Mitochondrial DNA polymorphisms and fertility in beef cattle

Theriogenology 57 (2002) 1603±1610

Mitochondrial DNA polymorphisms and fertility in beef cattle Sutarnoa,b, J.M. Cumminsb, J. Greeffc, A.J. Lymberya,* a

Division of Veterinary and Biomedical Sciences, Murdoch University, South Street, Perth, Murdoch, WA 6150, Australia b Biology Department, FMIPA, Sebelas Maret University, Solo 57126, Indonesia c Agriculture Western Australia, P.O. Box 757, Katanning, WA 6317, Australia Received 5 January 2001; accepted 31 August 2001

Abstract Four hundred and twenty-two beef cattle of two different breeds (purebred Hereford and composite multibreed) were characterized by polymerase chain reaction±restriction fragment length polymorphism, using the restriction enzymes ApaI, AvaII, HindIII, PstI, SpeI, SspI and TaqI in two regions (the D-loop and the ND-5 gene) of mitochondrial DNA. The association between molecular haplotypes and records on calving rate, de®ned as the mean number of live calves born per year over 4 years, were examined by analysis of variance. A signi®cant association was found between calving rate and mitochondrial polymorphisms in both breeds. This may have implications for genetically improving cow fertility. # 2002 Elsevier Science Inc. All rights reserved. Keywords: PCR±RFLP; Bovine mitochondrial DNA; D-loop; ND-5; Fertility

1. Introduction Many studies have suggested that maternal lineage effects in¯uence growth, reproduction and production traits of livestock. In particular, analyses of the dam lines have indicated that maternal lineage effects can explain 2±10% of the variation in milk production and related traits in dairy cattle [1,2]. A potential cause of maternal lineage effects is variation in maternally inherited mitochondrial DNA (mtDNA). In dairy cattle, extensive mtDNA diversity has been found [3±6], and differences in mtDNA have been signi®cantly associated with milk-yield traits [7±9]. By contrast, mtDNA diversity has been less commonly reported in beef cattle and no signi®cant effects on milk-yield or preweaning growth traits have been found [10±13]. * Corresponding author. Tel.: ‡61-89-360-2729; fax: ‡61-89-310-4144. E-mail address: [email protected] (A.J. Lymbery).

0093-691X/02/$ ± see front matter # 2002 Elsevier Science Inc. All rights reserved. PII: S 0 0 9 3 - 6 9 1 X ( 0 2 ) 0 0 6 6 4 - 7

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Hiendleder et al. [14], however, suggested that the lack of evidence for cytoplasmic genetic effects on growth traits in beef cattle could be owing to the lack of mtDNA variation among the animals studied. Japanese Black cattle are highly variable in mtDNA and Mannen et al. [15] recently reported an association between mtDNA haplotypes and carcass traits in this breed. Mammalian mtDNA contains genes that encode 13 polypeptides involved in oxidative phosphorylation, as well as the 12S and 16S ribosomal RNAs and the 22 mitochondrial transfer RNAs necessary for mRNA expression. The noncoding D-loop region is the site of transcriptional and replicational control [16]. Nucleotide substitutions in mtDNA appear to accumulate approximately 5±10 times faster than similar mutations in nuclear DNA [17±19]. The aim of this study was to examine the effects of variation in the D-loop and ND-5 regions of mtDNA on production traits in beef cattle. The D-loop was chosen because it is the most rapidly evolving region of the mitochondrial genome [3]. Sequence variation in the D-loop has been observed both within and across maternal lineages of cattle [20,21]. The ND-5 region was chosen because it is one of the seven subunits of the NADH± dehydrogenase complex involved in oxidative phosphorylation [22]. Suzuki et al. [23] reported RFLP variation in the ND-5 region within and among breeds of African cattle. Variation in the D-loop and ND-5 regions were examined in two breeds of beef cattle by polymerase chain reaction±restriction fragment length polymorphism (PCR±RFLP) analysis. The cattle were maintained on a research station and detailed phenotypic records were available for a number of production traits. Here, we report the associations between mtDNA polymorphisms and calving rate in cows. 2. Materials and methods 2.1. Experimental animals Blood samples and phenotypic records were available from a total of 422 cattle of two breeds, purebred Hereford and composite multibreed (comprising approximately onefourth Brahman, Charolais and Friesian, and one-eighth Angus and Hereford). The phenotypic trait used in association analyses was calving rate, de®ned as the mean number of live calves born per year over a 4-year period, from 1991 to 1994. The cattle were part of a selection experiment, with management conditions described in detail by Meyer et al. [24]. Approximately, half the animals of each breed had been selected for increased growth rate, while the others were from a control line re-established from frozen embryos. Population size for each breed was maintained at approximately 300 cows and 12 bulls, with 20±50 new females introduced each year. Cows were culled on age (>8 years) and pregnancy test results. The age structure of the cow herd was 0.17 heifers: 0.17 ®rst calves: 0.67 adults. All cows were mated in single-sire groups over a 7±8-week period for Autumn calving. All calves were tagged, weighed and identi®ed with their dam at birth. Full pedigree information was available for all animals and parentage was con®rmed by DNA ®ngerprinting when dam or sire was in doubt. Cows were kept in 20-ha paddocks of approximately 25 cows each throughout the year. The pastures of subterranean clover and annual ryegrass produced abundant green feed

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throughout Winter and Spring and adequate dry feed until late Autumn. Supplements of hay and grain were fed throughout late Autumn and early Winter. 2.2. Extraction of mitochondrial DNA Mitochondrial pellets were prepared according to Welter et al. [25]. In brief, white blood cells (250 ml) were homogenized in a clean microcentrifuge tube containing 1 ml cold homogenization buffer (100 mM Tris±HCl, pH 7.4; 250 mM sucrose; 10 mM EDTA). Nuclei and cellular debris were removed by centrifugation at 1500  g for 10 min at 4 8C. The supernatant was transferred to a clean microcentrifuge tube and a crude mitochondrial pellet was prepared by centrifugation at 11; 000  g for 20 min at 4 8C. The mitochondrial pellet was resuspended in 1 ml TE buffer (10 mM Tris±HCl pH 7.5, 1 mM EDTA), placed on ice for 10 min and repelleted at 11; 000  g for 20 min at 4 8C. MtDNA was then puri®ed from the pellet using the Wizard Miniprep (Promega, Madison, WI, USA) protocol. 2.3. PCR±RFLP PCR primers [26] were used to amplify a 1142-bp region of extracted mtDNA between positions 15601 and 404, which includes all of the D-loop and ¯anking sequences at both ends, and a 453-bp region between positions 12058 and 12510, which includes part of the gene for NADH dehydrogenase subunit 5. All PCR ampli®cation reactions were performed in an Omnigene thermocycler (Hybaid, Ashford, UK). The reactions were performed in a 50 ml reaction mix consisting of 200 ng template DNA, 0.15 mM each oligonucleotide primers, 200 mM each dNTPs, 2 mM MgCl2, 10 buffer and 1.5 units Taq DNA polymerase (Biotech Sydney, Australia) in 0.6 ml PCR reaction tubes. The cycling reaction consisted of a denaturing step at 94 8C for 6 min, followed by 30 ampli®cation cycles of 94 8C for 45 s and 72 8C for 1 min, and a ®nal polymerization at 72 8C for 6 min. PCR products were digested with restriction endonucleases previously found to have polymorphic sites [26]: TaqI, PstI, SspI, ApaI and AvaII in the D-loop region, and SpeI and HindIII in the ND-5 region. A master mix of each restriction enzyme, its buffer and water was made, divided into each tube containing 5 ml aliquots of ampli®ed mtDNA fragments, and incubated as directed by the manufacturer. Bovine serum albumin at a ®nal concentration of 100 mg/ml was used for many enzymes as directed by the manufacturer. Agarose gel electrophoresis was carried out using 1±2% agarose (Promega) in TAE buffer (40 mM Tris±HCl; 20 mM acetate; 2 mM EDTA, pH adjusted to 7.9). Electrophoresis was performed using horizontal gels, in electrophoretic cells (Bio-Rad, Richmond, VA, USA). Ethidium bromide was included in the gel at a ®nal concentration of 0.5 mg/ml. After electrophoresis, DNA was visualized under UV-illumination and photographed using Polaroid type 57 ®lm with a red ®lter. Restriction sites were mapped by reference to the complete nucleotide sequence of bovine mtDNA [16]. 2.4. Association analyses Associations between molecular haplotypes and quantitative traits were estimated from two linear models:

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Model I: Yijklmn ˆm‡Bi ‡Sj ‡Lk ‡Dl ‡Xm ‡Gn ‡eijklmn where m is the mean value, Bi the effect of breed, Sj the effect of sex of the calf, Lk the effect of line (selected or control), Dl the effect of the age of the dam, Xm the effect of year of birth, Gn the effect of haplotype, and eijklmn the residual error. All effects in this model, except the residual, were considered as ®xed. Model II: Yijklmno ˆm‡Bi ‡Sj ‡Lk ‡Dl ‡Xm ‡Gn ‡Aijklmno ‡eijklmno where m is the mean value, Bi the effect of breed, Sj the effect of sex of the calf, Lk the effect of line (selected or control), Dl the effect of the age of the dam, Xm the effect of year of birth, Gn the effect of haplotype, Aijklmno a random animal effect, determined from the additive relationship matrix, and eijklmno the residual error. Differences in means between haplotype classes were determined by the F-test, with a comparison error rate of 0.05. Model I was implemented using the general linear model procedure of JMP (SAS Institute Inc.) and Model II using the mixed linear model procedure of Groeneveld and Kovac [27]. The models differed in the addition of the relationship matrix to Model II. The relationship matrix was determined from pedigree information, including sire, dam and grandparents. Addition of the relationship matrix eliminates confounding between the effects of marker- and nonmarker genes among relatives, which may increase the likelihood of ®nding a signi®cant association between molecular markers and quantitative traits where none actually exists. Kennedy et al. [28] showed how mixed model methods under an individual animal model could be used to separate the effects of a single gene from those of polygenes in¯uencing a trait. Preliminary analyses using the models identi®ed no signi®cant interaction effects between haplotype and other factors (P > 0:1), and interactions were, therefore, not included in ®nal analyses. 3. Results Five polymorphic sites were detected in the ampli®ed D-loop region, using the enzymes TaqI, PstI, SspI, ApaI and AvaII. Eight haplotypes were found with these ®ve polymorphic sites (see [26] for a full description), but here, we consider only the two most common: haplotype A, which is equivalent to the complete bovine mtDNA sequence published by Anderson et al. [16], and haplotype B, which has lost TaqI and PstI sites, and has additional ApaI, AvaII and SspI sites [26]. Two polymorphic sites were detected in the ampli®ed ND-5 region, with the enzymes SpeI and HindIII. Only two haplotypes were seen: haplotype A, equivalent to the sequence published by Anderson et al. [16], and haplotype B, with the loss of SpeI and HindIII sites [26]. The loci were strongly linked, as expected for the nonrecombining mitochondrial genome, with 96% of the animals genotyped showing either haplotype A for both loci or haplotype B for both loci. Associations with calving rate were, therefore, examined for haplotypes combined from both loci.

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Table 1 Estimated mean calving rate (S.E.) in cattle with different haplotypes at the D-loop and ND-5 loci Group

Haplotype A

All cattle Composite selected Composite control Hereford selected Hereford control

0.70 0.58 0.83 0.73 0.76

Probability of difference between haplotypes B

    

0.04 0.07 0.08 0.07 0.05

0.61 0.42 0.81 0.48 0.68

    

0.05 0.08 0.09 0.07 0.07

Model I

Model II

0.04 0.09 0.85 0.01 0.24

0.06 0.10 0.75 0.01 0.20

Four-year calving rate was signi®cantly affected by line (P < 0:01), age of dam (P < 0:05) and haplotype (P  0:05), but not by breed, sex of calf or year of birth. Table 1 shows the estimated least square means for calving rates for each haplotype when all animals were examined, and when breeds (Hereford or composite) and lines (selected or control) were examined separately. Across all breed  line combinations, there was a trend for calving rate to be greater in animals with haplotype A than in animals with haplotype B. This difference was signi®cant under both analytical models for Hereford selected cattle, and verging on signi®cant for both models when all cattle were examined together. For all comparisons of calving rate between haplotypes, there was little difference in the probability values under Models I and II, which suggests that polygenic background effects were not a major cause of similarity in calving rate between cows with the same mtDNA haplotype. 4. Discussion This study has provided evidence that mitochondrial DNA polymorphisms in the D-loop and ND-5 regions are signi®cantly associated with cow fertility as measured by calving rate. This is the ®rst report of a correlation between mitochondrial DNA and reproduction traits in beef cattle, although Schutz et al. [9] found that nucleotide substitutions in the Dloop were signi®cantly associated with days from calving to the next conception in Holstein Friesians. What is the likely cause of this association? Any effects of mtDNA polymorphisms on reproduction traits may be exerted indirectly, through milk-yield traits, because reproductive success is usually negatively correlated with yield levels, which may be related to the energy status of lactating cows [8]. Alternatively, the association of mitochondria with cellular energy metabolism suggests a possible direct effect of mtDNA polymorphisms on reproduction. Cattle de®cient in energy have lower conception rates [9]. Van Blerkom and Runner [29] suggested the need for elevated concentrations of adenosine triphosphate (ATP) for localized activities in the ooplasm. In cattle, the number of mitochondria are increased proportionally to the increase in cytoplasmic volume, at which stage oocytes require a ®xed amount of mitochondria per unit volume of cytoplasm to remain viable [30]. Mitochondrial DNA copy number appears to correlate with oocyte volume since the

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amount of mtDNA per cell increases from about 0.1 pg in primordial cells to 4.5 pg in the preovulatory oocyte [31]. This distribution of mitochondria in the bovine oocyte has also been suggested to be correlated with the hormonal patterns of both gonadotrophins and steroids [32,33]. While it is possible, therefore, that mitochondrial function has an important in¯uence on female fertility, we have no evidence that function is in¯uenced by the genetic differences between the mitochondrial haplotypes reported in this study. If there are direct effects of mtDNA polymorphisms on mitochondrial function and female fertility, these effects may be exerted through the D-loop and ND-5 regions. The Dloop is the site of transcriptional and replicational control and sequence polymorphism in the D-loop region of mtDNA may relate to the control of mtDNA function. The ND5 gene codes for an enzyme vital to the oxidative phosphorylation pathway and recent studies indicate that mitochondrial genes that contribute subunits to the enzymes involved in respiratory-chain activities could in¯uence growth in lambs via mitochondrial respiratory metabolism [14,34]. Alternatively, D-loop and ND-5 polymorphisms may serve as indirect markers for differences elsewhere on the mtDNA genome in coding regions of genes directly affecting the phenotypic expression of reproduction traits. The importance of ®nding DNA markers for fertility traits, such as calving rate, is that the heritability of these traits is low, and they are, therefore, dif®cult to improve genetically through traditional phenotypic selection [35]. The presence of a DNA marker may enable the rate of genetic improvement in fertility to be increased, which would be important in extensive grazing situations where calving rates may be low. Associations between fertility and genetic markers have been reported for the Boorola gene in sheep [36], the major histocompatibility complex in pigs [37] and the estrogen receptor gene in pigs [35]. Mitochondrial markers, such as those reported here, are less useful for marker-assisted selection than nuclear markers, because they are maternally inherited and selection intensities in the dam to cow pathway are currently very low. This may change, however, if new developments in reproductive technology and embryo manipulation become more wide-spread commercially [2]. Even now, differences in mtDNA could be usefully applied by selecting donors and recipients in embryo transfer breeding programs on their mitochondrial genetic value as well as their additive genetic value [9]. For the putative markers identi®ed in the current study to be useful in this context, their association with fertility needs to be con®rmed in other populations of cattle. In particular, it is important to note that the calving rates of cattle in the present study were relatively low (60±70%), as a result of the mating system constraints placed on the herd because of it's involvement in a selection experiment. Associations between the mtDNA markers and fertility should be reexamined in a herd with calving rates approaching commercial values of 90±95%. Acknowledgements This work was supported by the Cattle Industry Compensation Fund of Western Australia. Thanks to Patrick Donnelly and the staff at Wokalup Research Station for help with the cattle, David Groth for advice in the laboratory and Eric Taylor for reading the manuscript.

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