Chronic Undernutrition in Ovine Twin Pregnancies Abolishes Differences in Birth Weight Due to Sex: An Evaluation of the Role of Nutritional and Antioxidant Supplementation.

In twin pregnancies of discordant sex, the male fetus grows larger than the female co-twin. Our study aimed to determine the effect of the sex of co-twins on lambs' birth weight in ovine pregnancies developed under natural undernourishment. Additionally, we investigated whether the nutritional and/or antioxidant supplementation provided to ewes during pregnancy could modulate the potential effects associated with the sex of co-twins. Ninety-six birth records of twin pregnancies of sheep grazing the natural Patagonian prairies were analyzed. The animals were divided into four groups: control (no supplementation), N (concentrate supplementation, 100% NRC), A (antioxidant supplementation), and NA (concentrate + antioxidant supplementation). Supplementation occurred from day 35 of gestation onwards until lambing. There were no differences in female or male birth weight in the control undernourished group. However, in group N, females or males with sex-discordant co-twins had a higher birth weight than did those with co-twins of the same sex. Group A males with female co-twins had a higher birth weight compared to males whose co-twins were also males. In NA lambs, males had a higher birth weight compared to females, regardless of their co-twin's sex. Therefore, chronic undernutrition abolished the differences in birth weight due to fetal sex. Restoring maternal nutrition or antioxidant supplementation tends to normalize birth weight and restore the differences between females and males. This effect is enhanced with the combined supplementation of concentrated food and antioxidants.

Placental Development and Physiological Changes in Pregnant Ewes in Silvopastoral and Open Pasture Systems during the Summer.

This study aimed to analyze the reproductive and physiological changes in ewes subjected to heat stress during pregnancy at UTFPR-Brazil. Twenty-four pregnant crossbred ewes were kept in a silvopastoral system (SP) or an open pasture system (OP) throughout the final trimester of pregnancy. Both systems were stressful, but the SP system had lower air temperature than the OP system (26.0 ± 0.38 and 26.9 ± 0.41 °C, respectively; p = 0.0288). Moreover, the radiant thermal load of the two groups presented a difference of 34 Wm-2 (p = 0.0288), and the grass temperature was also lower in the SP system compared to that in the OP system (23.4 ± 0.37 and 25.6 ± 0.44 °C, respectively; p = 0.0043). The respiratory and heart rates of animals from the OP group were higher than those from the SP group (p < 0.001), but no difference was observed in the mobilization of white blood cells (p = 0.4777), and the neutrophil count was only affected by time (p < 0.0001). As regards placental biometry, placentas in twin pregnancies had a greater membrane area (p = 0.0223), but no differences between the systems were observed in placental weight (p = 0.1522) and the number of cotyledons (p = 0.5457). We concluded that the type of rearing system used affects the thermal comfort of pregnant ewes, and that an SP system can offer more amenable microclimatic conditions, which result in greater comfort for the ewes.

Multi-Omics Analysis Reveals the Potential Effects of Maternal Dietary Restriction on Fetal Muscle Growth and Development.

In terms of fetal muscle growth, development, and health, maternal nutrition is a crucial influence, although the exact biochemical mechanism by which this occurs is still not fully understood. To examine the potential impacts of maternal dietary restriction on fetal muscle development, the sheep maternal dietary restriction model was developed for this study. In our study, 12 pregnant ewes were evenly split into two experimental groups and fed either 75% or 100% of a maternal nutrient. In addition, a multi-omics analysis was used to study the embryonic longissimus dorsis on gestational days (GD) 85 and 135. The fetal weight at GD 135 was significantly below normal due to the maternal restricted diet (p < 0.01). When fetuses were exposed to the dietary deficit, 416 mRNAs and 40 proteins were significantly changed. At GD 85, the multi-omics analysis revealed that maternal dietary restriction led to a significant up-regulation of the cell cycle regulator CDK2 gene in the cellular senescence signaling pathway, and the results of the qRT-PCR were similar to the multi-omics analysis, which showed that SIX1, PAX7, the cell cycle factors CDK4 and CDK6, and the BCL-2 apoptosis factor were up-regulated and several skeletal muscle marker genes, such as MYF5 and MyoD were down-regulated. At GD 135, maternal dietary restriction blocks the muscle fiber differentiation and maturation. The multi-omics analysis revealed that the TEAD1 gene was in the Hippo signaling pathway, the muscle marker genes MYF5 and MyoG were significantly down-regulated, and the TEAD1 binding of the down-regulated VGLL3 gene might be potential mechanisms affecting myofiber differentiation and maturation. Knocking down the CDK2 gene could inhibit the proliferation of primary embryonic myoblasts, and the expression levels of cell cycle regulatory factors CDK4 and CDK6 were significantly changed. Under low nutrient culture conditions, the number of myoblasts decreased and the expression of CDK2, CDK6, MYF5, PAX7 and BCL-2 changed, which was in perfect agreement with the multi-omics analysis. All of the findings from our study helped to clarify the potential effects of maternal dietary restriction on fetal muscle growth and development. They also provided a molecular foundation for understanding the molecular regulatory mechanisms of maternal nutrition on fetal muscle growth and development, as well as for the development of new medications and the management of related metabolic diseases.

An MRI approach to assess placental function in healthy humans and sheep.

KEY POINTS: Human placental function is evaluated using non-invasive Doppler ultrasound of umbilical and uterine artery pulsatility indices as measures of resistance in placental vascular beds, while measurement of placental oxygen consumption ( VO2 ) is only possible during Caesarean delivery. This study shows the feasibility of using magnetic resonance imaging (MRI) in utero to measure blood flow and oxygen content in uterine and umbilical vessels to calculate oxygen delivery to and VO2 by the gravid uterus, uteroplacenta and fetus. Normal late gestational human uteroplacental VO2 by MRI was ∼4 ml min-1  kg-1 fetal weight, which was similar to our MRI measurements in sheep and to those previously measured using invasive techniques. Our MRI approach can quantify uteroplacental VO2 , which involves the quantification of maternal- and fetal-placental blood flows, fetal oxygen delivery and VO2 , and the oxygen gradient between uterine- and umbilical-venous blood, providing a comprehensive assessment of placental function with clinical potential. ABSTRACT: It has not been feasible to perform routine clinical measurement of human placental oxygen consumption ( VO2 ) and in vitro studies do not reflect true metabolism in utero. Here we propose an MRI method to non-invasively quantify in utero placental and fetal oxygen delivery ( DO2 ) and VO2 in healthy humans and sheep. Women (n = 20) and Merino sheep (n = 10; 23 sets of measurements) with singleton pregnancies underwent an MRI in late gestation (36 ± 2 weeks and 128 ± 9 days, respectively; mean ± SD). Blood flow (phase-contrast) and oxygen content (T1 and T2 relaxometry) were measured in the major uterine- and umbilical-placental vessels, allowing calculation of uteroplacental and fetal DO2 and VO2 . Maternal DO2 (ml min-1  kg-1 fetus) to the gravid uterus was similar in humans and sheep (human = 54 ± 15, sheep = 53 ± 21, P = 0.854), while fetal DO2 (human = 25 ± 4, sheep = 22 ± 5, P = 0.049) was slightly lower in sheep. Uteroplacental and fetal VO2 (ml min-1  kg-1 fetus; uteroplacental: human = 4.1 ± 1.5, sheep = 3.5 ± 1.9, P = 0.281; fetus: human = 6.8 ± 1.3, sheep = 7.2 ± 1.7, P = 0.426) were similar between species. Late gestational uteroplacental:fetal VO2 ratio did not change with age (human, P = 0.256; sheep, P = 0.121). Human umbilical blood flow (ml min-1  kg-1 fetus) decreased with advancing age (P = 0.008), while fetal VO2 was preserved through an increase in oxygen extraction (P = 0.046). By contrast, sheep fetal VO2 was preserved through stable umbilical flow (ml min-1  kg-1 ; P = 0.443) and oxygen extraction (P = 0.582). MRI derived measurements of uteroplacental and fetal VO2 between humans and sheep were similar and in keeping with prior data obtained using invasive techniques. Taken together, these data confirm the reliability of our approach, which offers a novel clinical 'placental function test'.

Polychlorinated biphenyls (PCBs) alter DNA methylation and genomic integrity of sheep fetal cells in a simplified in vitro model of pregnancy exposure.

Polychlorinated biphenyls (PCBs) are persistent organic pollutants ubiquitously detectable in the environment and in the food chain. Prenatal exposure to PCBs negatively affects fetal development and produces long-term detrimental effects on child health. The present study sought to evaluate the cytotoxic and genotoxic effects of chronic PCB exposure on fetal cells during pregnancy. To this aim, sheep embryonic fibroblasts (SEF) and amniocytes (SA) were cultured in vitro in the presence of low doses of PCBs for a period of 120days, comparable to the full term of ovine pregnancy. Cellular proliferation rates, global DNA methylation, chromosome integrity, and markers of DNA damage were evaluated at different time points. Moreover, SEF treated with PCBs for 60days were left untreated for one further month and then examined in order to evaluate the reversibility of PCB-induced epigenetic defects. PCB-treated SEF were more sensitive than SA treated with PCBs, in terms of low cell proliferation, and increased DNA damage and global DNA methylation, which were still detectable after interruption of PCB treatment. These data indicate that chronic exposure of fetal cells to PCBs causes permanent genomic and epigenetic instability, which may influence both prenatal and post-natal growth up to adulthood. Our in vitro model offer a simple and controlled means of studying the effects of different contaminants on fetal cells - one that could set the stage for targeted in vivo studies.