«Agricultural University of Szczecin, Department of Genetics and Animal Breeding, Poland DANIEL POLASIK, MAREK KMIEĆ, ARKADIUSZ TERMAN and FILIP ...»
Arch. Tierz., Dummerstorf 50 (2007) Special Issue, 200-204
Agricultural University of Szczecin, Department of Genetics and Animal Breeding, Poland
DANIEL POLASIK, MAREK KMIEĆ, ARKADIUSZ TERMAN and FILIP NAPIERAŁA
Restriction polymorphism FABGL/BbvI in herd of sows derived
from crossing Polish Large White x Polish Landrace breeds
The FABGL gene encodes a nicotinamide adenine dinucleotide (NAD)-dependent 17β-hydroxysteroid
dehydrogenase (17b-HSD) which is responsible mainly for immune response. It can also regulate the concentration of biologically active estrogens and androgens. It is appear as oxidative enzyme that inactivates estradiol, testosterone, and dihydrotestosterone as well as reductive enzyme by synthesis of estradiol from estrone. Investigations showed that FABGL gene is expressed within the ovaries and testes. Different variants of this gene may be associated with reproduction traits in pigs. The aim of this study was to determine polymorphism in the promoter region of FABGL gene as well as examine associations between particular genotypes and reproduction traits in Polish Large White x Polish Landrace sows. Polymorphism in FABGL/BbvI was determined by applying PCR-RFLP and following frequency of alleles were obtained: A - 0.45, B - 0.55.
Statistical analysis showed associations (P≤0.05, P≤0.01) between particular genotypes and some reproduction traits in investigated herd of sows.
Key Words: FABGL gene, sows, polymorphism, reproduction traits Zusammenfassung Titel der Arbeit: Restriktionspolymorphismus des FABGL/BbvI Gens bei Kreuzungssauen aus Große Polnische Weiße x Polnische Landrasse Das FABGL-Gen verschlüsselt die NAD-abhängige 17ß-Hydroxysteroid Dehydrogenase (17b-HSD), welche hauptsächlich für die Immunantwort verantwortlich ist. Da sie ein Oxydationsenzym ist, wird auch mit deren Hilfe die Konzentration des biologisch aktiven Östrogens und Androgens reguliert. Untersuchungen haben ergeben, dass das FABGL-Gen einer Expression in den Eierstöcken und Hoden unterliegt. Verschiedene Varianten dieses Gens könnten also mit dem Niveau von Wurfleistungsmerkmalen beim Schwein verbunden sein. Das Ziel vorliegender Arbeit war die Bestimmung des Polymorphismus im promotorischen Teil des FABGL-Gens sowie die Untersuchung von Zusammenhängen zwischen den einzelnen Genotypen und den Wurfleistungsmerkmalen bei Kreuzungssauen der Rassen Große Polnische Weiße x Polnische Landrasse. Bei Anwendung der PCR-RFLP-Technik konnte der FABGL7BbvI-Polymorphismus bestimmt und Zusammenhänge zwischen den Genotypen und einzelnen Wurfleistungsmerkmalen festgestellt werden.
Schlüsselwörter: FABGL-Gen, Sau, Polymorphismus, Wurfleistungsmerkmale Introduction Steroid hormones act through specific receptors effect activation of gene transcription.
The biological activityof these hormones is regulated at the pre-receptor level (MINDNICH et al., 2004). Several enzymes are committed in this process.
Nicotinamide adenine dinucleotide (NAD)-dependent 17β-hydroxysteroid dehydrogenases (17β-HSD) are key enzymes acting in the last step of formation of androgens and estrogens. Eleven 17β-HSDs coded by different not homologous genes have been discovered, which vary in tissue distribution, catalytic preferences, substrate specificity, subcellular localization and mechanism of regulation. These enzymes are mainly involved in conversion at position 17 of sex steroids. They can also metabolize different substrates including alcohols, bile acids, fatty acids and retinols (ADAMSKI and JAKOB, 2001).
17β-hydroxysteroid dehydrogenase VIII (17β-HSD8) protein efficiently catalyses the oxidation of estradiol, testosterone and dihydrotestosterone leading to their inactivation. It also appear reductive activity by synthesis of estradiol from estrone (FOMITCHEVA et al., 1996). Gene which codes this enzyme is known as FABGL gene as well as KE6 or RING2 gene. In swine it was assigned to chromosome 7. within the class II region of pig major histocompatibility complex. It is called SLA and located on the long arm in 7q1.1 (CHARDON et al., 1999). FABGL gene consist of nine exons with 86% and 82% homology to human and mouse mRNA sequences respectively (JACOBS et al., 2002). FOMITCHEVA et al. (1996) have demonstrated that FABGL gene is expressed within the ovaries and testes maintaining local level of sex steroids. Polymorphisms in this gene may be associated with the fertility and lipid metabolism in swine.
The aim of this study was to determine polymorphism in the promoter region of FABGL gene discovered by JACOBS et al., (2002) as well as examine associations between particular genotypes and reproduction traits in Polish Large White x Polish Landrace sows.
Materials and methods Investigation were carried out on herd 305 sows derived from crossing Polish Large White x Polish Landrace breeds. Conditions of rearing and feeding were equalized for all animals. DNA was extracted from whole blood by using standard kit (MasterPure™ DNA Purification Kit for Blood, Epicentre). Primers for PCR reaction were designed according to JACOBS et al., (2002). To estimate optimal annealing temperature gradient thermocycler was used (TGradient, Biometra). PCR was performed for each sample in total volume 15µl contains: 1xPCR buffer (C), 2mM MgCl2, 0.2mM dNTP mix, 15pmol of each primer, 0.75U Taq (Eurx), about 80ng of DNA, PCR grade water up to 15µl. Following thermal profile was applied: initial denaturation at 94°C for 5min, 35 cycles: 94°C/45s, 69°C/50s, 72°C/40s and the final extension at 72°C for 5min. 5µl of PCR product was checked on 1% agarose gel staining with ethidium bromide. After positive estimation it was digested by using 1U of BseXI (BbvI) restriction enzyme in 65°C overnight. Restriction fragments were separated on 2% agarose gels. To visualization and record gels Vilber Lourmat system was used.
The analysis of associations between FABGL/BbvI genotypes and total number born (TNB), number of weaned (NW) and number falls (NF) were performed applying
following models according to parity order:
first parity (I) Yijk = µ + ai + cj + dk + b(av) + eijk second and remaining (I) Yijk = µ + ai + cj + dk + el + b(av) + eijk
Yijk - analyzed trait µ - the overall mean ai - constant effect of FABGL/BbvI genotype (i = 1, 2, 3) cj - fixed effect of sire (j = 1,...38) dk - fixed effect of year-season (k = 1,…14) el - fixed effect of parity order (l = 2, …14) b(av) - the regression of age at first farrowing/value of trait eijk - the random error
Discussion There are many investigations concerning polymorphisms and their relationships with performance traits in domestic animals. In pigs mainly reproduction, carcass and meat quality traits are taking under consideration. Recent studies have proved that polymorphisms in some genes may be associated with reproduction traits in pigs. In sows have examined following genes: ESR1, FSHB (WANG et al., 2006, HUMPOLIČEK et al., 2006), RBP4 (WANG et al., 2006), CYP21 (ZIEMAK and GRZESIAK, 2006), GPX, FUT1, ESR2 (BUSKE et al., 2006) PRLR, LEP (TERMAN
2006) sFBP (VALLET et al., 2005), EPOR (VALLET et al., 2005), however in boars:
ESR1, ESR2 (TERMAN et al., 2006), GNRHR, PRL, PRLR, FSHB, LHB, FST, INHA, INHBA, INHBB (LIN et al., 2006) ACTN1, ACTN4, ACTG2 (WIMMERS et al., 2005).
In our study we investigated FABGL gene on account of role in reproduction which plays the product of this gene. We have found statistical significant associations between particular genotypes and total number born and number of weaned in second and remaining parities. Higher value for TNB characterized sows (P≤0.01) with genotype AA (10.32) in relation to genotype BB (9.77). Considering NW, higher value for this traits achieved sows with genotype AA (8.80) than sows with genotype BB (8.39) (P≤0.01) and AB (8.45) (P≤0.05). Differences in number of falls for FABGL/BbvI genotypes were also observed but they were not statistical significant.
There are no data in the literature concerning association study of FABGL gene. Only JACOBS et al. (2002) has given frequency of alleles in different breeds of pigs. In our study following frequency of alleles were obtained: A - 0.45, B - 0.55. Similar frequency were observed in two breeds: Czech Meat Pig A - 0.47, B - 0.53 and Landrace A - 0.42, B - 0.58. Higher frequency of allele A were found in Pietrain - 0.57 and Large White - 0.54. Allele B was not found in pigs belong to Meishan breed.
Results of our study indicates that genotype AA is favourable for TNB and NW. It may be suggested that FABGL gene is 'candidate gene' for reproduction traits but investigation should be verified on bigger population as well as on different breeds.
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