Heterosis or hybrid vigor is a biological phenomenon referring to the phenotypic superiority of hybrids over their parents. Despite its economic importance, the mechanisms of heterosis are still poorly understood. Reciprocal cross Brahman (B) � Angus (A) calves display significant heterosis in birth weight, but this effect is almost entirely due to the dramatic fetal overgrowth observed in Brahman male � Angus female offspring. The reciprocal is much less affected and similar to purebred Brahman calves (Brown et al. 1993 J. Anim. Sci. 71, 3273–3279). We have generated a defined A � A (n = 20), B � A (n = 21), A � B (n = 13), and B � B (n = 15; male parent listed first) day 153 (term = 280) fetal/placental resource from artificially inseminated, estrous cycle synchronized heifers to identify components and mechanisms of heterotic fetal growth regulation. An ANOVA showed that full uterus weight (P < 0.001), fetal weight (P = 0.01), umbilical cord length (P = 0.003) and weight (P = 0.04), placenta fetalis weight (P < 0.001), total caruncle weight (P = 0.002), empty uterus weight (P < 0.001), and combined amniotic/allantoic fluid weight (P < 0.001) were significantly affected by the 4 genetic groups after adjustment for fetal sex and dam weight where required. The weight of reciprocal hybrid fetuses was intermediate to the purebred fetuses and thus did not display heterosis defined as the difference between reciprocal cross and parental means. Full uterus weight and combined amniotic/allantoic fluid weight, in contrast, displayed heterosis of 6.6% (P = 0.02) and 9.0% (P = 0.01). As in neonate calves, the heterosis effects were due to the B � A group. The t-tests demonstrated that full uterus weight in B � A was significantly greater (19.84 � 0.43 kg) than in A � B (16.23 � 0.47 kg; P < 0.001), A � A (17.41 � 0.35 kg; P < 0.001), and B � B (16.76 � 0.49 kg; P = 0.001) crosses. Combined amniotic/allantoic fluids were 12.58 � 0.31 kg in B � A as compared to 10.93 � 0.39 kg in A � B (P = 0.001), 10.75 � 0.29 kg in A � A (P < 0.001), and 11.48 � 0.36 kg in B � B (P = 0.02) crosses. We found similar superiority of the B � A group for parameters that did not fulfil the formal heterosis criterion. These include umbilical cord, placenta fetalis, empty uterus, and total caruncle weights. All but 1 of these (combined amniotic/allantoic fluid weight) were significantly correlated (r = 0.43–0.70; P < 0.001) with fetal weight. We conclude that massive changes in placental parameters underly and precede the heterosis effects in birth weight observed in Brahman � Angus crosses. Although formally designated heterosis, placental and fetal overgrowth is present in only 1 of the hybrids (B � A). This natural overgrowth phenotype is clearly distinct from the early onset overgrowth phenotypes observed after IVF and nuclear transfer cloning (Hiendleder et al. 2004 Biol. Reprod. 71, 217–223) and will be useful in the dissection of factors contributing to fetal growth and development.

Major problems in breeding of modern dairy cows include increasing rates of stillbirth and dystocia, associated with elevated costs for veterinary intervention (caesarean section) and loss of production animals (cows and calves). Rates of stillbirth as high as 12% have been reported, primarily caused by fetal overgrowth and increased birth weight. In order to discover molecular markers for selection against stillbirth, we developed a model for the inheritance of fetal growth traits. A Fleckvieh bull segregating for paternal stillbirth was used for insemination of 36 cows that were slaughtered at Day 150 of pregnancy in order to recover and phenotype the fetuses. The mode of inheritance indicated involvement of imprinting. Mapping results suggested an imprinted region on chromosome 9 as candidate for the stillbirth QTL. Due to the complexity of the trait, we opted for a holistic approach that is not restricted to the QTL candidate region but allows identification of genes and networks that influence fetal growth. Transcriptome profiles of liver and cotyledon samples from the fetuses with the highest (n = 10) and the lowest total weight n = 10) were analyzed using Affymetrix Bovine GeneChips (Affymetrix, Inc., Santa Clara, CA, USA). Analysis with the program SAM showed 41 up- and 4 down-regulated genes in liver samples of the heavy-weight group. Most of these genes are involved in immune response. Interestingly, many of these genes are reported to be regulated by vitamin D. Furthermore, vitamin D is closely connected to the IGF1 system and thus the most important fetal growth regulation circuit. Seasonal effects on vitamin D levels could mostly be excluded by the experimental design and did not correlate with growth traits. Most likely, the mRNA levels of our candidate genes were influenced by alterations in the IGF1/vitamin D circuit and did not cause the observed weight differences. The imprinted candidate genes showed no correlation with fetal weight. Gene set enrichment analysis indicated enhanced metabolic activity in the liver of heavy-weight fetuses. Genes from the QTL region showed a clear enrichment in correlation with fetal weight, confirming their involvement in fetal growth. The gene with the best correlation, GHITM (growth hormone inducible transmembrane protein), could give an explanation for the enhanced metabolic activity, as it is reported to function as a metabolic regulator. Simultaneous analysis of the data sets for liver and placenta in a linear model (R-package LIMMA; Smyth 2004 Stat. Applic. Genet. Mol. Biol. 3, art. 3) yielded essentially the same differentially expressed genes for liver and a higher number of differentially expressed genes for placenta (89 up- and 114 down-regulated), with little overlap between the two tissues. This work was supported by Grant BMBF FUGATO-Fertilink.

Large offspring syndrome (LOS) in ruminants refers to various poorly defined organ pathologies that are associated with fetal overgrowth and are encountered after a range of embryo manipulations (Rhind et al. 2003 Nat. Rev. Genet. 4, 855–864). We have explored the effects of somatic cell nuclear transfer (NT) and in vitro fertilization (IVF) on phenotype and relative expression levels of 2 imprinted genes important for fetal growth, insulin-like growth factor 2 (IGF2) and its receptor (IGF2R). Viable bovine fetuses were recovered near the end of the first trimester of pregnancy, and skeletal muscle, liver, and lung were sampled for real-time RT-PCR analyses. We compared NT-fetuses (n = 23), IVF-fetuses (n = 24), and fetuses generated by artificial insemination (controls, n = 24) in order to separate abnormalities specific to cloning from effects of in vitro gamete and embryo manipulation. Nuclear transfer and IVF-fetuses, both derived from embryos cultured with 10% estrous cow serum, demonstrated significant fetal overgrowth. The increase in body weight relative to controls was similar for both groups (+22%, P &lt; 0.001, and +19%, P &lt; 0.001, respectively), but further analyses clearly separated the NT phenotype from the IVF phenotype. The NT-fetuses were characterized by a shorter crown-rump length but larger thorax circumference, which consequently produced a significantly reduced fetus length-to-thorax circumference ratio in comparison with IVF-fetuses and controls (-9% each, P &lt; 0.0001). Absolute liver weight was significantly increased in NT- and IVF-fetuses (+62%, P &lt; 0.0001, and +20%, P &lt; 0.0001, respectively), but relative liver weight was increased only in NT-fetuses (+30%, P &lt; 0.0001). Heart (P&lt; 0.0001) and kidney (P = 0.0003) mass also showed disproportionate organomegaly in NT-fetuses only, but relative lung weight (NT, P = 0.263; IVF, P = 0.317) was not affected by either embryo technique. Transcript abundance for IGF2 and IGF2R genes were strongly correlated in muscle (r = 0.835, P &lt; 0.0001), liver (r = 0.922, P &lt; 0.0001), and lung (r = 0.772, P &lt; 0.0001). The IGF2 and IGF2R transcript levels in muscle tissue from NT-fetuses were markedly reduced in comparison with both IVF-fetuses (-31%, P &lt; 0.0001, and -41%, P &lt; 0.0001, respectively) and controls (-31%, P &lt; 0.0001, and -41%, P &lt; 0.0001, respectively). In liver tissue, however, transcript levels for NT-fetuses were similar to those of controls, and IVF-fetuses showed markedly elevated, albeit non-significant, IGF2 (+86%, P = 0.0591) and IGF2R (+54%, P = 0.1305) mRNA levels relative to controls. Our data demonstrate that seemingly similar syndromes caused by NT or IVF procedures can be clearly partitioned with respect to phenotype and IGF2/IGF2R expression.

No abstract

No abstract