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ANIMAL GENETICS |
* Agriculture and Life Sciences Division, PO Box 84, Lincoln University, Canterbury 7647, New Zealand; and
Christchurch School of Medicine and Health Sciences, PO Box 4345, Christchurch, New Zealand
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Abstract |
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 Top Abstract INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Key Words: ß3-adrenergic receptor • gene marker • mortality • polymorphism • sheep • survival
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INTRODUCTION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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), and these animals have impaired thermoregulatory and UCP-1 mRNA responses to cold-exposure (Lowell et al., 2000).
Neonatal lamb mortality represents a large loss to the New Zealand sheep industry. Not only is the industry affected by the direct loss of the dead lambs, but also by the reduction in productivity of hypothermic lambs that survive and the reduced selection potential incurred by having fewer lambs surviving until mating. Although completely eliminating lamb deaths due to inadequate cold-tolerance is impossible with outdoor lambing, previous studies have shown that genetic variation exists in the cold-tolerance of neonatal lambs (Slee et al., 1987; Slee and Simpson 1991; Forrest et al., 2003). Consequently, gains in lamb survival could be possible by including cold tolerance as a selection criterion in breeding programs, and the development of gene markers for cold tolerance would improve the accuracy of selection.
Subsequent to the characterization of the ovine ß3-adrenergic receptor gene (ADRB3; Forrest and Hickford, 2000), variation in this gene has been associated with cold-related mortality in Merino lambs (Forrest et al., 2003, 2006). This large mixed-breed study confirms the findings of Forrest et al. (2003, 2006) and supports the use of variation at the ADRB3 locus as a gene marker for marker-assisted breeding of relatively cold-tolerant lambs.
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MATERIALS AND METHODS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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All procedures involving animals were approved by the Lincoln University Animal Ethics Committee.
Lambs (total n = 13,420; Borderdale, n = 351; Corriedale, n = 4,260; Coopworth, n = 1,225; Dorset Down, n = 663; Dorset Down x Coopworth, n = 264; Hampshire, n = 230; Merino, n = 4,488; Merino x Coopworth, n = 329; Merino x Polwarth, n = 226; Polwarth, n = 928; Poll Dorset, n = 241; and Suffolk, n = 215) born on 22 farms throughout the South Island of New Zealand were used in this study. The lambs were born to mixed aged, adult ewes that were all greater than 2 yr of age and were multiparous ewes. Two hundred thirty-seven sires were used in total (Table 1), with the sires from different farms being unrelated. The relatedness of sires within each farm was unknown.
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The diagnosis of cold-related lamb death was based on field observations, using a technique similar to that used by Purser and Young (1964). Dead lambs were classified as having died from cold-exposure if they died within 5 d of birth and no other cause of death was obvious, such as being rejected by its mother, having a swollen head (dystocia), having membranes over the nose and mouth (suffocation), unbroken feet membranes (indicating that the animal had not walked), and the occurrence of birth defects, such as being poorly developed or mummified. When a lamb died, the dam’s udder was checked for abnormalities or infection to ensure the death was not the fault of the ewe.
For each live lamb, blood samples were collected onto an FTA card (Whatman BioSciences Ltd, Abington, Cambridge, UK) at tail docking or weaning. The blood was allowed to air-dry and was stored in darkness at room temperature.
DNA Preparation and Genotyping
For each blood sample, a disc 1.2 mm in diameter was punched from the FTA card and placed into a 200-µL tube. The DNA on the card was purified according to the manufacturer’s instructions. For each dead lamb, DNA was extracted from a 200-mg ear tissue sample (wool removed). The ear tissue was diced and incubated overnight at 55°C with 500 µL of lysis buffer (50 mM Tris, pH 8.0; 25 mM Na2EDTA; 100 mM NaCl; and 1% Triton X-100) and 25 µL of proteinase K solution (20 mg/mL; Quantum Scientific, Queensland, Australia). The DNA was then isolated in the aqueous phase by mixing the incubated mixture with 500 µL of phenol and 500 µL of chloroform:isoamyl alcohol (24:1, vol/vol) and centrifuging at 11,357 x g for 10 min. The aqueous phase was pipetted into a fresh tube, and the DNA was precipitated using 2 volumes of ice-cold ethanol (100%). The DNA was spooled onto a glass hook and washed in 70% ethanol overnight, before air-drying and dissolving in 500 µL of sterile water.
Each lamb was genotyped using PCR-single strand conformational polymorphism (PCR-SSCP), according to the method described in Forrest et al., 2003.
Statistical Analysis
All analyses were performed using SPSS, version 13 (Chicago, IL). The ADRB3 allele frequencies were calculated for the 13,420 lambs studied. The percentage of cold-related and total mortality within 5 d of birth was determined for each farm. Two data sets were analyzed to test the association of each of the ADRB3 alleles with cold-related mortality and total lamb mortality. The first used all data (n = 13,420), whereas in the second those farms that had less than 10% mortality were removed on the assumption that the lower death rate was due to an insufficient cold challenge. This second set of analyses utilized data from 7,390 lambs. Pearson 
2 tests and odds ratios were used to explore the univariate association between the presence of each of the ADRB3 alleles and cold-related and total mortality. Multivariate, binary, logistic regression analyses were used to assess the independent effects of each of the ADRB3 alleles. To determine which variables (farm, breed, sex, or birth rank) would be included in the multivariate models, a univariate Pearson 2 test was performed to explore the association between these variables and both cold-related mortality and total mortality.
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RESULTS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). Farm, breed, and birth rank were found to be associated with cold-related and total mortality (P < 0.001 for each association). Lambs from a multiple birth were associated with increased mortality (P < 0.001). Sex was not associated with cold-related mortality or total mortality (P = 0.640 and 0.796, respectively), and thus sex was not included in subsequent analyses.
Of the 13,420 lambs born, 237 carried alleles other than the 6 alleles (A – F) described by Forrest et al., 2003. Two new alleles were identified in these animals and they were designated G and H (GenBank accession DQ267939 and DQ269497, respectively). Alleles A, C, and E were the most common (Table 2). The allele frequencies for each farm are listed in Table 2 along with the overall allele frequencies for the entire data set. When the data from those farms that had greater than 10% total mortality were analyzed together, an increase in the frequencies of alleles A, B, C, D, and H along with a decrease in the frequencies of alleles E and G was observed when compared with the overall allele frequencies for the entire data set. The frequency of allele F remained constant (Table 2).
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). Of these, allele E showed a reduced risk of cold-related mortality, whereas alleles C and D were associated with a greater susceptibility to cold-related mortality. When the independent effects of each allele were assessed using a multivariate binary logistic regression model (n = 11,683) to correct for farm, breed, and birth rank, the strong association of allele D with an increased risk of cold-related mortality persisted. Although not significant, alleles A and E tended (P < 0.1) to be associated with a reduced risk of cold-related mortality, whereas allele C tended to be associated with an increased risk of cold-related mortality. A significant association (P = 0.048) of the F allele with a greater susceptibility to cold-related mortality was also observed.
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), and of these, allele E showed a reduced risk of lamb mortality, whereas alleles C and D showed a greater risk of lamb mortality. Unexpectedly, allele F was associated with a reduced risk of lamb mortality. Once farm, breed, and birth rank were corrected for, the strong association of allele D with a greater risk of lamb mortality persisted; however, the associations of alleles C, E, and F were not maintained.
Twelve farms (n = 7,390) had greater than 10% total mortality, with cold-related deaths ranging from 3.7 to 25% (Table 1). When these farms were analyzed together, similar univariate and multivariate associations with cold-related and total mortality were observed for alleles C, D, and F, to those observed for the entire data set (Table 4). Allele E no longer showed a significant univariate association with decrease cold-related mortality and was associated with an increase in total mortality (P < 0.05). However, once farm, breed, and birth rank were corrected for, allele E tended (P < 0.10) to be associated with a decreased risk of cold-related and total mortality. Allele A was associated with a decreased risk of both cold-related and total mortality in the univariate analyses, and once farm, breed, and birth rank were corrected for, still tended (P < 0.10) to be associated with a decreased risk of cold-related mortality.
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DISCUSSION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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It has long been accepted that lamb survival is associated with birth weight (Purser and Young, 1964; Hight and Jury, 1970; Sykes et al., 1976) and therefore, in the absence of birth weight data, birth rank was included in the multivariate statistical models, using the assumption that lambs from multiple births will be of a lower birth weight when compared with lambs from single births. This, however, will only in part correct for any birth weight effects. Farm was also included to allow for the variation in: the cold challenge experienced due to geographical location, farm management systems, and lamb death diagnoses. Sex of the lamb was not found to influence survival in this study.
The diagnosis of lamb death due to cold-exposure is complex due to interactions with the dam, infection, birth weight, dystocia, starvation, birth injury, and birth-coat, all of which can predispose lambs to death from cold-exposure (Alexander, 1984). This study used a technique that ruled out other causes of death using field observations, and although this technique may overestimate lamb deaths due to cold-exposure, it is still considered to be more accurate than the autopsy method originally developed by McFarlane (1965), which appears to underestimate cold-related deaths (Alexander, 1985) and overestimate deaths from dystocia (Haughey, 1980). Due to the difficulty of diagnosing cold death, total mortality analyses were performed along with those for cold-related mortality. Whereas one might have expected similar associations from each set of analyses (but with reduced significance due to the increase in deaths diluting any allele effect occurring in the cold-related deaths), new and strengthened associations suggest that these alleles may impact on other attributes that contribute to survival. Forrest et al. (2003) described an ADRB3 allele association with birth weight that may provide part of the explanation and is deserving of further investigation.
Variation in the ovine ADRB3 occurs in both the coding and noncoding regions (Forrest et al., 2003). Of particular interest is that sequence variation that occurs within exon-1 of allele D, predicts 2 amino acid substitutions, Val52
Ala and Leu322Val. These substitutions occur in positions that normally have residues common to all 3 ß-adrenergic receptor subtypes (i.e., ß1, ß2, and ß3) and that are thought to be involved in ligand binding (reviewed in Strosberg and Gerhardt, 2000). Although these substitutions are considered to be conservative (because the amino acids are similar), it is possible that even a small change in the size of the amino acid side chain may affect ligand binding. Thus it can be speculated that the association of allele D with an increased risk of cold-related mortality may be the result of altered ligand binding to the ADRB3, which subsequently affects receptor function. This could in turn provide an explanation as to why allele D has a low frequency in the New Zealand sheep population, with the D allele possibly being selected against naturally and by management practices (including farming outside and breeding strategies).
In order to assist in the selection for more cold-tolerant sheep (and to decrease cold-related mortality in lambs), we have developed a gene marker for cold tolerance based on variation in the ADRB3 gene. Removing the D allele, especially from the Merino population, would seem to be sound advice for sheep breeders, assuming that this allele does not beneficially impact any other desirable traits. Thus this gene marker test would be best employed after selection for all other important production traits, and alongside other assessments of survivability, to aid in the selection of rams that are more likely to have hardy progeny. In breeding, such a test may improve the accuracy of selecting sheep that are considered less likely to have progeny that will die from cold-exposure and the speed of genetic gain because sheep can be genotyped from birth and therefore preferentially selected at an earlier age.
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Footnotes |
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2 Corresponding author: forrestr{at}lincoln.ac.nz
Received for publication December 9, 2006. Accepted for publication July 2, 2007.
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LITERATURE CITED |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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This article has been cited by other articles:
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  S. W. P. Cloete, I. Misztal, and J. J. Olivier Genetic parameters and trends for lamb survival and birth weight in a Merino flock divergently selected for multiple rearing ability J Anim Sci, July 1, 2009; 87(7): 2196 - 2208. [Abstract] [Full Text] [PDF]
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