Effect of α-tocopherol on lactone formation in marbled beef and changes in lactone volatility during storage.

Dynamic-headspace sampling with a standard-addition method was employed to quantitatively analyze aliphatic lactones in rendered fat from marbled beef and to evaluate the effect of the matrix on volatility. Further, the effects of different levels of the antioxidant α-tocopherol on lactone formation were examined. The slopes of the linear regression curves from the standard-addition method were significantly changed (P < 0.05 or 0.01) for all lactones after storage, with the exception of γ-octalactone, indicating the volatility of the longer-chain lactones were increased after storage. The concentrations of γ-lactones were increased after 7 d of storage at 2 °C (P < 0.01), and the α-tocopherol content in the meat affected the formation of γ-octalactone (P < 0.05) and γ-nonalactone (P < 0.01). The greatest increase was observed for γ-nonalactone in the lowest α-tocopherol (2.9 ppm) group: the concentration of 51.4 ppb was 11.7-fold higher than that before storage. Meanwhile, δ-tetradecalactone in the highest α-tocopherol (28.8 ppm) group showed the highest concentration for the lactones at 415.8 ppb, which was 2.6-fold higher than the concentration before storage. The effect of α-tocopherol was unclear for the δ-lactones. The results indicate that most of the γ-lactones are produced by oxidation during storage but that the other lactones are also generated by other processes as well.

Cloned cows with short telomeres deliver healthy offspring with normal-length telomeres.

Dolly, the first mammal cloned from a somatic cell, had shorter telomeres than age-matched controls and died at an early age because of disease. To investigate longevity and lifetime performance in cloned animals, we produced cloned cows with short telomeres using oviductal epithelial cells as donor cells. At 5 years of age, despite the presence of short telomeres, all cloned cows delivered multiple healthy offspring following artificial insemination with conventionally processed spermatozoa from noncloned bulls, and their milk production was comparable to that of donor cows. Moreover, this study revealed that the offspring had normal-length telomeres in their leukocytes and major organs. Thus, cloned animals have normal functional germ lines, and therefore germ line function can completely restore telomere lengths in clone gametes by telomerase activity, resulting in healthy offspring with normal-length telomeres.

Relationships of survival time, productivity and cause of death with telomere lengths of cows produced by somatic cell nuclear transfer.

The reproductive ability, milk-producing capacity, survival time and relationships of these parameters with telomere length were investigated in 4 groups of cows produced by somatic cell nuclear transfer (SCNT). Each group was produced using the same donor cells (6 Holstein (1H), 3 Holstein (2H), 4 Jersey (1J) and 5 Japanese Black (1B) cows). As controls, 47 Holstein cows produced by artificial insemination were used. The SCNT cows were artificially inseminated, and multiple deliveries were performed after successive rounds of breeding and conception. No correlation was observed between the telomere length and survival time in the SCNT cows. Causes of death of SCNT cows included accidents, accident-associated infections, inappropriate management, acute mastitis and hypocalcemia. The lifetime productivity of SCNT cows was superior to those of the controls and cell donor cows. All SCNT beef cows with a relatively light burden of lactation remained alive and showed significantly prolonged survival time compared with the cows in the SCNT dairy breeds. These results suggest that the lifetime productivity of SCNT cows was favorable, and their survival time was more strongly influenced by environmental burdens, such as pregnancy, delivery, lactation and feeding management, than by the telomere length.

Characterization of a donor mitochondrial DNA transmission bottleneck in nuclear transfer derived cow lineages.

In embryos derived by nuclear-transfer (NT), fusion of donor cells with recipient oocytes resulted in varying patterns of mitochondrial DNA (mtDNA) transmission in NT animals. Distribution of donor cell mtDNA (D-mtDNA) found in offspring of NT-derived founders may also vary from donor cell and host embryo heteroplasmy to host embryo homoplasmy. Here we examined the transmission of mtDNA from NT cows to G(1) offspring. Eleven NT founder cows were produced by fusion of enucleated oocytes (Holstein/Japanese Black) with Jersey/ Holstein oviduct epithelial cells, or Holstein/Japanese Black cumulus cells. Transmission of mtDNA was analyzed by PCR mediated single-strand conformation polymorphism of the D-loop region. In six of seven animals sampled postmortem, heteroplasmy were detected in various tissues, while D-mtDNA could not be detected in blood or hair samples from four live animals. The average proportion of D-mtDNA detected in one NT cow was 7.6%, and those in other cows were <5%. Heteroplasmic NT cows (n = 6) generated a total 12 G(1) offspring. Four of 12 G(1) offspring exhibited high percentages of D-mtDNA populations (range 17-51%). The remaining eight G(1) offspring had slightly or undetectable D-mtDNA (<5%). Generally, a genetic bottleneck in the female germ-line should favor a homoplasmic state. However, proportions of some G(1) offspring maintained heteroplasmy with a much higher percentage of D-mtDNA than their NT dams, which may also reflect a segregation distortion caused by the proposed mitochondrial bottleneck. These results demonstrate that D-mtDNA in NT cows is transmitted to G(1) offspring with varying efficiencies.

Comparison of T cell subsets between somatic cloned and normal cow.

PROBLEM: Somatic cloning technology is beneficial for genetically producing excellent animals. However, many developmental problems of somatically cloned animals have been described. Some of them may cause disorders of the immune system, resulting in the fluctuation of the proportion of white blood cells (WBC), different from that of normal animals in peripheral blood. METHOD OF STUDY: In Holstein- cloned and normal cows, the fluctuation of granulocytes, monocytes, B cells and T cells, and further T cell subsets (CD4+, CD8+, gammadelta, CD8+gammadelta and WC1+gammadelta T cell) in peripheral blood were analyzed in early lactation stage (ELS) and mid to late lactation stage (MLS) by flow cytometry using specific monoclonal antibodies for cell surface markers. RESULTS: In both ELS and MLS, there were no significant differences in the proportions of granulocytes, monocytes, B cells and T cells between cloned and normal cows. In T cell subsets, gammadelta and WC1+gammadelta T cells in cloned cows were significantly less frequent than in normal cows in ELS. The decreased proportions of gammadelta and WC1+gammadelta T cells recovered to the level of normal cows in MLS. CONCLUSIONS: The population of granulocytes, monocytes, B cells and T cells, and T cell subsets except for gammadelta and WC1+gammadelta T cells in cloned cows fluctuated in a manner similar to those of normal cows during lactation. In ELS, the proportions of gammadelta and WC1+gammadelta T cells temporarily declined in cloned cows, suggesting that cloned cows may fall into an immunosuppressive state in ELS.

Remarkable differences in telomere lengths among cloned cattle derived from different cell types.

Regarding cloned animals, interesting questions have been raised as to whether cloning restores cellular senescence undergone by their donor cells and how long cloned animals will be able to live. Focusing our attention on differences in telomere lengths depending on the tissue, we had produced 14 cloned cattle by using nuclei of donor cells derived from muscle, oviduct, mammary, and ear skin. Here, we show remarkable variation in telomere lengths among them using Southern blot analysis with telomere-specific probe. Telomere lengths in cloned cattle derived from muscle cells of an old bull were longer than those of a donor animal but were within the variation in normal calves. On the other hand, those derived from oviductal and mammary epithelial cells of an equally old cow were surprisingly shorter than any found in control cattle. The telomere lengths of cloned cattle derived from fibroblasts and oviductal epithelial cells of younger cattle showed the former and the latter results, respectively. In both cases, however, less telomere erosion or telomere extension from nuclear transfer to birth in most cloned cattle was observed in comparison with telomere erosion from fertilization to birth in control cattle. Embryonic cell-cloned cattle and their offspring calves were also shown to have telomeres longer than those in age-matched controls. These observations indicate that cloning does not necessarily restore the telomere clock but, rather, that nuclear transfer itself may commonly trigger an elongation of telomeres, probably more or less according to donor cell type. Remarkable variations among cloned cattle are suggested to be caused by variation in telomere length among donor cells and more or less elongation of telomere lengths induced by cloning.