J. Anim. Sci. 2002. 80:2334-2339
© 2002 American Society of Animal Science
Examination of the relationship between the estrogen receptor gene and reproductive traits in swine1,2
B. J. Isler3,*,
K. M. Irvin*,
S. M. Neal
,
S. J. Moeller* and
M. E. Davis*
* Department of Animal Sciences, The Ohio State University, Columbus 43210 and
and
Agricultural Technical Institute, The Ohio State University, Wooster 44691
3 Correspondence:
Room 1, Animal Sciences Building, 2029 Fyffe Rd. (phone: (614) 292-6407; fax: (614) 292-2929; E-mail:
isler.12{at}osu.edu).
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Abstract
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The relationship between estrogen receptor (ESR) genotype and reproductive traits in a population of Yorkshire, Large White, and crossbred animals was studied. Reproductive tract and litter data were analyzed for associations with ESR genotype, parity, and breed. Forty-six Yorkshire, 31 Large White, and 70 crossbred females from the above population were mated to Hampshire boars and slaughtered at 75 d of gestation. Data collected included ovulation rate, uterine horn length, number of fetuses, fetal weight, uterine weight, number of mummies, fetal sex, fetal placement, fetal survival, and fetal space. Data were analyzed using a model that included the fixed effects of ESR genotype, breed, parity, and all significant two-way interactions. Litter data representing 212 litter records were analyzed in a model that included the fixed effects of ESR genotype of dam, parity, farrowing month, dam breed, sire breed, and all significant two-way interactions. The ESR genotype was significantly associated with the total litter weight of piglets born and total litter weight of piglets born alive. Dams with the AA genotype had significantly (P = 0.04) heavier litters at birth (14.44 ± 0.36 kg) than dams with the BB genotype (13.43 ± 0.47 kg). Ovulation rate was significantly (P < 0.05) different between animals of parity 1 (17.22 ± 0.41) and parity 
3 (19.92 ± 0.85). Significant breed effects were observed for fetal weight, with purebred Large White animals having a greater fetal weight per horn (3,909 ± 114 g) than purebred Yorkshire animals (3,553 ± 92 g). Notable, but nonsignificant, trends with respect to ESR genotype were also observed for number of piglets alive at weaning and total litter weight at weaning. The ESR gene is positively associated with several previously uninvestigated reproductive traits.
Key Words: Estrogen Receptors • Genetic Markers • Pigs • Reproductive Traits
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Introduction
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Most commonly, improvement of livestock species is accomplished using traditional methods of genetic selection. New discoveries in the field of molecular genetics now allow the isolation and study of specific regions of the genome that influence important traits. Animals that contain specific marker regions can then be selected for inclusion in a marker-assisted selection program. This approach has shown special promise for traits that are lowly heritable and act in a sex-limited manner, such as the reproductive traits. Some of the genes that have been shown to be associated with reproductive efficiency in swine include the estrogen receptor (ESR) gene (Rothschild et al., 1996), the follicle stimulating hormone-ß subunit gene (Zhao et al., 1998), and the prolactin receptor gene (Vincent et al., 1998).
The first of these genes to be studied, the ESR gene, was initially investigated due to the important role that estrogen plays in reproduction. Initial studies focused on the search for an association between the ESR gene and reproduction in animals of the Chinese Meishan breed, due to their large litter sizes (Rothschild et al., 1991). The advantageous allele at this locus was shown to have a positive additive effect on litter size, ranging from 0.4 to 0.6 pigs born alive per litter in Large White and Large White crosses to 1.25 pigs born alive per litter in Meishan crosses (Rothschild et al., 1996; Short et al., 1997). However, little attention has been focused on the relationship between the ESR gene and individual components of the reproductive system, such as ovulation rate and uterine weight. There has also been little study on the effect of the ESR gene on litter traits such as birth weight, number weaned, and weaning weight. The objective of the current study was to evaluate the effect of ESR genotype, breed, and parity on several of these previously uninvestigated reproductive and litter traits.
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Materials and Methods
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Animals
A population of Yorkshire (Y x Y), Large White (LW x LW), and crossbred (LW x Y, Y x LW) animals were utilized in this study. This unselected population of animals was created in 1992 from purebred Yorkshire and Large White seed stock. All animals were raised at the Western Branch of the Ohio Agricultural Research and Development Center (South Charleston, OH) in accordance with approved farm management practices.
ESR Genotype Analysis
For each animal, DNA was extracted from blood peripheral lymphocytes (Park, 1991), and ESR genotype was determined using the PCR procedure of Short et al. (1997). The PCR procedure used to determine ESR genotype has been patented by Iowa State University for use in improving reproductive efficiency in swine. Each sample prepared for the polymerase chain reaction included: 20 µL sterile deionized H2O, 3 µL 10x PCR buffer (Life Technologies, Grand Island, NY), 3 µL 10 mM dNTPs, 0.5 µL 10 µM forward primer, 0.5 µL 10 µM reverse primer, 2 µL genomic DNA, and 1 µL Taq DNA polymerase (1 U). All reactions were performed on a Perkin-Elmer Cetus DNA Thermal Cycler using the following temperature program: 31 cycles at 94°C for 45 sec, 55°C for 1 min, 72°C for 1 min; 1 cycle at 72°C for 5 min; hold at 4°C. Each sample was then digested using a mixture of 3.5 µL sterile deionized H2O, 3 µL 10x React 6 buffer (Life Technologies, Grand Island, NY), 0.5 µL PvuII (5U), and 23 µL PCR reaction product. Samples were digested at 37°C for 2 h, separated on a 4% agarose gel containing ethidium bromide, and visualized under UV light. Genotypes were determined as AA, AB, or BB for each animal as outlined previously (Short et al., 1997). Allelic and genotypic frequencies were calculated within each breed subgroup and allele frequencies were compared using a chi-square test to determine the presence of significant differences among breed subgroups.
Reproductive Tract Collection and Analysis
Collection.
From the original population, a subset of 147 females was included in the reproductive tract analysis. Females were of all four breed combinations and parities one to six. Females were mated to Hampshire boars and harvested at approximately 75 d of gestation in a commercial slaughter facility. Gravid uterine tracts were collected at the packing facility and analyzed approximately 1.5 h postslaughter. Data collected on these tracts included OR, UL, NF, FW, UW, NM, FSRV (= [NF / OR]•100), FSPC (= UL/[NF + NM]), fetal sex, and fetal placement (See Table 1
for all abbreviations). Parity was designated as one, two, or three and greater.
Statistical Analysis.
Reproductive tract data were analyzed using the General Linear Model procedures of SAS (SAS Inst. Inc., Cary NC). For OR, NF, and FSRV, the model included the fixed effects of ESR genotype, breed, parity, and all significant two-way interactions. For the traits UW, UL, and FW the model also included covariates to adjust for the number of days of gestation at slaughter and NF. The traits NM and FSPC were analyzed in a similar manner, with only NF included as a covariate. All two-way interactions were included in initial models. Nonsignificant interactions (P > 0.1) were then removed from the model for all subsequent analyses. Linear contrasts between model-adjusted least squares means were used to test for differences between purebred and crossbred animals. This contrast is an approximate test for the presence of heterosis as expressed in the numerator of the heterosis equation. In addition, linear contrasts among least squares means were used to determine the presence of paternal and maternal breed effects for animals with Large White sires or dams compared with animals having Yorkshire sires or dams, respectively.
Litter Data Collection and Analysis
Collection.
Litter data for 212 dams with a known ESR genotype were obtained and included in litter data analyses. Dams were of all four breed combinations and parities one to five. Sires of litters were Yorkshire, Large White, or Hampshire in origin. Litter data were collected for farrowing seasons ranging from August 1994 to July 1998. Animal data collected for use in litter data analysis included ESR genotype of dam, parity, farrowing month, sire breed, dam breed, TNB, NBA, WTNB, WNBA, NMUM, NS, NW, WNW, and age at weaning (see Table 1
for all abbreviations). Weaning age ranged from 5 to 26 d with an average weaning age of 20 d. Total number born was defined as the number of viable animals born plus the number of stillborn animals. Number born alive was defined as the number of viable animals born. Crossfostering was used to standardize litter size nursed. To maximize the number weaned, piglets from some litters were cross-fostered. Some multiparous dams included in the study produced both purebred and crossbred litters. Animals were grouped by parity using the same scheme as in the reproductive tract analysis.
Statistical Analysis.
All litter data were analyzed using the General Linear Model procedures of SAS. The traits TNB, NBA, NMUM, and NS were analyzed using a model that included the fixed effects of ESR genotype of dam, parity, farrowing month, breed of dam, breed of sire, and significant two-way interactions. The statistical model for NW included a covariate for age of the litter at weaning. The statistical model for WNW included NW and age of the litter at weaning as covariates. In a similar manner, WTNB and WNBA were also analyzed, except with the addition of TNB and NBA as a covariate, respectively. Nonsignificant two-way interactions were removed from the model using the same procedure as in the reproductive tract analysis. Linear contrasts were also used to determine the presence of differences between purebred and crossbred animals, maternal breed effects, and paternal breed effects as described previously.
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Results
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Allelic and genotypic frequencies for all animals included in the study are shown in Table 2. A chi-square test of allele frequencies between breed subgroups did not show any significant frequency differences. Allelic and genotypic frequencies for animals included in the reproductive tract and litter data analysis were similar to those of the total population (data not shown).
Estrogen receptor genotype was found to be significantly associated with WNBA (P = 0.01) and WTNB (P = 0.03). Females with the AA genotype produced a heavier WNBA and WTNB than females with the AB and BB genotypes (Table 3). No significant effects were found for the other reproductive traits studied. However, number of piglets alive at weaning and total litter weight at weaning showed nonsignificant trends with respect to ESR genotype. For both of these traits, animals of the BB genotype tended to have a greater reproductive efficiency, with a larger NW and a heavier WNW (Table 3).
Parity also significantly affected several traits (Table 4). Animals of a greater parity had a greater ovulation rate. This could reflect the increased reproductive efficiency of older animals, which produce more eggs per cycle and carry more piglets to farrowing (Hughes and Varley, 1980). However, in the present study, parity was not significantly associated with an increased NF (P = 0.19).
Breed effects were found for several of the traits studied (Table 5). Purebred Large White females had a greater FW (P < 0.05) than purebred Yorkshire females. For UL (Table 6), purebred Large White and Yorkshire animals had a greater performance than crossbred animals. The significant difference in values between purebred and crossbred animals is most likely an expression of negative heterosis for this trait. Maternal breed effects were also detected for several traits. Animals with Large White dams had a greater WNW (P = 0.005) and FSRV (P = 0.03) than animals with Yorkshire dams (Table 6). A significant effect of paternal breed of sire was observed for NM (P = 0.01). Females with Large White sires had a larger NM than females with Yorkshire sires.
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Table 6. Least squares means and standard errors for reproductive traits influenced by heterosis, maternal, or paternal breed
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Discussion
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The ESR B allele is present only in a select group of swine breeds (Rothschild et al., 1996) including the Meishan and Large White/Yorkshire. In PIC Large White lines, which had been previously included in selection schemes for increased reproductive efficiency, the frequency of the B allele ranges from 0.41 to 0.57 (Short et al., 1997). In comparison, Large White animals in our study were found to have a B allele frequency of 0.40 (Table 2). If the B allele does confer an increased reproductive efficiency, populations selected for increased efficiency (such as the PIC animals) would be expected to have a significantly greater B allele frequency than unselected populations, such as the one included in the current study.
Estrogen receptor genotype frequencies for American Yorkshire animals have not been published previously. In the current study, the ESR B allele was found to be present at similar frequencies in Yorkshire and Large White animals (Table 2). Previous research has attempted to determine the relatedness of these breeds (Kacirek et al., 1998). If these breeds are indeed distantly related members of the same ancestral population, as hypothesized, it would be expected that allele frequencies would be very similar, as is observed in the present study.
Previous studies have shown the ESR B allele to be associated with an increased number of piglets born and piglets born alive per litter (Short et al., 1997). In the present study, ESR genotype was significantly associated with only WNBA and WTNB (Table 3) and not with NF (P = 0.56), NBA (P = 0.43), or TNB (P = 0.77). If, however, the B allele does indeed produce an increased number of piglets born and piglets born alive per litter, we would expect females with more copies of the B allele to have more pigs early in gestation and increased fetal mortality, because of increased fetal crowding (Wu et al., 1989). The result of this crowding is observed in the results of the present study, in which it appears that increased fetal crowding results in reduced fetal weight for those fetuses that survive to farrowing. Indeed, ESR genotype was associated with both WNBA (P = 0.01) and WTNB (P = 0.03), and BB dams had a significantly lighter WNBA and WTNB than AA dams (Table 3). If the B allele originated in the Meishan breed, then perhaps this polymorphism is partly responsible for the small birth weights observed in this breed.
A previous study by van Rens et al. (2000) also focused on the association between the ESR gene and reproductive components. In their study, ESR genotype was shown to be associated with only the placental length of each fetus. No significant association was found between ESR genotype and either the total number of embryos or the weight of each embryo. However, all reproductive tract data were collected at only 35 d of gestation in Meishan/Landrace synthetic females. In contrast, in the present study, Large White and Yorkshire females were slaughtered at 75 d of gestation for collection of reproductive tract data. Also, van Rens et al. (2000) theorized that the effect of the ESR gene is not displayed early in gestation (< 35 d), but later in gestation, and that the effect of the gene is not on embryonic, as much as fetal, survival. By 35 d of gestation, most of the basic development of the fetus is complete. If the ESR gene exerts its influence after this time, it most likely influences the maternal-fetal interaction at the placental level. This effect could be manifested in the longer placenta observed at 35 d of gestation in BB animals, which would allow for the subsequent increase in total number of embryos (>35 d). The lengthening of the placentas and increased embryonic survival would, eventually, be constrained by the amount of total space available in the uterus and would result in a reduction of the weight of each gestating fetus. These same BB animals would therefore also be expected to have lighter piglets at birth than animals with the AA and AB genotypes, which is in fact what was observed in the current study.
Approximately 9,000 litter records have previously been utilized to study the effect of the ESR B allele (Short et al., 1997). In contrast, the current study utilized 147 reproductive tracts and 212 litter records to study this effect. The results presented here will allow for a greater understanding of the effects of the ESR gene and also allow researchers to make a better determination of how the ESR gene can be most efficiently utilized to improve reproductive performance.
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Implications
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The estrogen receptor gene is positively associated with several components of reproduction. We can now begin to construct a preliminary picture of how the estrogen receptor gene positively influences reproductive performance in the female pig. Additional copies of the estrogen receptor B allele in pregnant swine result in a decreased litter weight at farrowing. This decreased litter weight may be a result of an increased number of gestating fetuses and the resulting increased fetal crowding in animals with more copies of the B allele. Further investigation of the estrogen receptor gene and its association with these traits will allow us to further understand the true magnitude and significance of these associations.
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Footnotes
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1 Salaries and research support provided by state and federal funds appropriated to the Ohio Agricultural Res. and Dev. Center, The Ohio State University. 
2 Appreciation is expressed to H. C. Hines and J. Riggenbach for laboratory assistance and D. Owens and K. Black for animal care and data collection.
Received for publication August 3, 2001.
Accepted for publication May 24, 2002.
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Literature Cited
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Abstract
Introduction
