Identification of heat shock protein gene expression in hair follicles as a novel indicator of heat stress in beef calves

Heat shock proteins (HSPs) consist of highly preserved stress proteins that are expressed in response to stress. Two studies were carried out to investigate whether HSP genes in hair follicles from beef calves can be suggested as indicators of heat stress (HS). In study 1, hair follicles were harvested from three male Hanwoo calves (aged 172.2 ± 7.20 days) on six dates over the period of 10 April to 9 August 2017. These days provided varying temperature–humidity indices (THIs). In study 2, 16 Hanwoo male calves (aged 169.6 ± 4.60 days, with a BW of 136.9 ± 6.23 kg) were maintained (4 calves per experiment) in environmentally controlled chambers. A completely randomized design with a 2 × 4 factorial arrangement involving two periods (thermoneutral: TN; HS) and four THI treatment groups (threshold: THI = 68 to 70; mild: THI = 74 to 76; moderate THI = 81 to 83; severe: THI = 88 to 90). The calves in the different group were subjected to ambient temperature (22°C) for 7 days (TN) and subsequently to the temperature and humidity corresponding to the target THI level for 21 days (HS). Every three days (at 1400 h) during both the TN and HS periods, the heart rate (HR) and rectal temperature (RT) of each individual were measured, and hair follicles were subsequently collected from the tails of each individual. In study 1, the high variation (P < 0.0001) in THI indicated that the external environment influenced the HS to different extents. The expression levels of the HSP70 and HSP90 genes at the high-THI level were higher (P = 0.0120, P = 0.0002) than those at the low-THI level. In study 2, no differences in the THI (P = 0.2638), HR (P = 0.2181) or RT (P = 0.3846) were found among the groups during the TN period, whereas differences in these indices (P < 0.0001, P < 0.0001 and P < 0.0001, respectively) were observed during the HS period. The expression levels of the HSP70 (P = 0.0010, moderate; P = 0.0065, severe) and HSP90 (P = 0.0040, severe) genes were increased after rapid exposure to heat-stress conditions (moderate and severe levels). We conclude that HSP gene expression in hair follicles provides precise and accurate data for evaluating HS and can be considered a novel indicator of HS in Hanwoo calves maintained in both external and climatic chambers.     

Genetic variants and signatures of selective sweep of Hanwoo population (Korean native cattle)

Although there have been many studies of native Korean cattle, Hanwoo, there have been no selective sweep studies in these animals. This study was performed to characterize genetic variation and identify selective signatures. We sequencedthe genomes of 12 cattle, and identified 15125420 SNPs, 1768114 INDELs, and 3445 CNVs. The SNPs, INDELs, and CNVs were similarly distributed throughout the genome, and highly variable regions were shown to contain the BoLA family and GPR180, which are related to adaptive immunity. We also identified the domestication footprints of the Hanwoo population by searching for selective sweep signatures, which revealed the RCN2 gene related to BPV resistance. The results of this study may contribute to genetic improvement of the Hanwoo population in Korea. [BMB Reports 2013; 46(7):346-351]     

Estimates of genetic parameters for fatty acid compositions in the longissimus dorsi muscle of Hanwoo cattle

We estimated the heritabilities (h²) and genetic and phenotypic correlations among individual and groups of fatty acids, as well as their correlations with six important carcass and meat-quality traits in Korean Hanwoo cattle. Meat samples were collected from the longissimus dorsi muscles of 1000 Hanwoo steers that were 30-month-old (progeny of 85 proven Hanwoo bulls) to determine intramuscular fatty acid profiles. Phenotypic data on carcass weight (CWT), eye muscle area (EMA), back fat thickness (BFT), marbling score (MS), Warner–Bratzler shear force (WBSF) and intramuscular fat content (IMF) were also investigated using this half-sib population. Variance and covari.ance components were estimated using restricted maximum likelihood procedures under univariate and pairwise bivariate animal models. Oleic acid (C18:1n-9) was the most abundant fatty acid, accounting for 50.69% of all investigated fatty acids, followed by palmitic (C16:0; 27.33%) and stearic acid (C18:0; 10.96%). The contents of saturated fatty acids (SFAs), monounsaturated fatty acids (MUFAs) and polyunsaturated fatty acids (PUFAs) were 41.64%, 56.24% and 2.10%, respectively, and the MUFA/SFA ratio, PUFA/SFA ratio, desaturation index (DI) and elongation index (EI) were 1.36, 0.05, 0.59 and 0.66, respectively. The h² estimates for individual fatty acids ranged from very low to high (0.03±0.14 to 0.63±0.14). The h² estimates for SFAs, MUFAs, PUFAs, DI and EI were 0.53±0.14, 0.49±0.14, 0.23±0.10, 0.51±0.13 and 0.53±0.13, respectively. The genetic and phenotypic correlations among individual fatty acids and fatty acid classes varied widely (−0.99 to 0.99). Notably, C18:1n-9 had favourable (negative) genetic correlations with two detrimental fatty acids, C14:0 (−0.76) and C16:0 (−0.92). Genetic correlations of individual and group fatty acids with CWT, EMA, BFT, MS, WBSF and IMF ranged from low to moderate (both positive and negative) with the exception of low-concentration PUFAs. Low or near-zero phenotypic correlations reflected potential non-genetic contributions. This study provides insights on genetic variability and correlations among intramuscular fatty acids as well as correlations between fatty acids and carcass and meat-quality traits, which could be used in Hanwoo breeding programmes to improve fatty acid compositions in meat.     

Identification of BoLA-DRB3.2 alleles in Korean native cattle (Hanwoo) and Holstein populations using a next generation sequencer

Bovine leucocyte antigen (encoded by BoLA) has been widely studied to identify the association with many traits related to immunity. Exon2 of BoLA-DRB3 is extremely polymorphic, and more than 100 alleles have been identified. We investigated polymorphisms of BoLA-DRB3.2 in Korean native cattle and Holstein populations using a next generation sequencer of the GS-FLX Titanium system. We found 38 alleles including 11 new alleles (BoLA-DRB3*1303, *4702, *7101, *7501, *7201, *7301, *7601, *1104, *7701, *7401 and *50021) in Hanwoo, and nine alleles including one new allele (BoLA-DRB3*7601) in Holstein. The 454 sequencing method is a promising alternative technology for high throughput genotyping of BoLA-DRB3.2 because of its technical advantages that allow it to overcome the disadvantages of sequence-based typing methods.     

Performance of different SNP panels for parentage testing in two East Asian cattle breeds

The International Society for Animal Genetics (ISAG) proposed a panel of single nucleotide polymorphisms (SNPs) for parentage testing in cattle (a core panel of 100 SNPs and an additional list of 100 SNPs). However, markers specific to East Asian taurine cattle breeds were not included, and no information is available as to whether the ISAG panel performs adequately for these breeds. We tested ISAG's core (100 SNP) and full (200 SNP) panels on two East Asian taurine breeds: the Korean Hanwoo and the Japanese Wagyu, the latter from the Australian herd. Even though the power of exclusion was high at 0.99 for both ISAG panels, the core panel performed poorly with 3.01% false‐positive assignments in the Hanwoo population and 3.57% in the Wagyu. The full ISAG panel identified all sire–offspring relations correctly in both populations with 0.02% of relations wrongly excluded in the Hanwoo population. Based on these results, we created and tested two population‐specific marker panels: one for the Wagyu population, which showed no false‐positive assignments with either 100 or 200 SNPs, and a second panel for the Hanwoo, which still had some false‐positive assignments with 100 SNPs but no false positives using 200 SNPs. In conclusion, for parentage assignment in East Asian cattle breeds, only the full ISAG panel is adequate for parentage testing. If fewer markers should be used, it is advisable to use population‐specific markers rather than the ISAG panel.     

Whole-Genome Resequencing Analysis of Hanwoo and Yanbian Cattle to Identify Genome-Wide SNPs and Signatures of Selection

Over the last 30 years, Hanwoo has been selectively bred to improve economically important traits. Hanwoo is currently the representative Korean native beef cattle breed, and it is believed that it shared an ancestor with a Chinese breed, Yanbian cattle, until the last century. However, these two breeds have experienced different selection pressures during recent decades. Here, we whole-genome sequenced 10 animals each of Hanwoo and Yanbian cattle (20 total) using the Illumina HiSeq 2000 sequencer. A total of approximately 3.12 and 3.07 billion sequence reads were mapped to the bovine reference sequence assembly (UMD 3.1) at an average of approximately 10.71- and 10.53-fold coverage for Hanwoo and Yanbian cattle, respectively. A total of 17,936,399 single nucleotide polymorphisms (SNPs) were yielded, of which 22.3% were found to be novel. By annotating the SNPs, we further retrieved numerous nonsynonymous SNPs that may be associated with traits of interest in cattle. Furthermore, we performed whole-genome screening to detect signatures of selection throughout the genome. We located several promising selective sweeps that are potentially responsible for economically important traits in cattle; the PPP1R12A gene is an example of a gene that potentially affects intramuscular fat content. These discoveries provide valuable genomic information regarding potential genomic markers that could predict traits of interest for breeding programs of these cattle breeds.     

Identification of genetic markers for Korean native cattle (Hanwoo) by RAPD analysis

In order to develop the specific genetic marker for Korean native cattle (Hanwoo), randomly amplified polymorphic DNA (RAPD) analysis of 6 different cattle breeds was attempted by using 38 decamer primers. In comparison of RAPD patterns, two distinctive DNA bands specific for Hanwoo were detected. One was 296 bp of DNA fragment found to be specific only for female Hanwoo when primer GTCCACACGG was employed. In individual analysis of this RAPD marker was observed only in female individuals with the possibility of 85.3%. The other was 521 bp of RAPD marker amplified using TCGGCGATAG and AGCCAGCGAA primers, which showed 83.0% of genetic frequency in 85 male and 68 female individuals tested. Nucleotide sequencing of these genetic markers revealed that 296 bp marker has a short microsatellite-like sequence, ACCACCACAC, and a tandem repeat sequence of microsatellite GAAAAATG in the determined sequence. Two distinctive tandem repeats of microsatellite sequences, AAC and GAAGA, were also appeared in 521 bp DNA marker. In BLAST search, any gene having high homology with these markers was not found     

Targeted suppression of μ‐calpain and caspase 9 expression and its effect on caspase 3 and caspase 7 in satellite cells of Korean Hanwoo cattle

The calpains play an important role in cell death and cell signalling. Caspases catalyse wholesale destruction of cellular proteins which is a major cause of cellular death. The current study looks at the function of μ‐calpain and caspase 9, using RNAi (RNA interference)‐mediated silencing, and to observe the mRNA expression level of caspase genes during satellite cell growth. The satellite cells were treated with siRNA (small interfering RNA) of μ‐calpain and caspase 9 separately. There was reduction of 16 and 24% in CAPN1 (calpain1)‐siRNA2 and CAPN1‐siRNA3 transfected cells respectively, whereas it was 60 and 56% in CAPN1‐siRNA1 and CAPN1‐siRNA4 transfected cells respectively. CAPN1‐siRNA4 and CAPN1‐siRNA1 treated cells showed more reduction in caspase 3 and 7 gene expression. CARD9 (caspase recruitment domain 9)‐siRNA1 and CARD9‐siRNA2‐treated cells showed reduction of 40 and 49% respectively. CARD9‐siRNA1 and CARD9‐siRNA2 showed an increase in caspase 3 gene expression, whereas CARD9‐siRNA2 showed reduction in caspase 7 gene expression. These results suggest a strong cross‐talk between μ‐calpain and the caspase enzyme systems. Suppression of target genes, such as μ‐calpain and caspase 9, might have genuine potential in the treatment of skeletal muscle atrophy.