.09
0.01, 0.54
OR s odds ratio. a Risk factor codes are described in the text. b When replacing B12 by B12185 in the analysis, the final model included the variables ALB, CU and FLCPOS. This final model was ignored, as it provided no additional information. c After inclusion of BCSM- 3, NZ, W4, GR46 into this model, the factors FLCPOS and BCSM- 3 were dropped from the model as, if equal to null, they predicted perfectly the outcome CONC, and the number of observations was restricted to 30 hinds. Consequently, the resulting model is not presented in this table.
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Fig. 1. Relationship between mean pre-mating phosphorus concentrations of five adult hinds and conception rates Žbefore May 1 and overall conception. of adult hinds within each survey farm. Data from 1992 and 1993 combined.
Fig. 2. Relationship between mean pre-mating glutathione peroxidase from three hinds and yearling hind early conception rates within each survey farm. Data from 1992 and 1993 combined. Note: The line is a logarithm fitted line.
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Fig. 3. Relationship between geometric mean pre-mating serum vitamin B12 from five sentinel yearling hinds and yearling hind conception rates within each survey farm. Data from 1992 and 1993 combined. Note: The line is a logarithm fitted line.
Variables which were associated with an outcome with a p - 0.20 were those subjected to stepwise multiple logistic regression analysis to produce the final results presented in Tables 6 and 7, which are discussed. There was an inverse relationship between blood phosphorus concentration ŽP., Žrange 1.1–3.8 mmolrl, Table 2. and early conception and conception that year. However, this latter association became non-significant when individual hind variables
Fig. 4. Relationship between mean pre-mating albumin concentrations of five yearling hinds and conception rates of yearling hinds within each survey farm. Data from 1992 and 1993 combined. Note: The fitted line is an inverse exponential smoothing curve.
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WET and BCSM) 2.5 were included in the final model ŽTable 6.. There was a positive relationship between blood neutrophil percentage ŽNP. which ranged from 16–75% ŽTable 2. and CONC, but it became non-significant after the inclusion of WET and BCSM) 2.5 in the final model ŽTable 6.. In yearling hinds ŽTable 7., the serum copper concentration, which ranged from 1 to 24 mmolrl ŽTable 3., was negatively associated with the probability of early conception and conception that year. However, the association between CU and ADVC became non-significant when individual hind variable W4 was included in the final model. Mean glutathione peroxidase Žrange 21.4–29.24.1 nmolrl, Table 3. was positively associated with the probability of early conception. Furthermore, yearling hinds on farms with a mean GSHPx of more than 3 nmolrl had 11 times higher odds of conceiving early. Serum albumin Žrange 30–42.9 grl, Table 3. and vitamin B12 concentration Žrange 65–1500 pmolrl, Table 3. were positively associated with the probability of conception that year. Yearling hinds with lungworm larvae detected in faeces had about 11 times lower odds of conceiving that year than other hinds. Scatterplots ŽFigs. 1–4. show herd blood phosphorus in adult hinds, and mean GSHPx, individual blood albumin, and geometric mean serum vitamin B12 in yearling hinds, and blood phosphorus in adult hinds, plotted against conception rates. 4. Discussion This presentation is part of an extensive investigation of a large number of characteristics and outcomes from commercial red deer farms ŽAudige, ´ 1995.. It is the first to attempt to relate a range of deer blood characteristics and faecal parasite larvae output to measures of reproductive success. Previous analyses highlighted the importance of a wide range of individual hind characteristics such as weight and body condition score in yearling and adult hinds ŽAudige´ et al., 1999a,b,c.. In those analyses, 2600 adult and 700 yearling hinds were monitored for a limited range of individual characteristics which were simple, time efficient and inexpensive to measure or record. Much data used in this study was time-consuming to collect on the farm, and the laboratory analyses were costly to perform. For these reasons, it was necessary to limit the sample size to five hinds of each age category, from the 15 farms studied over the 2-year period. That only a small number of biological markers were found to be associated with the likelihood of conception or early conception in this study may have been due to sample size. Alternatively, the variation in many measurements may not have been large enough, or they were all within a normal range for physiological function, e.g., haematological and biochemical measurements such as HB, WCC, GGT, BUN and PEPS. Yet another explanation may be that the principal influences on individual deer conception, particularly in primiparous hinds, may be due to endocrine or behavioural factors not measured in this study. These warrant further investigation. Serum copper, vitamin B12 and glutathione peroxidase were analysed both as continuous and as categorical variables. All three markers have a minimum threshold level under which health problems may occur and production levels may be reduced. The minimum required blood level for copper in deer is thought to be 8 mmolrl ŽClark
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and Hepburn, 1986; Mackintosh et al., 1986.. Minimum levels for GSHPx in sheep are reported as 3 kIUrl ŽMAF Optigrowth Animal Health profile.. There are no reference data available for GSHPx in deer. Critical values of serum vitamin B12 in cattle are 38–76 pmolrl ŽSuttle, 1991., but concentrations below 185 pmolrl are regarded as deficient in sheep ŽClark and Millar, 1983.. No reference range exists for deer vitamin B12 concentrations ŽClark et al., 1986.. The threshold of 185 pmolrl has been chosen because of the sensitivity of the assay and the distribution of the data. These blood markers were also investigated as continuous variables because extrapolation of thresholds from other species may not be appropriate for deer. This study showed a negative relationship between serum phosphorus concentrations and adult hind ADVC. Many other studies failed to identify a significant relationship between phosphorus levels and fertility in cattle ŽRopstad, 1990; Forshell et al., 1991; Glauber et al., 1993.. However, there have been reports that high phosphorus concentrations could limit optimal fertility in cattle ŽHewett, 1975.. The non-significance of phosphorus levels on the onset of puberty and conception in yearling hinds is consistent with observations in heifers ŽWilliams et al., 1992.. The scatterplot of phosphorus data ŽFig. 1. suggests it is not obvious that phosphorus can be used to predict conception rates in adult hinds, although the relationship with overall conception rates was significant. It appears that low selenium concentrations as measured by GSHPx may impair early conception in yearling hinds. This effect was not evident in adults. Individual serum GSHPx activity was not investigated because only three hind samples per farm were analysed. However, because the variability of GSHPx is essentially due to farm difference Ž66.8% of the total variance ŽAudige, ´ 1995.., the mean GSHPx ŽMGSHPx. was used as an individual value for every hind within each farm. When MGSHPx was over 3 kIUrl, which has been proposed as the minimum threshold value in sheep ŽMAF Optigrow Animal Health profile., yearling hinds had 11 times higher odds of conceiving early. Fig. 2 demonstrates the possible negative effect of low selenium levels on yearling hind early conception, and suggests a threshold value may stand around 5–7 kIUrl, instead of 3 kIUrl as identified in sheep. Why this relationship in yearlings was not significant in adult hinds is not known, and requires further investigation. Furthermore, this finding questions the relevance of sheep reference ranges in yearling deer. More work is required to establish appropriate selenium reference ranges in deer. Many studies have failed to identify a significant relationship between blood selenium levels and fertility, and Se supplementation trials have failed to influence reproductive performance in cattle ŽSpears et al., 1986; Ropstad, 1990; Braun et al., 1991.. In sheep, however, the percentage of barrenness was significantly reduced by dosing with selenium prior to mating ŽScales, 1974., possibly due to reduced embryonic mortality. The negative relationship between serum copper concentrations and yearling-hind early conception and conception that year is intriguing and is difficult to explain. However, this association with ADVC became non-significant when W4 was forced into the model. The inclusion of yearling-hind individual characteristics into the model explaining CONC was not possible because of limited number of observations used for the regression analysis. The negative effect of CU may be a spurious result since it is
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contrary to observations in cattle where changes in oestrous cycle length, anoestrus, and an increased number of services per conception have been proven to be associated with low copper status, and embryonic death is suspected ŽCorah and Ives, 1991.. More intensive study of reproductive performance in deer herds of low copper status is required. Serum vitamin B12 was positively associated with the probability of yearling hinds conceiving. Cobalt is needed for optimal function of rumen micro-organisms and thus optimal forage digestibility ŽParagon, 1993.. The categorical variable B12 above 185 pmolrl was not significant, which suggests that 185 pmolrl may not be the appropriate threshold level in deer. This is also suggested by data in Fig. 3, supporting the observation of Clark et al. Ž1986., who reported no growth response following vitamin B12 supplementation trials in deer with B12 levels below 185 pmolrl. In sheep, vitamin B12 deficiency was not associated with lower conception rate, but lower lamb survival ŽFisher and McPherson, 1991.. Farm differences ‘explained’ 53.9% of the variability of pre-mating B12 in yearling hinds ŽAudige, ´ 1995.. There was a negative relationship between faecal larval count ŽFLC ) 0. and conception in yearling hinds. Hinds with positive FLC had 11 times lower odds of conceiving. The mechanism by which lung parasitism would affect reproductive performance directly is not clear, but it is believed to be mediated through the nutritional status of the animal ŽMorris and Meek, 1980.. This would be consistent with the observation that yearling hinds gaining body weight during mating were more likely to conceive that year ŽAudige´ et al., 1999c.. High serum albumin concentrations were associated with conception in individual yearling hinds, consistent with previous observation of the positive relationship with high growth rates during mating ŽAudige´ et al., 1999c.. This suggests serum albumin may be a useful marker to assess the likelihood of conception in yearling hinds. Many publications support the potential benefit of measuring serum albumin concentrations in dairy cattle ŽWilson et al., 1985.. Other authors referred to a reduction in the albumin:globulin ratio in infertile cows ŽRowlands et al., 1980.. Farm differences ‘explained’ 32.7% of the total variance of pre-mating serum albumin concentrations in yearling hinds ŽAudige, ´ 1995.. There was a statistically significant positive linear relationship between mean serum albumin concentrations and conception rates in this age group. Fig. 4 suggests a threshold value may stand around 35 grl, under which reproductive performance may be impaired. This will need to be further investigated.
5. Conclusion This study suggests that whereas no biological markers appeared useful in evaluating the risk of infertility in adult hinds, there is potential for the use of blood markers in yearling hinds. Measurement of selenium, serum vitamin B12, albumin concentrations, and faecal lungworm larval counts may be useful to trigger management decisions related to feeding, supplementation or treatment, to improve reproductive performance in that age group.
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Acknowledgements We wish to thank participating farmers for their active co-operation during this health and production research project. We thank the personnel of the Veterinary Faculty at Massey University for help and assistance. Financial assistance was provided by the New Zealand Game Industry Board, the French Government Žfor scholarship., CIRADEMVT, Deer Branch of the NZVA, Massey University, Pitman Moore Žnow Schering Plough., Rhone Cyanamid, AgResearch, Allflex New Zealand and Te Pari ˆ Merieux, ´ Products. References Asher, G.W., Fisher, M.W., Fennessy, P.F., 1996. Environmental constraints on reproductive performance of farmed deer. Anim. Reprod. Sci. 42, 35–44. Audige, ´ L., 1995. Deer herd health and production profiling. PhD thesis, Massey University, New Zealand, 550 pp. Audige, ´ L., Wilson, P.R., Morris, R.S., 1993. Deer herd health and production profiling the method. In: Wilson, P.R. ŽEd.., Proceedings of a Deer Course for Veterinarians. Deer Branch of the New Zealand Veterinary Association, No. 10, pp. 78–100. Audige, ´ L., Wilson, P.R., Morris, R.S., 1994. Deer-herd health and production profiling in New Zealand: 1. Study design. Vet. Res. 25 Ž2–3., 126–129. Audige, ´ L., Wilson, P.R., Morris, R.S., 1999a. Reproductive performance of farmed red deer Ž CerÕus elaphus. in New Zealand: I. Descriptive analysis. Anim. Reprod. Sci. 55, 127–141. Audige, ´ L., Wilson, P.R., Morris, R.S., 1999b. Reproductive performance of farmed red deer Ž CerÕus elaphus. in New Zealand: II. Risk factors for adult hind conception. Prev. Vet. Med. 40, 33–51. Audige, ´ L., Wilson, P.R., Morris, R.S., 1999c. Reproductive performance of farmed red deer Ž CerÕus elaphus. in New Zealand: III. Risk factors for yearling hind conception. Prev. Vet. Med. 40, 53–65. Braun, U., Forrer, R., Furer, W., Lutz, H., 1991. Selenium and vitamin E in blood sera of cows from farms with increased incidence of disease. Vet. Rec. 128 Ž23., 543–547. Clark, R.G., Hepburn, J.D., 1986. Deer liver and serum copper levels. Surveillance 13 Ž1., 11–14. Clark, R.G., Millar, K.R., 1983. Cobalt ŽCo.. In: Grace, N.D. ŽEd.., The Mineral Requirements of Grazing Ruminants. New Zealand Society of Animal Production, Palmerston North, No Occasional Publication No. 9, pp. 27–37. Clark, R., Burbage, J., Marshall, P.M., Valler, T., Wallace, D., 1986. Absence of a vitamin B12 weight gain response in two trials with growing red deer Ž CerÕus elaphus .. N. Z. Vet. J. 34 Ž11., 199–201. Corah, L.R., Ives, S., 1991. The effects of essential trace elements on reproduction in beef cattle. Vet. Clin. North Am. 7 Ž1., 41–57. Ellison, R.S., 1995. Trace elements in deer. In: Wilson, P.R. ŽEd.., Proceedings of a Deer Course for Veterinarians. Deer Branch of the New Zealand Veterinary Association, No. 12, pp. 57–68. Fisher, G.E.J., McPherson, A., 1991. Effect of cobalt deficiency in the pregnant ewe on reproductive performance and lamb viability. Res. Vet. Sci. 50 Ž3., 319–327. Forshell, K.P., Andersson, L., Pehrson, B., 1991. The relationships between the fertility of dairy cows and clinical and biochemical measurements, with special reference to plasma glucose and milk acetone. J. Vet. Med. Ser. A 38 Ž8., 608–616. Glauber, C.E., Lagger, J., Recoulat, A., 1993. Effect of supplementation with injectable phosphorus on the reproductive performance in dairy cows. Phosphataemia evaluation. Veterinaria ŽArgentina. 10 Ž97., 462–465. Henricksen, S.A., 1965. En forbedret teknik ved undersogelse for lungeormelarver I faeces. Nord. Vet. Med. 17, 446–454. Hewett, C., 1975. Importance of variations in the blood profile in cows in dairy herds. Svensk Veterinartidning 27 Ž16., 663–670.
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