Analiza povezanosti polimorfizma gena za laktoferin (LTF) sa zdravljem mlečne žlezde i proizvodnim karakteristikama krava holštajn-frizijske rase

Abstract

Brzi razvoj molekularne genetike tokom posledenje tri decenije omogućio je direktnu analizu genoma životinja, proučavanje strukture i funkcije gena, pa samim tim pomogao boljem razumevanju delovanja nasledne osnove. Molekularno-genetičke metode omogućile su uvid u nekodirajuće delove genoma, koje kod sisara čine više od 90%. DNK (dezoksiribonukleinska kiselina) zapisa. U slučаjevimа gde se prаte osobine koje su generаlno sа niskim heritibilitetom, kаo što je slučаj sа otpornošću nа mаstitis, MAS (mаrker аsistirаnа selekcijа) ili genomskа selekcijа pokаzаle su bolji rezultаt u odnosu nа konvencionаlni metod selekcije. Primаrni cilj u ispitivаnju kаndidаt-genа povezаnih sа pojаvljivаnjem mаstitisа je identifikаcijа genа koji su uključeni u proces imunog odgovorа mlečne žlezde. Laktoferin gen (LTF) se u većem broju istraživanja pominje kao validni molekularni marker u praćenju procesa otpornosti mlečne žlezde prema infekciji. Cilj ovog rada bio je ispitivanje polimorfizma gena za LTF i njegov uticaj na zdravstveni status mlečne žlezde i proizvodne karakteristike krava holštajnfrizijske rase u Srbiji. Ogled je obuhvatao 100 krava različite laktacione starosti (1.-4. laktacija) na farmi blizu Beograda. Životinje su bile ravnomerno raspoređene u grupama po laktacionoj starosti (χ2=0,578; p=0,902). Posle ekstrakcije DNK iz krvi amplifikovan je fragment gena za laktoferin na intronu 6 metodom PCR (polymerase chain reactionreakcija lančane polimeraze). Pomoću specifičnih prajmera, identifikovana su dva genotipa AA i AB od moguća tri, (BB nije identifikovan) metodom PCR-RFLP (Restriction Fragment Lenght Polymorphism-polimorfizam u dužini restrikcionih fragmenata). U cilju ispitivanja zdravstvenog statusa mlečne žlezde, kvalitativnog sastava i ukupne proizvodnje mleka, svakog meseca kroz period standardne laktacije od 305 dana, su uzimani uzorci mleka. Broj somatskih ćelija (SCC) određivan je mikroskopski a koncentracija proteina i mlečne masti utvrđivana je spektrofotometrijski (aparat Milkoscan). U zajedničkom uzorku od 90 krava, konstatovana je statistički vrlo značajno veća (χ2=40,894; p<0,001) zastupljenost genotipa AA ( 74 ili 82,22%) u odnosu na zastupljenost genotipa AB ( 16 ili17,78%). Prema Hardy-Weinberg jednačini odnos genotipova AA, AB i BB se nalazi u ravnoteži (p=0,3547). Distribucija A alela u posmatranoj populaciji bila je 91,11% a B alela 8,89%. Ovako visok broj homozigota ukazuje na smanjeni protok gena u populaciji i veći stepen genetske konzervisanosti zapata. Poizvodne karakteristike praćenih genotipova nisu se statistički razlikovale...

The rapid development of molecular genetics during last three decades has facilitate the direct analysis of the genome of animals and insight in gene structure and function, and thus helped to a better understanding basis of genetic inheritance. Molecular-genetic methods enabled access to the non-coding parts of the genome, which in mammals make up more than 90% DNA (deoxyribonucleic acid). MAS (marker assisted selection) or genomic selection showed a better result compared to the conventional method of selection when generally low heritability traits are followed, as is the case with resistance to mastitis. The primary objective in tests concerning appearance of mastitis is the identification of candidate genes that are involved in the immune response of the mammary gland. Lactoferin gene (LTF) is proved by many studies as a valid molecular marker in monitoring the process of resistance to infection of the mammary gland. The aim of this study was to investigate the polymorphism of LTF gene and its impact on the health status of the mammary gland and production characteristics of Holstein-Friesian breed in Serbia. The experiment included 100 cows of different lactation age (1.-4. lactation) on a farm near Belgrade. Due to some health issues that are not directly related to the research 10 cows were excluded. The animals were equally distributed in groups by lactation age (χ2 = 0.578; p = 0.902). DNA was extracted from the blood and intron 6 from lactoferrin gene was amplified by PCR (polymerase chain reaction). Using specific primers, two genotypes AA and AB of the three possible (BB was not found) were identified by PCR-RFLP (PCR-Restriction Fragment Length Polymorphism). In order to examine the health status of the mammary gland and qualitative composition of the total milk production, each month through standard lactation period of 305 days samples were taken from the milk. Somatic cell count (SCC) was determined microscopically and the concentration of protein and fat in milk was determined by spectrophotometry (MilkoScan). The pooled sample of 90 cows showed statistically significant (χ2 = 40.894; p <0.001) representation of the genotype AA (74 or 82.22%) compared to the prevalence of genotype AB (16 or 17.78 %). All genotypes (AA, AB and BB) were in the Hardy-Weinberg equilibrium (P = 0.3547). Distribution of A and B alleles in the observed population were 91.11% and 8.89%, respectively. This high number of homozygotes indicates reduced gene flow in the examined population and a higher degree of genetic conservation...