Category: Hybrid Fetuses

Here we demonstrate the development of a B. gaurus/B. taurus interspecies model for the analysis of genomic imprinting and DNA methylation in midgestation clones produced by somatic cell nuclear transfer. Interspecies NT has previously been used to generate live offspring in the equine (Equus asinus/Equine cabullus), ovine (Ovis orientalis musimo/Ovine aries), feline (Felis silvestris/ Feline catus) (unpublished), and bovine (B. gaurus/B. taurus and Bos javenicus/Bos taurus). The status of imprinted genes in animals derived from nuclear transfer has been widely investigated in mice; however, to our knowledge, this is the first report on the status of imprinting in cattle derived from nuclear transfer. Results presented from these experiments demonstrate loss of imprinting (LOI) of the XIST locus within the chorion of all pregnancies (n = 3) generated and additionally revealed improper reprogramming of the satellite I and epidermal cytokeratin promoter in the chorion.

The interspecies model generated for this experiment is ideal for identifying imprinted genes and subsequent analysis in NT-derived fetuses. Interspecies models have been used extensively in mice for the identification of imprinted genes due to their increased levels of SNPs that facilitate parental discrimination of alleles in the offspring. The described B. gaums X B. taurus interspecies models is ideal in that it allows identification of SNPs in the absence of any embryonic and fetal abnormalities. Previously, we sequenced the coding regions of 20 genes reported as imprinted in humans and mice in DNA obtained from B. gaums and B. taurus. This facilitated the identification of SNPs in the IGF2, GTL2, XIST, and Wilms tumor 1 (WT1) loci. canadian neighbor pharmacy online

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RESULTSProduction of Day 40 and Day 72 Control B. gaurus/B. taurus fetuses

Bos gaurus and Bos taurus hybrid fetuses and placentas were obtained at Day 40 and Day 72 of gestation (Fig. 1). A total of three Day 40 hybrid pregnancies were obtained (one female and two males) and six Day 72 hybrid pregnancies (four females and two males). Table 1 summarizes weights and measurements obtained from each of the hybrid fetuses. Additionally, sex of the three Day 40 fetuses was determined by Y chromosome-specific PCR reactions (data not shown).

Production of Day 40 NT-Derived B. gaurus/B. taurus Hybrids

Two hundred oocytes were fused with B. gaurus/B. taurus lung fetal fibroblast cells derived from the donor fetus and led to the generation of 32 grade-one blastocysts (32/211; 15.2%). At Day 28 of gestation, recipient cows were checked for pregnancy and three recipients were determined pregnant (3/15; 20%). At Day 40 of gestation, cloned fetal and placental tissues were isolated (Fig. 1). Monocho-rionic twins were present in clones 1 and 2 and a singleton was present in pregnancy 3. Gross comparisons of the fetal and placental components revealed an absence of cotyledons in each cloned pregnancy in contrast with Day 40 controls which possessed 4, 16, and 25 cotyledons per pregnancy. No apparent differences were observed in fetal or placental weights or allantoic fluid volumes (not shown). Table 1 summarizes weights and cotyledon numbers obtained from each of the cloned and control pregnancies.

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Animal experimentation was conducted in accordance with the Guide for the Care and Use of Agricultural Animals in Agricultural Research and Teaching. All animal protocols were reviewed and approved by the University Lab Animal Care Committee.

Production of Control B. gaurus/B. taurus Hybrids

Heifers and mature (1.5- to 3-yr-old) Angus and Angus-cross cows were used to generate Day 40 and Day 72 control hybrid fetuses. Estrus was synchronized by serial injections of 25 mg Lutalyse (Pharmacia, Exton, PA) administered at 11-day intervals. Twelve hours (h) after detection of estrus, heifers were artificially inseminated with semen from a Gaur bull. Heifers were then checked at Day 28 of gestation for establishment of pregnancy using transrectal ultrasonography. canadian neighbor pharmacy viagra

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INTRODUCTION

The nuclear transfer (NT) process in livestock and experimental animals results in pregnancy rates much lower than in vivo- or in vitro-derived animals. In addition, the clones that develop to term possess a myriad of disorders including obesity, large offspring syndrome, respiratory failure, organ defects, diabetes, and arthritis. In cattle, NT is currently an inefficient process, often resulting in embryonic and fetal death as well as high incidences of abnormalities.

Hill et al. demonstrated in a series of experiments that more than 80% of cloned pregnancies were lost in the bovine between Days 30-60 of gestation, and this was attributed to placental aberrations. Further examination of these animals indicated a reduction in the number of expected cotyledons and a marked decrease in chorio-allan-toic blood vessels. These findings are consistent with other reports in cloned cattle where no placentome formation was observed in the placenta of NT fetuses that died in utero between Days 35 and 55. Nuclear transfer-derived mice also exhibit abnormal placentation, most commonly an increase in placental size, and this has been correlated with an expansion of the spongiotrophoblast layer. Combined, these results suggest that improper establishment of the placenta gives rise, either entirely or partially, to the abnormalities and low rates of pregnancies observed in NT cattle.

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