In mammals, epigenetic marks on the X chromosomes are involved in dosage compensation. Specifically, they are required for X chromosome inactivation (XCI), the random transcriptional silencing of one of the two X chromosomes in female cells during late blastocyst development. During natural reproduction, both X chromosomes are active in the female zygote. In somatic-cell cloning, however, the cloned embryos receive one active (Xa) and one inactive (Xi) X chromosome from the donor cells. Patterns of XCIhave been reported normal in cloned mice, but have yet to be investigated in other species. We examined allele-specific expression of the X-linked monoamine oxidase type A (MAOA) gene and the expression of nine additional X-linked genes in nine cloned XX calves. We found aberrant expression patterns in nine of ten X-linked genes and hypomethylation of Xist in organs of deceased clones. Analysis of MAOA expression in bovine placentae from natural reproduction revealed imprinted XCI with preferential inactivation of the paternal X chromosome. In contrast, we found random XCI in placentae of the deceased clones but completely skewed XCI in that of live clones. Thus, incomplete nuclear reprogramming may generate abnormal epigenetic marks on the X chromosomes of cloned cattle, affecting both random and imprinted XCI.

Juvenile calf oocytes represent an untapped source of germ plasm for reproduction. Reports on the developmental competence of calf oocytes have been controversial. In this research, oocytes were recovered after gonadotropin stimulation from Holstein calves (N = 10) at 2-3 mo of age (2-mo cycle) and again at 4-5 mo of age (4-mo cycle). The in vitro developmental competence was measured, and prestimulation follicle numbers (for 2-mo cycle) and poststimulation follicle numbers (both cycles) were obtained. The number of antral follicles doubled after stimulation (23.4 +/- 6.1 vs. 55.1 +/- 16.1) for the 2-mo cycle and for the 4-mo cycle (47.4 +/- 12.4). The number of follicles observed prior to stimulation in the 2-mo cycle was found to be highly correlated with the poststimulation oocyte recovery for both collection cycles (r = 0.95, 2-mo cycle; r = 0.81, 4-mo cycle). The majority (90-96%) of recovered oocytes were found to be usable for in vitro maturation and fertilization; of these, 41-42% cleaved and 10-11% developed to morulae or blastocysts. Eighty-four in vitro-produced embryos were transferred to synchronized recipients and resulted in 11 pregnancies, leading to 7 live (4 males, 3 females) and 2 dead (one male, one female) calves at full term. No significant differences were observed between the 2-mo and 4-mo collection cycles; however, 73% of the total pregnancies resulted from the 2-mo cycle. All pregnancies resulted from embryos of high-responding donors. The high correlation between the number of follicles prior to stimulation and the poststimulation response suggests the possibility of screening calves prior to stimulation for routine embryo production.

Cumulus-oocyte complexes, 5596, were cultured for 24 h in either TCM-199 or Ham's F-10 with or without gonadotrophins and supplemented with either 20% buffalo oestrous serum (BES) or fetal calf serum (FCS). The maturation rates of oocytes cultured in TCM-199 or Ham's F-10 medium supplemented with 20% BES were 47.4 +/- 17.8 and 44.8 +/- 25.6, respectively. Addition of luteinizing hormone (LH) (5 micrograms ml-1) significantly improved the maturation rate in the Ham's F-10 medium supplemented with 20% BES (76.8 +/- 18.3), but follicle-stimulating hormone (FSH) (0.5 micrograms ml-1) and oestradiol (1 microgram ml-1) failed to synergize with LH (71.7 +/- 19.5). In the TCM-199 system, LH failed to enhance the maturation rate but the addition of FSH and oestradiol significantly enhanced the proportion of mature oocytes (42.7 +/- 1.4 and 81.7 +/- 14.5, respectively; P less than 0.05). Frozen-thawed spermatozoa prepared in Bracket and Oliphant (BO) medium and treated with 5 mmol caffeine 1(-1) + 10 micrograms heparin showed a higher fertilization rate (29.8%) than those treated in Hepes-Talp and treated with 10 micrograms heparin ml-1 (19.6%). Fertilization rate was significantly improved when fresh ejaculated spermatozoa treated with 5 mmol caffeine 1-1 and 10 micrograms heparin in BO medium (50%) was used. Rate of cleavage and development were also higher when in vitro fertilization was carried out with fresh ejaculated spermatozoa treated with caffeine and heparin (34.1 and 36.8%, respectively) than with frozen-thawed spermatozoa (27.0 and 22.0%, respectively). Development rate was enhanced when fertilized ova were cultured in ligated rabbit oviduct (28.0%) than when co-cultured on oviductal cell monolayers (8.2%). The results indicate that oocytes cultured in medium supplemented with BES and gonadotrophins reveal high rates of maturation and development to the blastocyst stage after fertilization with fresh ejaculated spermatozoa.

Cattle are the species used most frequently for the development of assisted reproductive technologies, such as nuclear transfer. Cattle cloning can be performed by a large number of laboratories around the world, and the efficiency of nuclear transfer in cattle is the highest among 25 all species in which successful cloning has been achieved. However, an understanding of the expression of imprinted genes in this important species is lacking. In the present study, real time reverse-transcription polymerase chain reaction (RT-PCR) was utilized to quantify the expression of the bovine Igf2, Igf2r and H19 genes in eight major organs (brain, bladder, heart, kidney, liver, lung, spleen and thymus) of somatic cell cloned calves that died shortly after birth, 30 in three tissues (skin, muscle and liver) of healthy clones that survived to adulthood, and in corresponding tissues of control animals from natural reproduction. We found that, deceased bovine cloned calves exhibited abnormal expression of all three genes studied in various organs.Large variations in the expression levels of imprinted genes were also seen among these clones, which were produced from the same genetic donor. In surviving adult clones, however, the 35 expression of these imprinted genes was largely normal, except for the expression of the Igf2 gene in muscle, which was highly variable. Our data showed disruptions of expression of imprinted genes in bovine clones, which is possibly due to incomplete reprogramming of donor cell nuclei during nuclear transfer, and these abnormalities may be associated with the high neonatal mortality in cloned animals; clones that survived to adulthood, however, are not only 40 physically healthy but also relatively normal at the molecular level of those three imprinted genes.3

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