Cloning whole animals with somatic cells as parents offers the possibility of targeted genetic manipulations in vitro such as ''gene knock-out'' by homologous recombination. However, such manipulation requires prolonged culture of nuclear donor cells. Previous successes in cloning have been limited to the use of cells collected either fresh or after short-term culture. Therefore, demonstration of genetic totipotency of cells after prolonged culture is pivotal to combining site-specific genetic manipulations and cloning. Here we report birth of six clones of an aged (17-year-old) Japanese Black Beef bull using ear skin fibroblast cells as nuclear donor cells after up to 3 months of in vitro culture (10 -15 passages). We observed higher developmental rates for embryos derived from later passages (10 and 15) as compared with those embryos from an early passage (passage 5). The four surviving clones are now 10 -12 months of age and appear normal, similar to their naturally reproduced peers. These data show that fibroblasts of aged animals remain competent for cloning, and prolonged culture does not affect the cloning competence of adult somatic donor cells. G enetic manipulation of mouse embryonic stem cells has revolutionized mouse genetic research. However, embryonic stem cells are not available in other species. Fortunately, animal cloning using cultured somatic cells offers the possibility of targeted genetic manipulations like those performed in the mouse, should those somatic cells remain competent for cloning after prolonged culture. Live clones have been obtained from adult somatic cells in sheep (1), mice (2), and cows (3, 4). Furthermore, transgenic animals have been produced by cloning gene-transfected fetal somatic donor cells (5, 6). However, to date, successful somatic cell cloning has been largely limited to the use of the donor cells either fresh (2) or after short-term (under 10 passages) in vitro culture (1, 3-6), which would not allow targeted gene manipulations.A recent report (7) indicates that Dolly, the cloned sheep, inherited the shortened telomeres of the adult nuclear donor animal. Moreover, the telomeres of Dolly were further shortened during the brief in vitro culture of the donor cells. These observations raise the questions of whether healthy clones may be obtained from aged donor animals, particularly after longterm cultures of the ''aged'' donor cells. This study was conducted to test the cloning competence of skin fibroblast cells after prolonged in vitro culture, using an aged (17-year-old) elite bull. In this paper, we report that normal live clones were produced from cultured adult somatic cells in a cattle model after up to 3 months of culture (passage 15). Our finding offers promise for producing site-specific genetically modified animals such as ''gene knockout'' animals by somatic cell cloning. Additionally, success in cloning live, aged animals opens the possibility to compare the telomere lengths, aging, and the ''biological age'' of the cloned animals. Materials and Methods...

Successful somatic cloned animal production has been reported in various domesticated species, including cattle; however, it is associated with a high rate of pregnancy failure. The low cloning yield could possibly arise from either an abnormal and/or poorly developed placenta. In comparison to control cows, fewer placentomes were found in somatic cell nuclear recipient (NT) cows at day 60 of gestation, suggesting a retardation of fetal/placental growth in these animals. NT cows not only had fewer numbers of chorionic villi but also had poorly developed caruncles. Macroscopic examination revealed atypical development of the placentome in terms of shape and size. Histological disruption of chorionic villi and caruncular septum was found in NT cows. Of particular interest was that the expression of genes, as well as proteins in the placentome, was disparate between NT and artificially inseminated cows, especially placental lactogen (PL) and pregnancy-associated glycoprotein (PAG). In contrast, prolactin-related protein-1 (PRP-1) signals were comparable across cows, including NT cows carrying immotile fetuses. The expression of extracellular matrix degrading molecule, heparanase (HPA), in NT cows was divergent from that of control cows. Microarray data suggest that gene expression was disorientated in early stages of implantation in NT cows, but this was eliminated with progression of gestation. These findings strongly support a delay in trophoblast development during early stages of placentation in NT cows, and suggest that placental specific proteins, including PLs, PAGs, and HPA, are key indicators for the aberration of gestation and placental function in cows.

The aims of this study were to develop a sensitive and specific assay for bovine inhibin A using europium and to investigate the endocrine role of inhibin A in various reproductive conditions by characterizing the relationship between profiles of inhibin A, FSH, and estradiol and follicle growth during the postpartum period, during the intact estrous cycle, and in cows with follicular cysts. The time-resolved immunofluorometric assay (Tr-IFMA) for bovine inhibin A, using purified polyclonal antibodies to alpha and beta(A) subunits, was specific for bovine inhibin A and did not cross-react with bovine activin A, activin AB, activin B, pro-alphaC or human recombinant inhibin B. The detection limit of the IFMA was 3.3 pg/ml expressed in terms of bovine 32-kDa inhibin A. Dose-response curves of plasma samples obtained from intact and FSH-stimulated cows and cystic cows were parallel to the standard without any preassay processing of samples. Plasma inhibin A levels increased (P < 0.01) concomitant with emergence of nonovulatory or ovulatory follicular waves during the postpartum period. In cystic cows, plasma inhibin A was sustained at high levels for a longer period, associated with growth of persistent dominant follicles. The highest levels of inhibin A were noted during the growth phase of normal and persistent dominant follicles; however, inhibin A levels declined (P < 0.01) as these dominant follicles ceased to grow or ovulated. An inverse relationship between patterns of plasma inhibin A and FSH existed during each follicular wave in the three physiologic conditions. Increases in plasma inhibin A levels were associated with increases in plasma estradiol levels during most follicular waves; however, there was no increase in plasma estradiol level and no relationship between patterns of estradiol and FSH during follicular waves observed during the early postpartum period or midluteal phase of the estrous cycle. In conclusion, the Tr-IFMA does not require pretreatment of samples and can be used for precise measurement of bovine inhibin A without interference with free inhibin alpha subunits. Inhibin A, produced primarily during growth of the dominant follicle, functions as a negative feedback regulator for FSH secretion throughout the postpartum period and the estrous cycle, whereas estradiol appears to have a minor role in regulation of FSH compared with inhibin A, especially during the early postpartum period and midluteal phase of the estrous cycle. The results also indicate that a persistent dominant follicle sustains inhibin A production for a longer period than the dominant follicle emerging in the estrous cycle and establishes long-term dominance by suppressing emergence of a new follicular wave.

We used a half-sib family of purebred Japanese Black (Wagyu) cattle to locate economically important quantitative trait loci. The family was composed of 348 fattened steers, 236 of which were genotyped for 342 microsatellite markers spanning 2,664 cM of 29 bovine autosomes. The genome scan revealed evidence of 15 significant QTL (<5% chromosome-wise level) affecting growth and carcass traits. Of the 15 QTL, six QTL were significant at the 5% experiment-wise level and were located in bovine chromosomes (BTA) 4, 5, and 14. We analyzed these three chromosomes in more detail in the 348 steers, with an average marker interval of 1.2 cM. The second scan revealed that the same haplotype of the BTA 4 region (52 to 67 cM) positively affected LM area and marbling. We confirmed the QTL for carcass yield estimate on BTA 5 in the region of 45 to 54 cM. Five growth-related QTL located on BTA 14, including slaughter and carcass weights, were positively affected by the same region of the haplotype of BTA 14 (29-51 cM). These data should provide a useful reference for further marker-assisted selection in the family and positional cloning research. The research indicates that progeny design with moderate genotyping efforts is a powerful method for detecting QTL in a purebred half-sib family.

ResearchThe bovine placenta secretes multiple molecules during implantation and placentation, many of which are produced by binucleate cells. In this study, production of prolactin-related protein I (PRP-I), a member of the nonclassical prolactin-related family, was investigated during the implantation period in cows. Expression of bovine PRP-I (bPRP-I) in the placentome was examined during the preimplantation (days 17-19), implantation (days 20-25) and post-implantation (days 30-60) periods by immunohistochemistry, immunofluorescence and in situ hybridization. During the preimplantation period, both bPRP-I and bovine placental lactogen (bPL) were