Effect of ß-hydroxybutyrate acid on gene expression levels of antioxidant biomarkers and growth hormone-related genes in liver cell culture.

Introduction: In dairy cattle, oxidative stress is a predominant problem associated with diseases and reproductive health issues. This study aimed to detect the variation in the antioxidant biomarkers by adding different concentrations of ß-hydroxybutyric acid (BHBA) and sought to elucidate its effects on the gene expression levels of growth hormone (GH) and antioxidant biomarkers in bovine hepatocytes. Material and Methods: Four antioxidant biomarkers, namely malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH Px) were evaluated using commercially available bovine ELISA kits. The expression levels of the bovine GH, its receptor (GHR), insulin-like growth factor (IGF), IGF-1, IGF-1 receptor, CAT, SOD, GSH-Px and ß-actin (as a reference) genes in liver cell culture were determined by reverse transcriptase-PCR assay. Results: With the increase of BHBA concentration and culture time, the activities of SOD, CAT, and GSH Px biomarkers in hepatocytes decreased. However, the content of MDA in hepatocytes increased gradually with the increase of hepatocyte culture time and BHBA concentration. The qPCR results revealed that after adding BHBA, gene expression levels of GSH-Px, SOD and IGF biomarkers in hepatocytes began to differ in the culture groups at 12 h, whereas the gene expression level of the CAT and GHR biomarkers in hepatocytes began to differ at 6 h. Conclusion: Quantitative PCR results showed that the BHBA significantly downregulated the expression levels of the GHR gene and CAT, GSH Px and SOD antioxidant biomarker genes.

Elevated tissue status of omega-3 fatty acids protects against age-related telomere attrition in fat-1 transgenic mice.

BACKGROUND & AIMS: Leukocyte telomere length (LTL) is a biomarker of aging that may be influenced by dietary factors. Omega-3 fatty acids (n-3 FA) have been suggested to affect LTL. However, research on this effect has been inconclusive. The aim of the study was to test the hypothesis about the positive effect of n-3 FA on LTL. METHODS: Fat-1 transgenic mice, which can convert omega-6 fatty acids (n-6 FA) to n-3 FA and have elevated levels of endogenous n-3 FA in their tissues, were used to study the effects of n-3 FA on LTL at different ages. Blood samples from 10-month-old wild-type (WT) mice (n = 10) and fat-1 mice (n = 10) and 3-month-old WT mice (n = 5) and fat-1 mice (n = 5) were used to measure relative and absolute LTL. The levels of proteins critical for telomere maintenance were examined by Western blot analysis. RESULTS: Fat-1 transgenic mice had longer leukocyte telomeres than their WT siblings, suggesting a slower rate of age-related telomere shortening in fat-1 mice. In animals aged 10 months, the LTL was significantly longer in fat-1 than in WT mice (mean ± SEM; relative LTL: WT = 1.00 ± 0.09 vs. fat-1: 1.25 ± 0.05, P = 0.031; absolute LTL: WT = 64.41 ± 6.50 vs. fat-1: 78.53 ± 3.86, P = 0.048). The difference in LTL observed in three-month-old mice was insignificant, however the mean LTL was still longer in fat-1 mice than in the WT mice. Fat-1 mice also had abundant levels of two shelterin proteins: TRF1 (27%, P = 0.028) and TRF2 (47%, P = 0.040) (telomeric repeat binding factor 1 and 2) compared to WT animals. CONCLUSION: This study, for the first time in a unique animal model free of dietary confounders, has demonstrated that increased levels of n-3 FA in tissues can reduce telomere attrition. The data presented indicate the possibility of using omega-3 fatty acids to reduce accelerated telomere attrition and, consequently, counteract premature aging and reduce the risk of age-related diseases.

Effects of Heat Stress on Bovine Oocytes and Early Embryonic Development-An Update.

Heat stress is a major threat to cattle reproduction today. It has been shown that the effect of high temperature not only has a negative effect on the hormonal balance, but also directly affects the quality of oocytes, disrupting the function of mitochondria, fragmenting their DNA and changing their maternal transcription. Studies suggest that the induction of HSP70 may reduce the apoptosis of granular layer cells caused by heat stress. It has been shown that the changes at the transcriptome level caused by heat stress are consistent with 46.4% of blastocyst development disorders. Cows from calves exposed to thermal stress in utero have a lower milk yield in their lifetime, exhibit immunological disorders, have a lower birth weight and display a shorter lifespan related to the expedited aging. In order to protect cow reproduction, the effects of heat stress at the intracellular and molecular levels should be tracked step by step, and the impacts of the dysregulation of thermal homeostasis (i.e., hyperthermy) should be taken into account.

Molecular Characterisation of Uterine Endometrial Proteins during Early Stages of Pregnancy in Pigs by MALDI TOF/TOF.

The molecular mechanism underlying embryonic implantation is vital to understand the correct communications between endometrium and developing conceptus during early stages of pregnancy. This study's objective was to determine molecular changes in the uterine endometrial proteome during the preimplantation and peri-implantation between 9 days (9D), 12 days (12D), and 16 days (16D) of pregnant Polish Large White (PLW) gilts. 2DE-MALDI-TOF/TOF and ClueGOTM approaches were employed to analyse the biological networks and molecular changes in porcine endometrial proteome during maternal recognition of pregnancy. A total of sixteen differentially expressed proteins (DEPs) were identified using 2-DE gels and MALDI-TOF/TOF mass spectrometry. Comparison between 9D and 12D of pregnancy identified APOA1, CAPZB, LDHB, CCT5, ANXA4, CFB, TTR upregulated DEPs, and ANXA5, SMS downregulated DEPs. Comparison between 9D and 16D of pregnancy identified HP, APOA1, ACTB, CCT5, ANXA4, CFB upregulated DEPs and ANXA5, SMS, LDHB, ACTR3, HP, ENO3, OAT downregulated DEPs. However, a comparison between 12D and 16D of pregnancy identified HP, ACTB upregulated DEPs, and CRYM, ANXA4, ANXA5, CAPZB, LDHB, ACTR3, CCT5, ENO3, OAT, TTR down-regulated DEPs. Outcomes of this study revealed key proteins and their interactions with metabolic pathways involved in the recognition and establishment of early pregnancy in PLW gilts.

Effects of Dietary n-3 and n-6 Polyunsaturated Fatty Acids in Inflammation and Cancerogenesis.

The dietary recommendation encourages reducing saturated fatty acids (SFA) in diet and replacing them with polyunsaturated fatty acids (PUFAs) n-3 (omega-3) and n-6 (omega-6) to decrease the risk of metabolic disturbances. Consequently, excessive n-6 PUFAs content and high n-6/n-3 ratio are found in Western-type diet. The importance of a dietary n-6/n-3 ratio to prevent chronic diseases is linked with anti-inflammatory functions of linolenic acid (ALA, 18:3n-3) and longer-chain n-3 PUFAs. Thus, this review provides an overview of the role of oxylipins derived from n-3 PUFAs and oxylipins formed from n-6 PUFAs on inflammation. Evidence of PUFAs' role in carcinogenesis was also discussed. In vitro studies, animal cancer models and epidemiological studies demonstrate that these two PUFA groups have different effects on the cell growth, proliferation and progression of neoplastic lesions.

Effects of Three-Month Administration of High-Saturated Fat Diet and High-Polyunsaturated Fat Diets with Different Linoleic Acid (LA, C18:2n-6) to α-Linolenic Acid (ALA, C18:3n-3) Ratio on the Mouse Liver Proteome.

The aim of the study was to evaluate the effect of different types of high-fat diets (HFDs) on the proteomic profile of mouse liver. The analysis included four dietary groups of mice fed a standard diet (STD group), a high-fat diet rich in SFAs (SFA group), and high-fat diets dominated by PUFAs with linoleic acid (LA, C18:2n-6) to α-linolenic acid (ALA, C18:3n-3) ratios of 14:1 (14:1 group) and 5:1 (5:1 group). After three months of diets, liver proteins were resolved by two-dimensional gel electrophoresis (2DE) using 17 cm non-linear 3-10 pH gradient strips. Protein spots with different expression were identified by MALDI-TOF/TOF. The expression of 13 liver proteins was changed in the SFA group compared to the STD group (↓: ALB, APOA1, IVD, MAT1A, OAT and PHB; ↑: ALDH1L1, UniProtKB-Q91V76, GALK1, GPD1, HMGCS2, KHK and TKFC). Eleven proteins with altered expression were recorded in the 14:1 group compared to the SFA group (↓: ARG1, FTL1, GPD1, HGD, HMGCS2 and MAT1A; ↑: APOA1, CA3, GLO1, HDHD3 and IVD). The expression of 11 proteins was altered in the 5:1 group compared to the SFA group (↓: ATP5F1B, FTL1, GALK1, HGD, HSPA9, HSPD1, PC and TKFC; ↑: ACAT2, CA3 and GSTP1). High-PUFA diets significantly affected the expression of proteins involved in, e.g., carbohydrate metabolism, and had varying effects on plasma total cholesterol and glucose levels. The outcomes of this study revealed crucial liver proteins affected by different high-fat diets.

Gene promoter polymorphisms in boar spermatozoa differing in freezability.

Single nucleotide polymorphisms (SNPs) in the 5'-flanking regulatory regions of genes could affect their expression levels. This is a follow-up study aimed to identify polymorphic variants in the 5'-flanking regulatory regions of genes expressed in boar spermatozoa, and to predict the interactions of such variants with transcription factors (TFs) on the gene promoter activity, using bioinformatics. Five and six boars were classified as having good and poor semen freezability (GSF and PSF, respectively) according to post-thaw (PT) assessment of sperm motility and membrane integrity characteristics. The 5'-flanking region sequences of the 14 genes (FOS, NFATC3, EAF2, FGF-14, BAMBI, RAB33B, CKS2, LARS2, SLC25A16, ACADM, CPT2, CCT3, DTD2 and CCDC85A) were PCR amplified and analyzed by Sanger sequencing method. A total of 32 polymorphic variants were identified in the 5'-flanking regions of the genes, including 4 insertion/deletion (indel) polymorphisms, and 8 unknown (novel) SNPs. Multiple sequence alignment analysis revealed a 26-bp indel variant in the 5'-flanking region of the LARS2 gene, which showed greater protein expression in spermatozoa from boars of the PSF group. It was found that 17 polymorphic variants, observed in the differentially expressed (DE) genes, showed significant allele frequency differences between the GSF and PSF groups. Polymorphic variants in the 5'-flanking regulatory regions of the genes contributed to the decrease or increase in the binding affinity for different testis-specific TFs, such as SMAD1, NF-1, FOXMI, RXRA, STAT4 and C/EBPß. This study provides more insights into the mechanisms responsible for variations in transcriptional activity in promoters of genes expressed in boar spermatozoa. The allelic variants are promising genetic markers for predicting the freezability of boar spermatozoa.

Differentiation of Subclinical Ketosis and Liver Function Test Indices in Adipose Tissues Associated With Hyperketonemia in Postpartum Dairy Cattle.

Past studies suggested that during early lactation and the transition period, higher plasma growth hormone (GH) levels in subclinical ketosis (SCK) might involve the initiation of body adipose tissues mobilization, resulting in metabolic disorders in ruminants particularly hyperketonemia. The upregulated GH mRNA expression in adipose tissue may take part in the adipolysis process in SCK-affected cows that paves a way for study further. This study aimed to characterize the plasma levels of GH, ß-hydroxybutyrate acid (BHBA) and non-esterified fatty acid (NEFA) and glucose (GLu) in ketotic cows and healthy control (CON) cows; to measure the liver function test (LFT) indices in ketotic and healthy CON cows, and finally the quantitative real-time PCR (qRT-PCR) assay of candidate genes expressed in adipose tissues of ketotic and healthy CON cows during 0 to 7 week postpartum. Three experiments were conducted. Experiment-1 involved 21 Holstein cows weighing 500-600 kg with 2-5 parities. Results showed that GH, BHBA, and NEFA levels in ketotic cows were significantly higher and the GLu level significantly lower. Pearson's correlation analysis revealed a significant positive correlation of GH with BHBA, NEFA, and GLu in ketotic and healthy CON cows. In experiment-2, dynamic monitoring of LFT indices namely, alanine aminotransferase (ALT), aspartate aminotransferase (AST), γ-glutamyl transpeptidase (GGT), total bilirubin (TBIL), direct bilirubin (DBIL), total protein (TP), albumin (ALB), globulin (GLOB) and albumin/globulin (A/G) were examined. The TBIL, DBIL, and GGT indices were significantly higher in ketotic cows and TP was significantly lower. In experiment-3, mRNA expression levels of GHR and peroxisome-proliferator-activated receptor alpha (PPARα) genes in adipose tissue were significantly upregulated in ketotic cows. However, the mRNA expression of insulin-like growth factor-I (IGF-1), insulin-like growth factor-I receptor (IGF-1R), and sterol regulatory element-binding protein-1c (SREBP-1c) genes in adipose tissue were downregulated in ketotic cows. Our study concluded that during postpartum, higher plasma GH levels in SCK cows might involve the initiation of body adipose tissue mobilization, resulting in hyperketonemia.

Regulatory Potential of Long Non-Coding RNAs (lncRNAs) in Boar Spermatozoa with Good and Poor Freezability.

Long non-coding RNAs (lncRNAs) are suggested to play an important role in the sperm biological processes. We performed de novo transcriptome assembly to characterize lncRNAs in spermatozoa, and to investigate the role of the potential target genes of the differentially expressed lncRNAs (DElncRNAs) in sperm freezability. We detected approximately 4007 DElncRNAs, which were differentially expressed in spermatozoa from boars classified as having good and poor semen freezability (GSF and PSF, respectively). Most of the DElncRNAs were upregulated in boars of the PSF group and appeared to significantly affect the sperm's response to the cryopreservation conditions. Furthermore, we predicted that the potential target genes were regulated by DElncRNAs in cis or trans. It was found that DElncRNAs of both freezability groups had potential cis- and trans-regulatory effects on different protein-coding genes, such as COX7A2L, TXNDC8 and SOX-7. Gene Ontology (GO) enrichment revealed that the DElncRNA target genes are associated with numerous biological processes, including signal transduction, response to stress, cell death (apoptosis), motility and embryo development. Significant differences in the de novo assembled transcriptome expression profiles of the DElncRNAs between the freezability groups were confirmed by quantitative real-time PCR analysis. This study reveals the potential effects of protein-coding genes of DElncRNAs on sperm functions, which could contribute to further research on their relevance in semen freezability.

Identification of Differentially Expressed Gene Transcripts in Porcine Endometrium during Early Stages of Pregnancy.

During the early stages of pregnancy, the uterine endometrium undergoes dramatic morphologic and functional changes accompanied with dynamic variation in gene expression. Pregnancy-stage specific differentially expressed gene (DEG)-transcript-probes were investigated and identified by comparing endometrium transcriptome at 9th day (9D), 12th day (12D) and 16th day (16D) of early pregnancy in Polish large-white (PLW) gilts. Endometrium comparisons between 9D-vs-12D, 9D-vs-16D and 12D-vs-16D of early pregnancy identified 6049, 374 and 6034 highly significant DEG-transcript-probes (p < 0.001; >2 FC). GO term enrichment analysis identified commonly shared upregulated endometrial DEG-transcript-probes (p < 0.001; >2 FC), that were regulating the gene functions of anatomic structure development and transport (TG), DNA-binding and methyltransferase activity (ZBTB2), ion-binding and kinase activity (CKM), cell proliferation and apoptosis activity (IL1B). Downregulated DEG-transcript-probes (p < 0.001; >2 FC) were involved in regulating the gene functions of phosphatase activity (PTPN11), TC616413 gene-transcript and Sus-scrofa LOC100525539. Moreover, blastn comparison of microarray-probes sequences against sus-scrofa11 assembly identified commonly shared upregulated endometrial DEG-transcript-probes (E < 0.06; >2 FC), that were regulating the gene functions of reproduction and growth (SELENOP), cytoskeleton organization and kinase activity (CDC42BPA), phosphatase activity (MINPP1), enzyme-binding and cell-population proliferation (VAV3), cancer-susceptibility candidate gene (CASC4), cytoskeletal protein-binding (COBLL1), ion-binding, enzyme regulator activity (ACAP2) Downregulated endometrial DEG-transcript-probes (E < 0.06; >2FC) were involved in regulating the gene functions of signal-transduction (TMEM33), catabolic and metabolic processes (KLHL15). Microarray validation experiment on selected candidate genes showed complementarity to significant endometrial DEG-transcript-probes responsible for the regulation of immune response (IL1B, S100A11), lipid metabolism (FABP3, PPARG), cell-adhesion (ITGAV), angiogenesis (IL1B), intercellular transmission (NMB), cell-adhesion (OPN) and response to stimuli (RBP4) was confirmed by RT-PCR. This study provides a clue that identified pregnancy-stage specific microarray transcript probes could be considered as candidate genes for recognition and establishment of early pregnancy in the pig.

Pituitary Hormones (FSH, LH, PRL, and GH) Differentially Regulate AQP5 Expression in Porcine Ovarian Follicular Cells.

This study aimed to examine the effect of follicle-stimulating hormone (FSH), luteinizing hormone (LH), prolactin (PRL), and growth hormone (GH) on Aquaporin 5 (AQP5) expression in granulosa (Gc) and theca cells (Tc) from medium (MF) and large (LF) ovarian follicles of pigs. The results showed that GH significantly decreased the expression of AQP5 in Gc from MF in relation to the control. In the Gc of large follicles, PRL stimulated the expression of AQP5. However, the increased expression of AQP5 in the Tc of LF was indicated by GH and PRL in relation to the control. A significantly higher expression of the AQP5 protein in the Gc from MF and LF was indicated by FSH and PRL. In co-cultures, an increased expression of AQP5 was observed in the Gc from LF incubated with LH, PRL, and GH. A significantly increased expression of AQP5 was also observed in co-cultures of Tc from all type of follicles incubated with LH, whereas PRL stimulated the expression of AQP5 in Tc from MF. Moreover, AQP5 protein expression increased in the co-culture isolated from MF and LF after treatment with FSH, LH, PRL, and GH. AQP5 immunoreactivity was observed in the cytoplasm, mainly in the perinuclear region and endosomes, as well as in the cell membranes of Gc and Tc from the LF and MF.

A porcine gluteus medius muscle genome-wide transcriptome analysis: dietary effects of omega-6 and omega-3 fatty acids on biological mechanisms.

BACKGROUND: The level of omega-6 and omega-3 polyunsaturated fatty acids can affect many cellular systems and function via nuclear receptors or the bioactive lipid regulation of gene expression. The objective of this study was to investigate changes in the muscle transcriptome and the biological functions regulated by increased consumption of omega-3 and omega-6 fatty acids in the pig gluteus medius muscle. RESULTS: The transcriptome of the gluteus medius muscle was studied for pigs subjected to either a control diet or a diet supplemented with linseed and rapeseed oil to increase polyunsaturated fatty acid content. Next-generation sequencing (NGS) was used to generate the muscle tissue transcriptome database pointing differentially expressed genes (DEG). Comparative expression analyses identified 749 genes significantly differing at least in the twofold of change between two groups of animals fed with divergent level of omega-3 and omega-6 fatty acids. The expression of 219 genes was upregulated, and the expression of 530 genes was downregulated in the group of pigs supplemented with omega-3 and omega-6 fatty acids in relation to control group pigs. Results of RNA-seq indicated a role of fatty acid in the regulation of the expression of genes which are essential for muscle tissue development and functioning. Functional analysis revealed that the identified genes were important for a number of biological processes including inflammatory response, signaling, lipid metabolism, and homeostasis. CONCLUSIONS: Summarizing, obtained results provide strong evidence that omega-6 and omega-3 fatty acids regulate fundamental metabolic processes in muscle tissue development and functioning.

From genotype to phenotype in bovine functional genomics.

In past 5 years, the promise that came with genome sequencing has revolutionized the functional genomics research field at unprecedented manner. It would soon know what all known genes do, particularly genes involved in genetic improvement of animal health and increase food animal production. With the availability of full bovine genomic sequence, yet we still have a lot of daunting tasks on 'genotype-to-phenotype problem' particularly about the phenotypic variations and trying to predict what genes are likely to be involved, and improved integrated interactive database. This article outlined and discussed about the current status of bovine functional genomics, recent development in bovine genome databases particularly in annotation of bovine genome, bovine quantitative trait loci database and its potential impact to unveil the from genotype-to-phenotype problem.

Populationwide investigation of two indel polymorphisms at the prion protein gene in Polish Holstein-Friesian cattle.

The allele, genotype, and haplotype frequencies among 837 Polish Holstein-Friesian cattle were determined at two regulatory indel polymorphisms of the PRNP gene. Allele frequencies at the 23 bp indel promoter polymorphism were 0.622 (del) and 0.378 (ins), with 0.613 and 0.387 in sires and 0.633 and 0.366 in dams. Allele frequencies at the 12 bp indel intron polymorphism were 0.527 (del) and 0.473 (ins), with 0.529 and 0.471 in sires and 0.543 and 0.456 in dams. Four haplotypes were identified in this population (23-12del, 23-12ins, 23del-12ins, and 23ins-12del). Haplotype 23-12del occurred most frequently in both sire and dam groups. Comparative analysis of Polish Holstein-Friesian and German Holstein populations revealed a similar genetic structure for the 23 bp indel polymorphism and a significantly different one for the 12 bp indel polymorphism. In allele and haplotype analysis, significant differences were observed between the Polish Holstein-Friesian population and a BSE-free German Holstein population.

Evaluation of reference genes for studies of gene expression in the bovine liver, kidney, pituitary, and thyroid.

Expression patterns of candidate genes with important functions in animal metabolism can help to identify potential molecular markers for cattle production traits. Reverse transcription followed by polymerase chain reaction is a method for rapid and accurate mRNA quantification. However, for exact comparison of mRNA quantity in various samples or tissues, it is important to choose appropriate reference genes. In cattle, little information is available on the expression stability of housekeeping genes (HKGs). The aim of the present study is to develop a set of reference genes that can be used for normalization of concentrations of mRNAs of genes expressed in the bovine liver, kidney, pituitary and thyroid. The study was performed on 6-, 9-, and 12-month-old bulls of dairy and meat cattle breeds. Six HKGs were investigated: ACTB, GAPDH, HPRTI, SDHA, TBP, and YWHAZ. The most stably expressed potential reference HKGs differed among tissues/organs examined: ACTB, TBP, YWHAZ, GAPDH, HPRTI, and SDHA in the liver; GAPDH and YWHAZ in the kidney; GAPDH and SDHA in the pituitary; and TBP and HPRTI in the thyroid. The results showed that the use of a single gene for normalization may lead to relatively large errors, so it is important to use multiple control genes based on a survey of potential reference genes applied to representative samples from specific experimental conditions.

DGAT1 K232A quantitative trait nucleotide polymorphism in Polish Black-and-White cattle.

The diacylglycerol o-acyltransferase 1 gene (DGAT1) was investigated in Polish Black-and-White cattle. The frequency of the K allele was 0.60, 0.68 and 0.48 for AI sires (n=150), young bulls (n=139) and cows (n=213), respectively. The method of selective genotyping for identification of the quantitative trait nucleotide was verified through identification of DGAT1 effect on milk production traits. Daughters of six heterozygous bulls were selectively genotyped based on their milk traits. The genotypic frequencies differed between high and low yield groups representing milk and fat contents. The Kruskal-Wallis test revealed a highly significant effect of DGAT1 K232A in cows with extremely low fat content and a significant effect in cows with extremely high protein content of milk. No significant effect of AI sires' genotypes on their breeding value was found.

Silent point mutation polymorphism of the bovine CD18 encoding gene.

We report on a PCR-RFLP procedure for recognising of a silent point mutation of ITGB2 CD18 subunit gene in cattle. Polymorphism screening was performed in a Polish Black-and-White cattle population (n=210). The genotype and allele frequencies were established in the sires and cows. Further research is needed to explain the possible applications of the CD18 silent point mutation as a potential molecular marker for high milk productivity.

Abnormal segregation of prion protein octapeptide-repeat alleles in cattle.

The study was conducted on full-families of Black-and-White cattle obtained as 25 AI sire families and 355 cows, as well as their progenies, mostly heifers at the age of 1-3 months. The sire group was composed by the casual qualification of 10 PRNP 6/6 and 15 PRNP 6/5 individuals on the basis of accessible young progenies. The randomly selected group of cows is characterised by a very high frequency of PRNP 6/6 (74.9%), followed by lower frequency of PRNP 6/5 (24.5%) and a very low frequency of PRNP 5/5 genotype (0.6%). The progenies represent all expected genotypes, such as: PRNP 6/6 (60.5%), PRNP 6/5 (35.8%) and PRNP 5/5 (3.7%), respectively. Taking into consideration the genotypes of parents and progenies, the segregation of PRNP 6 and PRNP 5 alleles was analysed. Results of the non-informative mating variant of male symbol PRNP 6/6 x female symbol PRNP 6/6 (n = 87) are affected by the PRNP 6/6 progeny genotype in all cases. Subsequently, the results of mating variants male symbol PRNP 6/6 x female symbol PRNP 6/5 (n = 29) and male symbol PRNP 6/5 female symbol PRNP 6/6 (n = 179) showed statistically non-significant differences in both above-mentioned alternations. The progeny group related from male symbol PRNP 6/5 x female symbol PRNP 6/5 parental mating obtained fully informative and most valuable results based on the presented research concept. In the common group of 58 calves, the genotype PRNP 6/6 is represented by 26 individuals (44.8%), PRNP 6/5 - by 19 individuals (32.8%) and PRNP 5/5 - by 13 individuals (22.4%). Therefore, the theoretical genotype rate (25% : 50% : 25%) is drastically deformed and the differentiation between the observed and expected numbers of animals is statistically highly significant (chi(2) = 12.72; 2 df.). These differences are affected by two times higher PRNP 6/6 homozygous (chi(2) = 9.12; 1 df.) and responsively by the low number of PRNP 6/5 heterozygous animals (chi(2) = 3.45; 1 df.). Further investigations are carried out to explain the genetic determination of abnormal PRNP octa-peptide repeat allele segregation, which suggests possible lethal cis-trans linkage effects.

Novel linkage mapping approach using DNA pooling in human and animal genetics. II. Detection of quantitative traits loci in dairy cattle.

Selective DNA pooling is an advanced methodology for linkage mapping of quantitative trait loci (QTL) in farm animals. The principle is based on densitometric estimates of marker allele frequency in pooled DNA samples of phenotypically extreme individuals from half-sib, backcross and F(2) experimental designs in farm animals. This methodology provides a rapid and efficient analysis of a large number of individuals with short tandem repeat markers that are essential to detect QTL through the genome - wide searching approach. Several strategies involving whole genome scanning with a high statistical power have been developed for systematic search to detect the quantitative traits loci and linked loci of complex traits. In recent studies, greater success has been achieved in mapping several QTLs in Israel-Holstein cattle using selective DNA pooling. This paper outlines the currently emerged novel strategies of linkage mapping to identify QTL based on selective DNA pooling with more emphasis on its theoretical pre-requisite to detect linked QTLs, applications, a general theory for experimental half-sib designs, the power of statistics and its feasibility to identify genetic markers linked QTL in dairy cattle. The study reveals that the application of selective DNA pooling in dairy cattle can be best exploited in the genome-wide detection of linked loci with small and large QTL effects and applied to a moderately sized half-sib family of about 500 animals.

Novel linkage mapping approach using DNA pooling in human and animal genetics. I. Detection of complex disease loci.

DNA pooling is a potential methodology for genetic loci with small effect contributing to complex diseases and quantitative traits. This is accomplished by the rapid preliminary screening of the genome for the allelic association with the most common class of polymorphic short tandem repeat markers. The methodology assumes as a common founder for the linked disease locus of interest and searches for a region of a chromosome shared between affected individuals. The general theory of DNA pooling basically relies on the observed differences in the allelic distribution between pools from affected and unaffected individuals, including a reduction in the number of alleles in the affected pool, which indicate the sharing of a chromosomal region. The power of statistic for associated linkage mapping can be determined using two recently developed strategies, firstly, by measuring the differences of allelic image patterns produced by two DNA pools of extreme character and secondly, by measuring total allele content differences by comparing between two pools containing large numbers of DNA samples. These strategies have effectively been utilized to identify the shared chromosomal regions for linkage studies and to investigate the candidate disease loci for fine structure gene mapping using allelic association. This paper outlines the utilization of DNA pooling as a potential tool to locate the complex disease loci, statistical methods for accurate estimates of allelic frequencies from DNA pools, its advantages, drawbacks and significance in associate linkage mapping using pooled DNA samples.