Genetic Parameter Estimates of Carcass Traits under National Scale Breeding Scheme for Beef Cattle.

Carcass and price traits of 72,969 Hanwoo cows, bulls and steers aged 16 to 80 months at slaughter collected from 2002 to 2013 at 75 beef packing plants in Korea were analyzed to determine heritability, correlation and breeding value using the Multi-Trait restricted maximum likelihood (REML) animal model procedure. The traits included carcass measurements, scores and grades at 24 h postmortem and bid prices at auction. Relatively high heritability was found for maturity (0.41±0.031), while moderate heritability estimates were obtained for backfat thickness (0.20±0.018), longissimus muscle (LM) area (0.23±0.020), carcass weight (0.28±0.019), yield index (0.20±0.018), yield grade (0.16±0.017), marbling (0.28±0.021), texture (0.14±0.016), quality grade (0.26±0.016) and price/kg (0.24±0.025). Relatively low heritability estimates were observed for meat color (0.06±0.013) and fat color (0.06±0.012). Heritability estimates for most traits were lower than those in the literature. Genetic correlations of carcass measurements with characteristic scores or quality grade of carcass ranged from -0.27 to +0.21. Genetic correlations of yield grade with backfat thickness, LM area and carcass weight were 0.91, -0.43, and -0.09, respectively. Genetic correlations of quality grade with scores of marbling, meat color, fat color and texture were -0.99, 0.48, 0.47, and 0.98, respectively. Genetic correlations of price/kg with LM area, carcass weight, marbling, meat color, texture and maturity were 0.57, 0.64, 0.76, -0.41, -0.79, and -0.42, respectively. Genetic correlations of carcass price with LM area, carcass weight, marbling and texture were 0.61, 0.57, 0.64, and -0.73, respectively, with standard errors ranging from ±0.047 to ±0.058. The mean carcass weight breeding values increased by more than 8 kg, whereas the mean marbling scores decreased by approximately 0.2 from 2000 through 2009. Overall, the results suggest that genetic improvement of productivity and carcass quality could be obtained under the national scale breeding scheme of Korea for Hanwoo and that continuous efforts to improve the breeding scheme should be made to increase genetic progress.

Mutations in calpastatin and µ-calpain are associated with meat tenderness, flavor and juiciness in Hanwoo (Korean cattle): molecular modeling of the effects of substitutions in the calpastatin/µ-calpain complex.

The objective of this study was to evaluate the effects of seven single nucleotide polymorphisms (SNPs) in Calpain 1 and Calpastatin genes previously associated with meat tenderness attributes in other cattle breeds in Korean Hanwoo cattle. The Hanwoo resource population was used to study association of 7 SNPs with beef tenderness, flavor, juiciness, intramuscular fat and shear force. In this association study, CAST:c.182A>G (+0.14, P=0.04) and CAST:c.1985G>C (-0.12, P=0.02) had significant effects on juiciness, but no effects on other traits. In contrast, CAPN1:c.1589G>A was associated with meat tenderness (P=0.01) and juiciness (P=0.04). The CAPN1:c.1589G>A (Val530Ile) SNP marker displayed significant effect on the meat tenderness score which is strongly supported by molecular modeling of the CAPN1:c.1589G>A (Val530Ile) variant that inhibits CAST protein from binding more strongly than the wild-type protein, which may explain its effect on meat tenderness.

Expressed sequence tags for bovine muscle satellite cells, myotube formed-cells and adipocyte-like cells.

BACKGROUND: Muscle satellite cells (MSCs) represent a devoted stem cell population that is responsible for postnatal muscle growth and skeletal muscle regeneration. An important characteristic of MSCs is that they encompass multi potential mesenchymal stem cell activity and are able to differentiate into myocytes and adipocytes. To achieve a global view of the genes differentially expressed in MSCs, myotube formed-cells (MFCs) and adipocyte-like cells (ALCs), we performed large-scale EST sequencing of normalized cDNA libraries developed from bovine MSCs. RESULTS: A total of 24,192 clones were assembled into 3,333 clusters, 5,517 singletons and 3,842contigs. Functional annotation of these unigenes revealed that a large portion of the differentially expressed genes are involved in cellular and signaling processes. Database for Annotation, Visualization and Integrated Discovery (DAVID) functional analysis of three subsets of highly expressed gene lists (MSC233, MFC258, and ALC248) highlighted some common and unique biological processes among MSC, MFC and ALC. Additionally, genes that may be specific to MSC, MFC and ALC are reported here, and the role of dimethylarginine dimethylaminohydrolase2 (DDAH2) during myogenesis and hemoglobin subunit alpha2 (HBA2) during transdifferentiation in C2C12 were assayed as a case study. DDAH2 was up-regulated during myognesis and knockdown of DDAH2 by siRNA significantly decreased myogenin (MYOG) expression corresponding with the slight change in cell morphology. In contrast, HBA2 was up-regulated during ALC formation and resulted in decreased intracellular lipid accumulation and CD36 mRNA expression upon knockdown assay. CONCLUSION: In this study, a large number of EST sequences were generated from the MSC, MFC and ALC. Overall, the collection of ESTs generated in this study provides a starting point for the identification of novel genes involved in MFC and ALC formation, which in turn offers a fundamental resource to enable better understanding of the mechanism of muscle differentiation and transdifferentiation.

Efficacy of four synchronization protocols on the estrus behavior and conception in native Korean cattle (Hanwoo).

Ineffective estrus detection is the foremost limiting factor in the fertility of farmed cattle worldwide. Failure to detect estrus or erroneous diagnosis of estrus results in great economic losses in Korea each year. This study was carried out in order to comprehensively describe the estrus behaviors and conception rates of different estrus synchronization protocols applied to 40 cycling native Korean cattle (Hanwoo). The cows were grouped into four (n = 10) and treated with the following protocols: (1) Day -15: controlled intravaginal drug-releasing device (CIDR) for 12 days; Day -5: prostaglandin F2α (PGF2α), (2) ovulation synchronization (OVS): Day -15: GnRH; Day -6: PGF2α; Day -4: GnRH, (3) Day -15: progesterone-releasing intravaginal device for 12 days; Day -5: PGF2α; and (4) Day -15: PGF2α; Day -4: PGF2α. Artificial insemination was performed 12 hours after the detection of estrus using frozen-thawed semen. Estrus signs were compared using a charge-coupled device camera (CCDC) and a control method (direct visual observation). The pregnancy of the cows was determined by transrectal ultrasonography at Days 25 to 30 postinsemination. The results indicated that the day of estrus return was significantly earlier using the CCDC method compared with direct visualization (P < 0.05). Mounting of other cows was the most predominant sign of estrus among the flock (P < 0.05), as analyzed using the CCDC. In the OVS group, a lower rate of mounting was observed than in the other three groups. Moreover, significantly fewer estrus behaviors were noticed in the OVS protocol group (P < 0.05). Both first service conception and overall conception rates were significantly higher (P < 0.05) in the CIDR and OVS treatment groups. In conclusion, the CIDR and OVS protocols appear to be the best practice for the synchronization of estrus for reproductive competence through the CCDC in Hanwoo cows. However, CIDR has a practical advantage over OVS with respect to estrus detection.

Transthyretin is a key regulator of myoblast differentiation.

Transthyretin (TTR) is a known carrier protein for thyroxine (T4) and retinol-binding protein in the blood that is primarily synthesized in the liver and choroid plexus of the brain. Herein, we report that the TTR gene is expressed in skeletal muscle tissue and up-regulated during myotube formation in C2C12 cells. TTR silencing (TTRkd) significantly reduced myogenin expression and myotube formation, whereas myogenin silencing (MYOGkd) did not have any effect on TTR gene expression. Both TTRkd and MYOGkd led to a decrease in calcium channel related genes including Cav1.1, STIM1 and Orai1. A significant decrease in intracellular T4 uptake during myogenesis was observed in TTRkd cells. Taken together, the results of this study suggest that TTR initiates myoblast differentiation via affecting expression of the genes involved during early stage of myogenesis and the genes related to calcium channel.

Induction of Ski Protein Expression upon Luteinization in Rat Granulosa Cells.

Ski protein is implicated in proliferation/differentiation in a variety of cells. We had previously reported that Ski protein is present in granulosa cells of atretic follicles, but not in preovulatory follicles, suggesting that Ski has a role in apoptosis of granulosa cells. The alternative fate of granulosa cells other than apoptosis is to differentiate to luteal cells; however, it is unknown whether Ski is expressed and has a role in granulosa cells undergoing luteinization. Thus, the aim of the present study was to locate Ski protein in the rat ovary during luteinizationto predict the possible role of Ski. In order to examine the expression pattern of Ski protein along with the progress of luteinization, follicular growth was induced by administration of equine chorionic gonadtropin to immature female rats, and luteinization was induced by human chorionic gonadtropin treatment to mimic luteinizing hormone (LH) surge. While no Ski-positive granulosa cells were present in preovulatory follicle, Ski protein expression was induced in response to LH surge, and was maintained after the formation of the corpus luteum (CL). Though Ski protein is absent in granulosa cells of preovulatory follicle, its mRNA (c-Ski) was expressed and the level was unchanged even after LH surge. Taken together, these results demonstrated that Ski protein expression is induced in granulosa cells upon luteinization, and suggests that its expression is regulated post-transcriptionally.

Cloning and functional characterization of pig CMP-N-acetylneuraminic acid hydroxylase for the synthesis of N-glycolylneuraminic acid as the xenoantigenic determinant in pig-human xenotransplantation.

In the present study, the pig CMP-N-acetylneuraminic acid hydroxylase gene (pcmah), a key enzyme for the synthesis of NeuGc (N-glycolylneuraminic acid), was cloned from pig small intestine and characterized. The ORF (open reading frame) of pcmah was 1734 bp, encoding 577 amino acids and consisting of 14 exons. Organ expression pattern analysis reveals that pcmah mRNA is mainly expressed in pig rectum, tongue, spleen and colon tissues, being the most highly expressed in small intestine. In the ectopic expression of pcmah, when pig kidney PK15 cells and human vascular endothelial ECV304 cells were transfected with the cloned pcmah, the NeuGc contents of these transfectants were greater in comparison with vector transfectants used as controls. In addition, in the functional analysis of NeuGc, HSMC (human-serum-mediated cytotoxicity) was elevated in the ectopic NeuGc-expressing pcmah-transfected cells compared with controls. Moreover, binding of human IgM to the pcmah-transfected cells was significantly increased, whereas binding of IgG was slightly increased, indicating that the human IgM type was a major anti-NeuGc antibody. Furthermore, pcmah silencing by shRNA (short hairpin RNA) resulted in a decrease in NeuGc content and xenoantigenicity in PK15. From the results, it was concluded that the pcmah gene was capable of synthesizing the NeuGc acting as a xenoantigen in humans, confirming the NeuGc-mediated rejection response in pig-human xenotransplantation.

Osmolarity at early culture stage affects development and expression of apoptosis related genes (Bax-alpha and Bcl-xl) in pre-implantation porcine NT embryos.

This study examined whether high osmolarity of culture medium at the early culture stage affects development and expression of apoptosis related genes (Bax-alpha and Bcl-xl) of porcine nuclear transfer (NT) and in vitro fertilization (IVF) embryos. NT and IVF embryos were divided into three groups and the basic medium was PZM-3 (260-270 mOsmol, control group). The control group of embryos was cultured in PZM-3 for whole culture period. Other two groups of embryos were cultured in a modified PZM-3 with 0.05 M sucrose (300-320 mOsmol, sucrose group) or increased NaCl to 138 mM (300-320 mOsmol, NaCl group) for the first 2 days, and then cultured in PZM-3 for 4 days. NT embryos cultured in NaCl group showed a significantly higher developmental rate to the blastocyst stage with a decreased apoptosis rate compared to the control (P < 0.05). There was no difference in blastocyst formation and apoptosis incidence among the three culture treatments for IVF-derived embryos. Bax-alpha mRNA expression was significantly higher in the control than sucrose or NaCl group for both NT and IVF embryos (P < 0.05). Moreover, the relative abundance of Bax-alpha/Bcl-xl was higher in the control than the treatment groups. These results indicate that the higher osmolarity at the early embryonic stage of porcine NT and IVF embryos can improve the in vitro development with reduced apoptosis through regulating the Bax-alpha/Bcl-xl gene expression.

Development of vitrified-thawed bovine oocytes after in vitro fertilization and somatic cell nuclear transfer.

Cryopreservation could be a useful technique for providing a steady source of oocytes for nuclear transfer and in vitro embryo production. The purpose of this study was to develop a method for cryopreservation of bovine oocytes while maintaining the developmental potential following subsequent in vitro fertilization (IVF) or somatic cell nuclear transfer (SCNT). Following vitrification-thawing, the surviving oocytes were (a) used for parthenogenetic activation, (b) examined for pronuclear formation after IVF, (c) examined for embryo development after IVF, and (d) used for SCNT employing fetal fibroblasts transfected with green fluorescent protein (GFP) gene. While most of the oocytes survived vitrification when the microdrop method was used (92.50%), the cleavage and blastocyst formation rates after parthenogenetic activation were lower (46.5% and 11.1%) than that in the non-vitrified control (86.6% and 13.5%). After IVF, the pronuclear formation (2PN) of fertilized embryos was lower in the vitrified group than in the control (21.7% and 59.9%). After SCNT, fusion rates were similar in control (58.33%) and vitrified-thawed oocytes (53.19%). However, the cleavage (73.1% and 46.3%) and blastocyst formation rates (22.2%, 7.4%; p<0.05) differed between control and vitrified-thawed oocytes. In vitrified-thawed or control oocytes, all embryos reconstructed using fetal fibroblasts transfected with GFP gene showed GFP expression. To evaluate the complete developmental potential, embryos derived from vitrified-thawed and fresh control oocytes were non-surgically transferred to 27 recipients (16 for control and 11 for vitrified-thawed). In the vitrified-thawed group, two pregnancies were detected at day 60, and one of them lasted until day 222. While in the fresh group, one pregnancy maintained to term. In conclusion, vitrified-thawed bovine oocytes could support development into the subsequent stages after IVF and SCNT. In addition, this study showed the possibility of the vitrified-thawed bovine oocytes in the production of transgenic cloned animals. In addition, further studies are required to increase the efficiency of oocyte vitrification for the practical uses and production of live offspring.

Vitrification of immature bovine oocytes by the microdrop method.

This study was conducted to determine the optimal vitrification conditions for immature bovine oocytes using the microdrop method. In experiment 1, the optimal pre-equilibration period for microdrop vitrification was examined. The maturation rate of vitrified oocytes with a 3 min first pre-equilibration period (41.1%) was higher than that of vitrified oocytes with a 0 min first pre-equilibration period (21.4%), and the values of those with a 1 (33.9%) or 5 min (27.4%) first pre-equilibration period were intermediate. The value for a 1 min second pre-equilibration period (44.4%) was significantly higher (P<0.05) than those for a 0.5 (28.6%) and 2 min (21.4%) second pre-equilibration period. In experiment 2, the distribution of microtubules in matured oocytes was investigated. There was no difference among the first pre-equilibration times in terms of the rates of normal spindles in vitrified oocytes. However, this value was significantly higher (P<0.05) in the 1 min group (52.8%) compared with the 0.5 (16.7%) and 2 min groups (12.3%). In experiment 3, we investigated the developmental capacity of immature bovine oocytes vitrified under optimal pre-equilibration conditions (3 min and 1 min for the first and second pre-equilibrations, respectively). Although the total fertilization rates were significantly lower (P<0.05) in the vitrified oocytes (65.6%) compared with the control oocytes (92.4%), there was no difference in the rate of normal fertilization (2PN) between the vitrified (78.6%) and control (82.0%) oocytes. Cleavage and blastocyst rates were significantly lower (P<0.05) in vitrified oocytes (55.7 and 2.3%) than in control oocytes (84.4 and 34.7%). Thus, these results indicated that immature bovine oocytes can survive after microdrop vitrification and subsequently can be cultured to mature oocytes capable of undergoing fertilization in vitro and developing into blastocysts.

Development and apoptosis of pre-implantation porcine nuclear transfer embryos activated with different combination of chemicals.

Artificial activation of oocytes is a pre-requisite for successful cloning by nuclear transfer (NT). This study investigated effect of different combination of activation chemicals such as electric pulse (E), thimerosal (Thi) + dithiothreitol (DTT), 6-dimethylaminopurine (6-DMAP), or cycloheximide (CH) on the developmental ability and the frequency of apoptosis of porcine NT embryos during the culture in vitro. NT embryos activated with chemicals showed significantly higher developmental rate to blastocyst stage compared to embryos activated with E alone (21.5%-26.6% vs. 15.7%, respectively). Of chemicals, Thi + DTT supported higher development to blastocyst stage as compared to 6-DMAP or CH (26.6% vs. 21.5%-23.4%, respectively). Apoptosis of NT embryos were analyzed by using a terminal deoxynucleatidyl transferase-mediated deoxyuridine 5-triphosphate nick-end labeling (TUNEL) assay. The onset of apoptosis of embryos activated E alone was on Day 4, whereas embryos activated with chemicals showed apoptosis on Day 3 post-activation NT embryos exposed to chemicals for activation had higher frequency of apoptosis compared to that of embryos exposed to E alone from Day 3 to Day 7 during the culture. In conclusion, this study shows that chemical activation after fusion could increase not only the developmental ability of porcine NT embryos but also the mean cell number with an increased ratio of inner cell mass (ICM) to trophectoderm (TE) cells. However, the chemical activation also could increase the frequency of apoptosis and induced apoptosis earlier in porcine NT embryos.

Fragmentation and development of preimplantation porcine embryos derived by parthenogenetic activation and nuclear transfer.

Fragmentation occurs during early developmental stages of electrically activated oocytes and nuclear transfer (NT) embryos. It might contribute to the low developmental rate of porcine NT embryos. The present study was conducted to investigate whether the addition of sugars such as sorbitol or sucrose suppresses fragmentation and supports the development of electrically activated oocytes and NT embryos. The activated oocytes were cultured in Porcine Zygote Medium-3 (PZM-3) supplemented with sorbitol or sucrose for 2 days after electric activation, and then cultured in the PZM-3 for the remaining 4 days. The osmolarities of PZM-3, PZM-3 supplemented with 0.05 or 0.1 M sorbitol, and PZM-3 with 0.05 M sucrose were 269 +/- 6.31, 316 +/- 3.13, 362 +/- 4.37, and 315 +/- 5.03 mOsm, respectively. When parthenogentically activated oocytes were cultured in PZM-3 supplemented with 0.05 M sorbitol or sucrose for the first 2 days and then cultured in PZM-3 without sugar, a significantly higher (P < 0.05) cleavage rate and blastocyst rate were observed. Interestingly, addition of sugar to PZM-3 for 2 days reduced the fragmentation rate compared to PZM-3 without sugar. In NT embryos, sugar addition into PZM-3 increased the fusion rate (84.2% +/- 6.07 vs. 95.1% +/- 2.52), cleavage rate (67.6% +/- 5.80 vs. 77.3% +/- 3.03), and developmental rate to the blastocyst stage (10.2% +/- 0.79 vs. 19.4% +/- 1.77). There was no significant difference between treatments for the number of the blastocysts. In addition the fragmentation rate was reduced compared to PZM-3 without sorbitol (26.1 +/- 4.30 vs. 14.5 +/- 1.74). In conclusion, increasing the osmolarity of PZM-3 through addition of either sorbitol or sucrose for 48 hr increased the cleavage and developmental rate to the blastocyst stage by reducing the fragmentation rate through increasing osmolarity.