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Igor Valockyl*, Eudmila Tkacikova2, Joana Gruszczynska3, Frantisek Novotnyl, Vladimir Hural, Jan Bilekl, Martin Boldizarl, Jan Posivakl,
Martin Mihalyl ©
¡Clinic of Horses, 2Laboratory of Biomedicine Microbiology and Immunology, University of Veterinary Medicine, Komenskeho 73, 04181 Kosice, Slovak Republic 3Department of Genetics and Animal Breeding, Warsaw Agricultural University, ul.Ciszewskiego 8, 02,-786 Warszawa, Poland
FERTILITY GENE (FECB) IN THE IMPROVED VALACHIAN SHEEP
Abstract
A cohort of 139 ewes of the Improved Valachian breed was subjected to genetic analysis, aimed at determination of genotypes and their association with fecundity. 9 0arAE101 alleles ranged from 97 to 125 bp and 6 BM1329 alleles ranged from 162 to 180 bp. The frequency of 0arAE101 97 bp was the highest whereas; BM1329 164 bp allele was the most frequently occurring allele in the population. When the sheep population was divided into two groups ewes giving recurrent singletons or recurrent twins, an association between twinning and BM1329 164 bp allele was found, however, an association between 97 bp allele 0arAE101 and twinning was not confirmed.
Key words: Improved Valachian sheep, FecB gene, fecundity, 0arAE101, BM1329
Introduction
The endeavour to increase economic profitability (Willingham and Waldron, 2000; Gootwine et al., 2001) in sheep breeding necessitates the detection of fecundity markers. Most domesticated sheep breeds deliver one or two lambs yearly. Sheep derived from Booroola Merino strain carry a major autosomal mutation that increases ovulation rate and litter size (Montgomery et al., 1994). On average one copy of autosomal mutation of Booroola fecundity (FecB) gene influences the increase in ovulation rate and affects litter size to the extent of one or two extra lambs (Parsons et all., 1996). High fertility in sheep which are FecB gene carriers is the result of mutation in BMPIB receptor, and high fecundity in Inverdale sheep (FecX(I)) is the result of mutation in BMP15 oocyte - derived growth factor gene. Davies et al. (2002) have also explored the effect of these two major genes affecting fecundity in Garole sheep of India. This breed is the genetic predecessor of Australian Booroola Merinos (Davis et al., 2002). Inverdale fecundity gene FecX was identified in prolific New Zealand sheep, which are descendants of Romney females that produced 33 lambs from 11 lambings (Davis et al., 1991). Higher ovulation rate and larger litter size in Booroola lines of Australian Merino sheep are due to the influence of the FecB(B) allele of FecB gene. FecB (Booroola) prolificacy gene is localized between
© Igor Valocky, Eudmila Tkacikova, Joana Gruszczynska, Frantisek Novotny, Vladimir Hura, Jan Bilek, Martin Boldizar, Jan Posivak, Martin Mihaly, 2008
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markers OarAElOl and BM1329 in the centromeric part (10cM) of ovine chromosome 6 (OOV6) (Lord et al., 1998; Mulsant et al., 1998). Growth differentiation factors - 5 (GDF5) and BMP4, two natural ligands of BMPR-IB (bone morphogenetic protein) are responsible for inhibition of progesterone secretion by thecal granulosa cells, which is essential for differentiation of granulosa cells and ovulatory follicle maturation. Granulosa cells (cultured in vitro) in ewes with expression of FecBB/FecBB alleles secrete significantly more progesterone than ewes with the expression of FecB+/ FecB+ (Mulsant et al., 2001). Granulosa cells with the expression of FecBB/FecBB are less susceptible to inhibitory effects of GDF5 and BMP4 due to altered function of BMPR-IB receptor, which results in progressive differentiation of granulosa cells and escalation of follicular development in animals (Smith et al., 1996).
Task, the aim of the article
The aim of this study was to analyse polymorphism in microsatellites OarAE101 and BM1329 and determine the association of the allele frequencies and recurrent twinning in the population of Improved Valachian breed.
Material and methods
Experimental animals
The experiment was conducted on the Improved Valachian breed adapted to harsher mountain conditions. The Improved Valachian breed belongs to mountain sheep group, herd book established 1956. It is an autochthonous breed appreciated for being a strong walker. It produces 1.22 lambs per ewe per season (Hosko and Machynova, 2005). Improvement in wool quality has been achieved through import of Texel breed. Improvement for the purpose of production of heavy, quality lambs with the ewe showing trouble-free lambing and requiring minimum care has been implemented by import of Hampshire Down. Further amelioration of wool quality, increase in milk production and number of lambs per ewe has been achieved by introgression of Leicester Longwool, Lincoln Longwool, East Friesian and Lacaune (Ochodnicky 1986, Davis et al 2002).
4 - 5 year-old ewes (n=139) with average body weight 38.5 ± 1.72 kg produced on Litmanova farm situated in a mountain region of Slovakia. All the ewes were provided with standard food. The ewes were bred in the breeding season and the number of lambs born was recorded during three lambing seasons (the latter half of February - March).
The cohort of ewes with recurrent twinning in three consecutive seasons (n=29) differed in litter size from the cohort of ewes recurrently producing singletons (n=110) in three consecutive seasons (p<0.05).
Analysis of microsatellite markers Statistical significance of incidence of frequencies of the microsatellite alleles 0arAE101 and BM1329 in the ewes with recurrent twinning (n=29) and ewes giving birth to a single lamb recurrently (n=110) was calculated. Genomic DNA was isolated from leucocytes separated from peripheral blood by using the method
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described by Sambrook et al. (1989). Amplification of OarAElOl and BM1329 microsatellites was carried out with the help of the polymerase chain reaction (PCR). Primers and amplification conditions were set as per Montgomery et al. (1993) and Crawford et al. (1995), however, annealing temperature for microsatellite OarAElOl amplification was altered to 59°C and that for microsatellite BM1329 was standardized at 50°C. Moreover, primers were labelled with Cy5 (Generi Biotech Ltd., Czech Republic). Electrophoresis of amplified OarAElOl and BM1329 gene fragment was performed on ALF express II (Amersham Pharmacia Biotech) sequencer on 5% native polyacrylamide gel in 0.5x TBE buffer under conditions of 400 V, 40 mA, 20W, for 4 hours. The length of the analysed gene fragment was estimated using Alfwin Fragment Analyser l.03 software (Amersham Pharmacia Biotech).
Statistical analysis
The frequency of microsatellite alleles and heterozygosity coefficient were calculated according to the formula developed by Ott (l992). The polymorphic information content was estimated according to Botstein et al. (l980). The significance of the differences between the two groups of traits was established by Student's t - test.
Results of researches
The autochthonous Improved Valachian breed has been subjected for first time to analysis aimed at revelation of microsatellite allele frequency within FecB gene at the level of it is microsatellite alleles OarAElOl and BMl329. Twenty-nine ewes with recurrent twinning and llO ewes with recurrent single births were observed. Within the scope of investigation of microsatellite polymorphism 9 OarAElOl and 6 BMl329 microsatellites were found in these cohorts. These two markers were used to identify FecB carriership. The most frequent OarAElOl alelle 97 bp with a frequency of O,55 was identified in the group of ewes with a history of recurrent single births (Tab. l). This allele displayed a frequency of O,36 in the twinning mothers. A moderate frequency of O.2 of lO5bp allele and O.l of l2lbp allele was found in the mothers with singletons as well. In the twinning ewes a moderate frequency of O.O9 of lO5bp allele, O.l2 of ll3 bp and O.28 of l23 bp allele was observed with the last allele differing in frequency (p<O.O5) within the cohorts compared. A low frequency of O.O5 of 99 bp allele, O.O5 of l23bp allele as well as O.O5 of l25 bp allele was found in the ewes with singletons. A low frequency of O.O3 of lOl bp allele, O.O7 of lO3 bp allele and O.O7 of l2l bp allele was noted in the twinning ewes. In the mothers with singletons no incidence of lOl bp, lO3bp and ll3 bp alleles within polymorphism of OarAElOl microsatellite was observed, no occurrence of 99 bp and l25 bp was noted in the twinning ewes.
The most frequently occurring BMl329 l64 bp allele with a frequency of O,45 was identified in the group of twinning mothers (Tab. 2). In the same group BMl329 l62 bp allele with a frequency of O,O3, l66 bp with a frequency of O,2l, l68 bp with a frequency of O,26 and l7O bp with a frequency of O,O7 were observed.
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In the ewes with singletons BM1329 microsatellites with a moderate frequency of incidence were noted i.e. 164 bp with a frequency of 0,2, 166 bp with a frequency of 0,25, 170 bp with a frequency of 0,1, 180 with a frequency of 0,05 and 168 bp allele with a higher frequency of 0,40.
Within polymorphism of BM1329 microsatellite 180 allele was not identified in the twinning ewes or 162 allele in the ewes with singletons.
In the whole population expected heterozygosity coefficient (HE), observed heterozygosity coefficient (HO), and polymorphic information content (PIC) values were observed to be 0.76, 0.46, 0.66 respectively for OarAE 101 and 0.72, 0.85 and 0.66 respectively for BM 1329 microsatellite markers.
Montgomery et al. (1994) specified in the ovine microsatellite 0arAE101 marker, 8 alleles 99, 101,103, 109, 111, 115, 117 and 121 bp in Booroola half - sib pedigrees with the frequency of incidence 0.24, 0.01, 0.03, 0.01, 0.28, 0.05, 0.30, 0.08 respectively. The 0arAE101 103 bp and 121 bp alleles found in Montgomery's work were also observed in our study in ewes with twins, however the frequencies of these alleles were low. The novel alleles namely 97 bp and 123 bp were frequently occurring microsatellite alleles in our study in ewes with twins.
Chu et al. (2001) identified 5 0arAE101 alleles and 4 BM1329 alleles in the Dorset, Charolais, Hu, Ujumquin and Small Tail Han sheep breeds. Small Tail Han sheep was found as an excellent indigenous Chinese breed with the significantly higher prolificacy trait (2,6 lambs per ewe). Chu et al. (2002) recognized polymorphism of 5 microsatellite loci 0arAE101, BM1329, BMS2508, TGLA54 and TGLA68 that were closely linked to fecundity genes FecB and FecXI. They further recorded the number of alleles for BM1329, 0arAE101, TGLA54, TGLA68 and BMS2508, respectively 6, 9, 5, 2 and 6. In Improved Valachian sheep we observed 6 different alleles in BM1329 and 9 alleles in 0arAE101. The range of allele size for BM1329, 0arAE101, TGLA54, TGLA68 and BMS2508 was 160 - 180 bp, 97 - 135 bp, 116 - 136 bp, 98 - 100 bp and 93 - 115 bp, respectively. In our study the alleles were ranged between 162-180 bp (BM1329) and 97-125 bp (0arAE101). The highest allele frequencies for BM1329, 0arAE101, TGLA54 and BMS2508 were 164 bp, 97 bp, 134 bp and 99 bp respectively (Chu et al., 2002). In Improved Valachian breed the highest frequency of 164 bp allele of microsatellite BM1329 was identified in the ewes with recurrent twinning, which is consistent with the finding of Chu et al. (2002).
Chu et al. (2001) reported a significantly positive correlation between alleles 97 bp of microsatellite 0arAE101, 146bp of microsatellite BM1329 and litter size in Small Tail Han sheep as well as significantly negative correlation between 109 bp and 111bp alleles of microsatellite 0arAE101, 148 bp of microsatellite BM1329 and litter size in Small Tail Han sheep.
The present study indicates a positive correlation between the alleles 164 bp and 168 bp of BM1329, and litter size. In the case of 0arAE101 we found a positive association between 123 bp and litter size, however, this association was not significantly confirmed in the case of 97 (0arAE101) with twinning as mentioned by Chu et al. (2001).
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When both microsatellite polymorphisms were compared we observed the presence of 164/168 heterozygotes (BM1329) having also 123 bp allele of OarAElOl dominantly in the ewes with twins.
Conclusions
Nine alleles ranging from 97 - 125 bp on OarAElOl marker and 6 alleles 162 - 180 bp on BM1329 were identified in Improved Valachian. Alleles indicating the presence of the high prolificacy gene FecB in the Improved Valachian breed with the highest frequency of incidence were 123 bp for OarAE101 and 164 bp for BM1329 with a frequency of 0.55 and 0.45 respectively.
Although the authors have not succeed in showing explicitly the significance of the observed markers as a selection criterion of recurrent twinning trait, the Improved Valachian breed has the potential for prolificacy .
Acknowledgement
This work was supported in part by the Slovak Grant Agency AV 4/0009/07
Reference
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Summary
Nine alleles ranging from 97 - 125 bp on 0arAE101 marker and 6 alleles 162 - 180 bp on BM1329 were identified in Improved Valachian. Alleles indicating the presence of the high prolificacy gene FecB in the Improved Valachian breed with the highest frequency of incidence were 123 bp for 0arAE101 and 164 bp for BM1329 with a frequency of 0.55 and 0.45 respectively.
Although the authors have not succeed in showing explicitly the significance of the observed markers as a selection criterion of recurrent twinning trait, the Improved Valachian breed has the potential for prolificacy.
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