Agnieszka Korwin-Kossakowska

Genetic basis of mastitis resistance in dairy cattle - a review* *

Abstract Mastitis is one of the most important mammary gland diseases impacting lactating animals. Resistance to this disease could be improved by breeding. There are several selection methods for mastitis resistance. To improve the natural genetic resistance of cows in succeeding generations, current breeding programmes use somatic cell count and clinical mastitis cases as resistance traits. However, these methods of selection have met with limited success. This is partly due to the complex nature of the disease. The limited progress in improving udder health by conventional selection procedures requires applying information on molecular markers of mastitis susceptibility in marker-assisted selection schemes. Mastitis is under polygenic control, so there are many genes that control this trait in many loci. This review briefly describes genome-wide association studies which have been carried out to identify quantitative trait loci associated with mastitis resistance in dairy cattle worldwide. It also characterizes the candidate gene approach focus on identifying genes that are strong candidates for the mastitis resistance trait. In the conclusion of the paper we focus our attention on future research which should be conducted in the field of the resistance to mastitis. Streszczenie Zapalenie wymienia (mastitis) jest jedną z najważniejszych chorób bydła mlecznego. Poprawa odporności na tę chorobę jest możliwa dzięki wykorzystaniu metod hodowlanych. Aby poprawić odporność krów mlecznych na mastitis, dopuszczalne jest wykorzystanie kilku metod selekcyjnych, które zostały omówione pokrótce w niniejszej pracy. Programy hodowlane, obecnie stosowane na świecie, wykorzystują jako kryterium selekcyjne zwierząt odpornych na mastitis, liczbę komórek somatycznych w mleku krów lub częstość występowania przypadków klinicznych. Programy te jednak tylko częściowo przyczyniły się do poprawy odporności krów na zapalenie wymienia. Jednym z powodów małej ich efektywności jest złożona etiologia tej choroby. Aby zwiększyć efektywność programów hodowlanych w odniesieniu do odporności na mastitis, w ostatnich latach podjęto badania nad markerami genetycznymi tej choroby i ich wykorzystaniem w selekcji wspomaganej markerami. Na zapalenie wymienia wpływa wiele genów. W niniejszej pracy dokonano przeglądu badań nad poszukiwaniem markerów związanych z odpornością na mastitis i genów do niej kandydujących. W podsumowaniu wskazano kierunki dalszych badań.

Association between interleukin 8 receptor α gene (CXCR1) and mastitis in dairy cattle.

The innate immune response plays an important role in the course of bacterial infections. Innate immunity effectiveness relies on the expression of many genes, connected, among others, to the activity of neutrophils. Interleukin 8 (IL-8) receptor α, coded by the CXCR1 gene, is present on the neutrophil surface and binds pro-inflammatory IL-8 with high affinity. This is why the bovine CXCR1 gene carries a potential for use as a dairy cattle mastitis marker. To date, several studies on the CXCR1 polymorphism brought out contradictory results. The aim of this study was to analyse the association between two SNPs of the CXCR1 gene, which is potentially important for the protein function and animal phenotype for mastitis susceptibility. A total of 554 Polish Holsteins were genotyped, and 140 among them were bacteriologically tested. The differences between animals carrying different genotypes and haplotypes of CXCR1 in test day somatic cell count (SCC) and Staphylococcus aureus mastitis susceptibility were estimated. We found that test day SCC was significantly related to CXCR1+472 SNP but not to CXCR1+735 SNP. No statistically significant association between CXCR1 polymorphism and susceptibility to S. aureus mastitis was found in the studied herd.

Health status and potential uptake of transgenic DNA by Japanese quail fed diets containing genetically modified plant ingredients over 10 generations

Abstract The hypothesis assumes that feed containing GMOs affects animal health and results in the transgene product accumulating in the body. Therefore, the objective of the study was to evaluate the impact of genetically modified (GM) ingredients used in poultry diets on aspects of bird health status and accumulation of transgenic DNA in eggs, breast muscle and internal organs. A total of 10 generations of Japanese quail were fed three types of diets: group A – containing GM soya (Roundup Ready) and non-GM maize, group B – containing GM maize (MON810) and non-GM soya, and group C – containing non-GM soya and maize. Bird performance traits were monitored throughout the trial. In 17-week-old animals of each generation, health examination took place on birds from each group including post-mortem necropsy and histological organ evaluation. For the purpose of transgenic DNA detection, samples of selected important tissues were taken. A molecular screening method of PCR amplification was used. The analysis of the sectional examination of birds used in the current experiment did not indicate the existence of the pathological changes caused by pathogens, nutritional factors or of environmental nature. The histopathological changes occurred in all three dietary groups and there were no statistically significant differences between the groups. There was no transgene amplification – neither CaMV35S promoter sequence nor nos terminator sequence, in the samples derived from breast muscle, selected tissues and germinal discs (eggs). According to the obtained results, it was concluded that there was no negative effect of the use of GM soya or maize with regard to bird health status or to the presence of transgenic DNA in the final consumable product.

Interrelationships between Morphological, Densitometric and Mechanical Properties of Eggs in Japanese Quails (Coturnix Japonica)

Eggshell quality in birds results from mineral density and composition determining its mechanical endurance. The aim of the study was to determine interrelationships between morphological, densitometric and mechanical properties of eggs in Japanese quails. Twenty four eggs randomly collected from 17-week-old quails were subjected to morphological, denstiometric and mechanical evaluation using dual-energy X-ray absorptiometry (DEXA), quantitative computed tomography (QCT) and three-point bending test. Weight, height and width of eggs were positively correlated with the densitometric parameters obtained using DEXA (egg mineral density (EMD) and egg mineral content (EMC)) and QCT (total egg volume (TEvol) and total eggshell volume (TESvol)). Positive correlations were stated between TEvol and TESvol (r=0.52; P<0.05) and EMD and EMC r=0.83; P<0.05). Egg mineral density revealed positive correlations with TEvol and mean volumetric eggshell mineral density (MvESMD), while EMC was positively correlated with TEvol, TESvol and MvESMD (all P<0.05). Eggshell breaking strength was positively correlated with MvESMD (r=0.53; P<0.05) and negatively correlated with eggshell thickness (r=−0.50; P<0.05). In conclusion, the results obtained in this study showed numerous interrelationships between morphological, densitometric and mechanical properties of eggs in Japanese quails. Both DEXA and QCT were shown to be valuable tools for evaluation of whole egg and eggshell quality with superior prognostic value of QCT for eggshell mechanical endurance prediction. The elaborated experimental model may serve for further investigations on physiological, pharmacological, environmental, nutritional and toxicological factors influencing egg quality.

Association between lactoferrin single nucleotide polymorphisms and milk production traits in Polish Holstein cattle

Abstract. Bovine lactoferrin exhibits strong potential for further applications as a mastitis resistance marker. Since selection for mastitis resistance should not interfere with dairy performance, we investigated the association between bovine lactoferrin gene polymorphism and production traits in Polish Holsteins. The associations between four SNPs, localized in the 5’-flanking region and in exons 4 and 9 of the lactoferrin gene, and dairy performance were examined. SNPs were associated with almost all test-day milk performance traits. Significant associations were found between lactoferrin genotypes and the estimated breeding values for those traits. To find out whether the discrepancies between the lactoferrin gene SNP’s influence on phenotype (test-day milk performance) and on estimated breeding values originate from the impact of other factors, we explored the genotype by environment interaction. Substantial impacts of SCC, lactation stage and parity were found. This paper suggests that the genotype by environment interaction may significantly change associations between genes and traits. It is important to include similar analyses to the studies on disease markers before using them in the selection.

Polymorphisms of the COL9A2, AOAH and FRZB Genes in the Horse Genome and their Association with the Occurrence of Osteochondrosis

Abstract The aim of the study was to evaluate the chosen polymorphisms of the COL9A2, AOAH and FRZB genes and find their potential effect on the occurrence of osteochondrosis in Polish sport horses population. During two successive years, all 198 performance tested horses were checked for osteochondrosis. The health status of the horses was assessed based on 10 x-ray images of three joints: fetlock, hock and stifle, and scored on a 0-3 scale. The methodology of analysis of selected candidate genes using the PCR-RFLP technique was developed. The analysis of variance was performed to evaluate significance of the effect of the COL9A2, AOAH and FRZB genotype on the occurrence of osteochondrosis in individual joints. Fixed effects of breed, gender and training centre were taken into account in the analysis. The results showed a significant influence (P≤0.05) of the COL9A2 genotypes on the occurrence of osteochondrosis in fetlock and hock joints. Polymorphism of this gene, even not proved a causal mutation, appears to have effect on symptoms of the disease. In genes AOAH and FRZB there was no significant effect for investigated SNPs. Further analysis of the discussed genes/polymorphisms seems to be important.

Polymorphisms of the osteopontin gene and level of its expression in the reproductive tract of sows.

Abstract The aim of the study was to find osteopontin gene (OPN) polymorphisms as potential mutations affecting the expression level of genes in the ovaries, uterus and oviduct of sows. The material consisted of 71 F1 sows (Polish Large White × Polish Landrace). In the first stage several polymorphisms in the promoter region, intron 6, exon 6 and 7 of the OPN gene were found. The parameters estimated were the frequency of alleles and genotypes, observed heterozygosity and gene diversity, PIC, and chi2 factors. Chi2 values allow for assessment of genetic equilibrium in the population. Thus, the loci OPNp3-4 and OPNe6-1 were in genetic disequilibrium while locus OPNe6-Knoll showed genetic equilibrium. Also real-time PCR analysis to determine the expression dynamics of the OPN gene in examined tissues was performed in relation to “housekeeping” genes. A comparison was made for relative expression in different tissues and different mutations. The highest expression pattern was observed in the oviduct. Based on the novel polymorphisms a significant correlation between the OPN genotype and OPN expression (mRNA) level in the ovary, oviduct, uterine body and uterine horn was observed. In the second stage, the levels of expression of the OPN gene in individual tissues, traits of reproductive performance and reproductive tract traits of sows were also compared. The expression levels in the uterine body and oviduct were related to the age of mating, cervical length, litter weight at birth, number of active nipples, age at slaughter and body weight at mating. Streszczenie Celem badań było znalezienie polimorfizmu genu osteopontyny (OPN) jako potencjalnego miejsca mutacji mającej związek z ekspresją tego genu na poziomie mRNA w jajnikach, macicy i jajowodzie. Materiał stanowiło 71 loch ras wbp × pbz. W pierwszym etapie badań opracowano kilka polimorfizmów w obrębie promotora, intronu 6 oraz eksonów 6 i 7 genu osteopontyny. Oszacowano frekwencję genotypów i alleli, heterozygotyczność oczekiwaną i spodziewaną,, współczynniki PIC oraz chi2. Stwierdzono, że w obrębie dwóch loci badana populacja nie znajdowała się w stanie równowagi genetycznej, natomiast w trzecim locus, tj. OPNe6-Knoll taka równowaga została zachowana. Wykonano również analizę real time PCR w celu określenia poziomu ekspresji w badanych tkankach w odniesieniu do genów ulegających stabilnej ekspresji w układzie rozrodczym. Dokonano porównania poziomu relatywnej ekspresji w różnych tkankach i w odniesieniu do różnych mutacji. Najwyższy poziom ekspresji odnotowano w jajowodzie. Bazując na nowo opracowanych mutacjach zaobserwowano wiele istotnych zależności pomiędzy genotypem OPN a poziomem ekspresji w jajniku, jajowodzie, rogach i trzonie macicy. W drugim etapie badań porównywano poziom ekspresji w poszczególnych częściach układu rozrodczego i poziom cech związanych z rozrodem i budową układu rozrodczego. Poziom ekspresji w jajowodzie miał związek z wiekiem krycia, liczbą aktywnych sutków, długością szyjki macicy, masą miotu przy urodzeniu, i masą ciała przy uboju. Natomiast poziom ekspresji w trzonie macicy miał związek z długością szyjki macicy, masą miotu przy urodzeniu i wiekiem uboju, a także wiekiem krycia i masą ciała przy kryciu.

Lactoferrin gene variants, their expression in the udder and mastitis susceptibility in dairy cattle

Lactoferrin gene (LF) is regarded as one of the potential markers of mastitis susceptibility/resistance in dairy cattle. The study’s aim was therefore, to investigate the feasibility of two single nucleotide polymorphisms (SNP), placed in the 5′-flanking region and 3′-untranslated region of the LF gene, to serve as mastitis markers. The associations between these SNP and the expression of LF, both on mRNA and protein level, were estimated in the milk of Polish Holstein-Friesian cows. The relationships between polymorphisms and cows’ estimated breeding values (EBV) for somatic cell count were also calculated. It was shown that both polymorphisms have a significant impact on lactoferrin content in milk, and that LF+32 SNP is associated with the cow’s EBV for somatic cell count. No association between SNP chosen for the study and lactoferrin mRNA abundance in milk somatic cells was observed. We propose LF+32 SNP for a molecular marker of mastitis resistance in dairy cows.