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.

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.

Although it is well known that mRNA is present in mammalian spermatozoa, the relevance of mRNA to capacitation and early embryo development in the pig remains unclear. In the present study, we investigated differences in the abundance of selected mRNAs coding for MYC, CYP19, ADAM2, PRM1 and PRM2 in purified porcine spermatozoa depending on embryo cleavage rate and capacitation (n=20 semen samples). Semen samples were used in IVF procedures, with subsequent embryo development classified into one of two groups based on cleavage rate (i.e. high (>75%) and low (<75%) cleavage groups) and mRNA abundance in purified spermatozoa compared between these two groups. In addition, mRNA abundance was compared between capacitated and non-capacitated spermatozoa. Comparison of mRNA levels between porcine spermatozoa revealed that the abundance of MYC, CYP19, ADAM2, PRM1 and PRM2 mRNA was significantly greater in the high cleavage group (n=10 high cleavage group semen samples) than in the low cleavage group (n=10; P<0.05). Significant downregulation of MYC mRNA was observed in capacitated spermatozoa (n=12; P<0.05). The results of the present study suggest that the amount of specific mRNAs could be used for estimating the quality of spermatozoa in the pig.

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.

Abstract. 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 preequilibration period (41.1%) was higher than that of vitrified oocytes with a 0 min first preequilibration period (21.4%), and the values of those with a 1 (33.9%) or 5 min (27.4%) first preequilibration 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 preequilibration 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 preequilibration 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.

The present study aimed to evaluate the effect of methyl-β-cyclodextrin (MβCD) as a cholesterol loader to change oocyte plasma membrane and increase its tolerance toward cryopreservation. The first and second experiments were conducted to investigate if MβCD could improve nuclear and cytoplasmic maturation after oocyte exposure to cold stress for 10 or 30 min, respectively. No differences (P>0.05) in either experiment in the metaphase II (MII) rate of oocytes exposed to MβCD and cold stress; but these oocytes presented lower maturation rates than control groups. In the second experiment, a lower percentage of oocytes showed degenerated chromatin (P<0.05) after exposure to 2mg/mL of MβCD compared to the group exposed to 0mg/mL. However, no differences among treatments were observed in cytoplasmic maturation. Groups exposed to cold stress demonstrated a lower (P<0.05) capacity for embryonic development compared to the control groups. In the third experiment immature oocytes were exposed to MβCD and then, vitrified (cryotop). After warming, we observed that the ability to reach MII and chromatin degeneration were altered (P<0.05) by MβCD. The blastocysts rate (P<0.05) on D7 was higher in the 2 mg/mL MβCD group, but an identical finding was not observed on D8 (P>0.05). Chromatin degeneration was higher in the vitrification groups. We conclude that MβCD improved nuclear maturation by reducing oocyte degeneration after cold stress or vitrification; however, more studies are required to clarify the usefulness of MβCD use in oocyte cryopreservation.

Purpose We analyzed the sex chromosome-encoding ZFX-ZFY genes and tested molecular sexing using the amplification patterns of intron 9 of ZFX-ZFY in the horse. Methods and resultsThe amplification of the ZFX-ZFY produced two distinct patterns, reflecting sexual dimorphism based on a length difference between the X and Y chromosomes. The amplification products from foals showed two distinct bands: one was common to all foals and mares, indicating that this band was amplified from ZFX, while the other was specific to some foals, indicating that it was from ZFY. The result based on the PCR assay was identical to the results of amplification of the Y chromosome-specific SRY gene and those of investigations of the phenotypic gender in three different horse populations. Conclusion We suggest that this PCR strategy for determining sexes by comparing the amplification patterns of ZFX-ZFY genes is a convenient and precise method for discriminating sexes in horses.Most mammals have an X-Y chromosome sex determination system. Sexual differentiation is determined primarily by the presence or absence of the Y chromosome, and the sex determining region Y (SRY) gene plays a key role in inducing male development, while inhibiting female differentiation [1][2][3].Control of the sex ratio of animals is desirable in the livestock industry. In mammals, the X-Y homologous sequences for the X-and Y-linked genes zinc finger, amelogenin-X and -Y (AMELX-AMELY), and SMCX/SMCY have been compared [4][5][6]. PCR assays for molecular sexing are often more useful because they provide sensitive, precise, rapid, and reliable results. To date, PCR-based sex typing has been used for embryonic sexing in cattle, mice, and pigs [7][8][9][10][11][12]. To establish a rapid and precise molecular sexing method using PCR amplification for horse breeding, we analyzed the sequence characteristics and sexual dimorphism of the ZFX and ZFY genes and compared the results to those obtained from Y chromosome-specific SRY gene amplification and phenotypic investigation.Total of 128 genomic DNA samples were collected from three horse populations (61 Thoroughbreds, 20 Tsushima native horses from Japan, and 47 Jeju native horses from South Korea). To amplify the intron 9 of ZFX and ZFY genes, the primer pair was designed from exon 8 and 10 of the sequences of human and cattle [13]. The primer Capsule Length difference was found in PCR amplification products between equine ZFX and ZFY genes. This sexual dimorphism may be a diagnostic molecular marker for determining the sexes in the horse.

Genetic polymorphisms in the glycogen debrancher enzyme (AGL) gene were assessed with regard to their association with growth and carcass traits in the F2 population crossbred Landrace and Jeju (Korea) Black pig. Three genotypes representing the insertion and/or deletion (indel) polymorphisms of short interspersed nuclear element were detected at frequencies of 0.278 (L/L), 0.479 (L/S), and 0.243 (S/S), respectively. The AGL S allele-containing pigs evidenced significantly heavier body weights at birth, the 3rd week, 10th week, and 20th week during developmental stages and higher average daily gains during the late period than were noted in the L/L homozygous pigs (P < 0.05), respectively. However, average daily gains during the early period were not significantly associated with genotype distribution (P > 0.05). With regard to the carcass traits, the S allele pigs (S/-) evidenced significantly heavier carcass weights and thicker backfat than was measured in L/L homozygous pigs (P < 0.05). However, body lengths, meat color, and marbling scores were all found not to be statistically significant (P > 0.05). Consequently, the faster growth rate during the late period and backfat deposition rather than intramuscular fat deposition cause differences in pig productivity according to genotypes of the AGL gene. These findings indicate that the AGL genotypes may prove to be useful genetic markers for the improvement of Jeju Black pig-related crossbreeding systems.