A. K. Jones

Transabdominal ultrasound for detection of pregnancy, fetal and placental landmarks, and fetal age before Day 45 of gestation in the sheep

Detection of pregnancy during early gestation is advantageous for flock breeding management. Transabdominal ultrasound is a practical and efficient approach for monitoring pregnancy and fetal growth in small ruminants. However, there is limited information using the transabdominal technique before Day 45 of gestation in sheep. Therefore, our objective was to determine how accurately transabdominal ultrasound could be used to detect pregnancy, to identify pregnancy landmarks, and to quantify fetal length before Day 45 in ewes. Multiparous Western White-faced ewes (n = 99) were estrus synchronized and exposed to one of four Dorset rams. The day a ewe was marked by a ram was considered Day 0 of gestation. Ewes not remarked by Day 20 were separated for ultrasonography. To detect pregnancy and landmarks, ewes were scanned three times per week between Day 26.0 ± 0.3 (mean ± standard error) and Day 40.0 ± 0.2. A single technician performed all scans in the right nonhaired abdominal pit using a real-time portable Eazi-Scan machine and a 5-MHz linear rectal transducer. All data were analyzed using the MIXED procedure in SAS (with repeated measures where appropriate). Because of rebreeding activity, 113 ultrasound periods were initiated. The specificity and positive predictive value were 100% during the entire study. The accuracy, sensitivity, and negative predictive value of ultrasound scanning were greater than 90% beginning at Day 33 ± 1. On average, pregnancy (n = 85) was detected at Day 28.7 ± 0.4 and nonpregnancy (n = 28) at Day 25.5 ± 0.6. Three early fetal losses were identified at Day 39.7 ± 0.7. In pregnant ewes (n = 82), the overall accuracy of fetal counting was 78%. The first observance of an enlarged uterus (P = 0.05) and pregnancy (P = 0.03) was detected earlier when multiple fetuses were developing compared with singletons. Placentome evagination was first observed earlier in triplets compared with twins and singletons (P = 0.02). Fetal length increased with day of gestation (P < 0.0001) but not fetal number (P = 0.72). A fetal number by day of gestation interaction (P = 0.01) indicated differences in fetal length at Day 29 ± 1 and Day 32 ± 1. These data demonstrate that a portable ultrasound using the transabdominal technique can be used to accurately determine pregnancy, identify landmarks indicative of gestation, and estimate fetal age, before Day 45 of gestation in sheep.

Fetal and organ development at gestational days 45, 90, 135 and at birth of lambs exposed to under- or over-nutrition during gestation

Abstract To determine the effects of poor maternal nutrition on offspring body and organ growth during gestation, pregnant Western White-faced ewes (n = 82) were randomly assigned into a 3 × 4 factorial treatment structure at d 30.2 ± 0.2 of gestation (n = 5 to 7 ewes per treatment). Ewes were individually fed 100% (control), 60% (restricted) or 140% (over) of NRC requirements for TDN. Ewes were euthanized at d 45, 90 or 135 of gestation or underwent parturition (birth) and tissues were collected from the offspring (n = 10 to 15 offspring per treatment). Offspring from control, restricted and overfed ewes are referred to as CON, RES and OVER, respectively. Ewe data were analyzed as a completely randomized design and offspring data were analyzed as a split-plot design using PROC MIXED. Ewe BW did not differ at d 30 (P ≥ 0.43), however restricted ewes weighed less than overfed and overfed were heavier than controls at d 45, and restricted weighed less and overfed were heavier than controls at d 90 and 135 and birth (P ≤ 0.05). Ewe BCS was similar at d 30, 45 and 90 (P ≤ 0.07), however restricted ewes scored lower than control at d 135 and birth (P ≤ 0.05) and over ewes scored higher than control at d 135 (P ≤ 0.05) but not at birth (P = 0.06). A maternal diet by day of gestation interaction indicated that at birth the body weight (BW) of RES offspring was less than CON and OVER (P ≤ 0.04) and heart girth of RES was smaller than CON and OVER (P ≤ 0.004). There was no interaction of maternal diet and day of gestation on crown-rump, fetal, or nose occipital length, or orbit or umbilical diam. (P ≥ 0.31). A main effect of maternal diet indicated that the RES crown-rump length was shorter than CON and OVER (P ≤ 0.05). An interaction was observed for liver, kidney and renal fat (P ≤ 0.02). At d 45 the liver of RES offspring was larger than CON and OVER (P ≤ 0.002), but no differences observed at d 90, 135 or birth (P ≥ 0.07). At d 45, the kidneys of OVER offspring were larger than CON and RES (P ≤ 0.04), but no differences observed at d 90, 135 or birth (P ≥ 0.60). At d 135, OVER had more perirenal fat than CON and RES (P ≤ 0.03), and at birth RES had more perirenal fat than CON and OVER (P ≤ 0.04). There was no interaction observed for offspring heart weight, length or width, kidney length, adrenal gland weight, loin eye area or rib width (P ≥ 0.09). In conclusion, poor maternal nutrition differentially alters offspring body size and organ growth depending on the stage of gestation.

PHYSIOLOGY AND ENDOCRINOLOGY SYMPOSIUM:The effects of poor maternal nutrition during gestation on offspring postnatal growth and metabolism

Poor maternal nutrition during gestation has been linked to poor growth and development, metabolic dysfunction, impaired health, and reduced productivity of offspring in many species. Poor maternal nutrition can be defined as an excess or restriction of overall nutrients or specific macro- or micronutrients in the diet of the mother during gestation. Interestingly, there are several reports that both restricted- and over-feeding during gestation negatively affect offspring postnatal growth with reduced muscle and bone deposition, increased adipose accumulation, and metabolic dysregulation through reduced leptin and insulin sensitivity. Our laboratory and others have used experimental models of restricted- and over-feeding during gestation to evaluate effects on early postnatal growth of offspring. Restricted- and over-feeding during gestation alters body size, circulating growth factors, and metabolic hormones in offspring postnatally. Both restricted- and over-feeding alter muscle growth, increase lipid content in the muscle, and cause changes in expression of myogenic factors. Although the negative effects of poor maternal nutrition on offspring growth have been well characterized in recent years, the mechanisms contributing to these changes are not well established. Our laboratory has focused on elucidating these mechanisms by evaluating changes in gene and protein expression, and stem cell function. Through RNA-Seq analysis, we observed changes in expression of genes involved in protein synthesis, metabolism, cell function, and signal transduction in muscle tissue. We recently reported that satellite cells, muscle stem cells, have altered expression of myogenic factors in offspring from restricted-fed mothers. Bone marrow derived mesenchymal stem cells, multipotent cells that contribute to development and maintenance of several tissues including bone, muscle, and adipose, have a 50% reduction in cell proliferation and altered metabolism in offspring from both restricted- and over-fed mothers. These findings indicate that poor maternal nutrition may alter offspring postnatal growth by programming stem cell populations. In conclusion, poor maternal nutrition during gestation negatively affects offspring postnatal growth, potentially through impaired stem and satellite cell function. Therefore, determining the mechanisms that contribute to fetal programming is critical to identifying effective management interventions for these offspring and improving efficiency of production.

Muscle growth in young horses: Effects of age, cytokines, and growth factors

Success as equine athletes requires proper muscle growth in young horses. Muscle hypertrophy occurs through protein synthesis and the contribution of muscle satellite cells, which can be stimulated or inhibited by cytokines and growth factors present during exercise and growth. The hypotheses of this study were that 1) the LM area in young horses would increase over 1 yr, and 2) specific cytokines and growth factors (IL-1β, IL-6, tumor necrosis factor [TNF]-α, IGF-I, and fibroblast growth factor [FGF]-2) would alter proliferation and differentiation of satellite cells isolated from young horses. Fourteen horses were divided into 3 age groups: weanlings ( = 5), yearlings to 2 yr olds ( = 4), and 3 to 4 yr olds ( = 5). The area, height, and subcutaneous fat depth of the LM were measured using ultrasonography, and BW and BCS were taken in October (Fall1), April (Spring), and October of the following year (Fall2). Satellite cells obtained from 10-d-old foals ( = 4) were cultured in the presence of IL-6, IL-1β, TNF-α, IGF-I, or FGF-2 before evaluation of proliferation and differentiation. Data were analyzed using PROC MIXED in SAS. Body weight increased from Fall1 to Spring in weanlings ( < 0.001) and increased in all horses from Spring to Fall2 ( ≤ 0.02). Area and height of the LM increased over time ( < 0.001) and with increasing age group of horse ( ≤ 0.03), although there was no interaction of time and age ( > 0.61). There was a significant increase in LM area in all animals from Spring to Fall2 ( < 0.001) but not from Fall1 to Spring. Interleukin-6 and TNF-α decreased satellite cell proliferation by 14.9 and 11.5%, respectively ( ≤ 0.01). Interleukin-6 increased fusion 6.2%, whereas TNF-α decreased fusion 8.7% compared with control cells ( ≤ 0.001). Interleukin-1β had no effect on proliferation ( = 0.32) but tended to decrease fusion ( = 0.06). Satellite cell proliferation was increased 28.8 and 73.0% by IGF-I and FGF-2, respectively ( < 0.0001). Differentiation was decreased 13.1% in the presence of FGF-2 but increased 3.5% in the presence of IGF-I ( ≤ 0.01). In summary, the LM area increases over the course of a year in young horses with the most growth occurring in summer. By stimulating or inhibiting proliferation and differentiation of satellite cells, IL-6, TNF-α, IL-1β, IGF-I, and FGF-2 may alter muscle growth in young horses, thereby impacting athletic potential.

Gestational restricted- and over-feeding promote maternal and offspring inflammatory responses that are distinct and dependent on diet in sheep

Abstract Inflammation may be a mechanism of maternal programming because it has the capacity to alter the maternal environment and can persist postnatally in offspring tissues. This study evaluated the effects of restricted- and over-feeding on maternal and offspring inflammatory gene expression using reverse transcription (RT)-PCR arrays. Pregnant ewes were fed 60% (Restricted), 100% (Control), or 140% (Over) of National Research Council requirements beginning on day 30.2 ± 0.2 of gestation. Maternal (n = 8–9 ewes per diet) circulating nonesterified fatty acid (NEFA) and expression of 84 inflammatory genes were evaluated at five stages during gestation. Offspring (n = 6 per diet per age) inflammatory gene expression was evaluated in the circulation and liver at day 135 of gestation and birth. Throughout gestation, circulating NEFA increased in Restricted mothers but not Over. Expression of different proinflammatory mediators increased in Over and Restricted mothers, but was diet-dependent. Maternal diet altered offspring systemic and hepatic expression of genes involved in chemotaxis at late gestation and cytokine production at birth, but the offspring response was distinct from the maternal. In the perinatal offspring, maternal nutrient restriction increased hepatic chemokine (CC motif) ligand 16 and tumor necrosis factor expression. Alternately, maternal overnutrition increased offspring systemic expression of factors induced by hypoxia, whereas expression of factors regulating hepatocyte proliferation and differentiation were altered in the liver. Maternal nutrient restriction and overnutrition may differentially predispose offspring to liver dysfunction through an altered hepatic inflammatory microenvironment that contributes to immune and metabolic disturbances postnatally. Summary Sentence Restricted- and over-feeding differentially alter maternal inflammation and metabolism and indirectly promote systemic and hepatic inflammation in the perinatal offspring, which may contribute to liver dysfunction postnatally.

Evaluation of the Nova Vet Meter for sheep-side monitoring of β-hydroxybutyric acid (BHBA) and description of ewe BHBA during late gestation in three flocks from the Northeastern U.S.

Prevention of metabolic diseases in small ruminants may improve production efficiency and profitability, yet ewes carrying multiples or who are in poor body condition are at increased susceptibility to develop ketosis. This study evaluated the hand-held Nova Vet Meter to accurately detect β-hydroxybutyric acid (BHBA) concentrations in ewes and determined the percentage of ewes at moderate (0.8 to 1.5 mmol/L BHBA) and greatest (≥1.6 mmol/L BHBA) risk to develop ketosis during late gestation. To validate the Nova Vet Meter, BHBA concentrations of 104 paired blood samples were measured using the Nova Vet Meter and gold-standard laboratory analysis. Receiver operating characteristics were calculated. The accuracy and sensitivity of detecting BHBA concentrations at 0.8 to 1.5 mmol/L were 94.2% and 97.3%, respectively. The accuracy and sensitivity of detecting BHBA concentrations ≥ 1.6 mmol/L were 98.0% and 50.0%, respectively. Ewe body weight (BW), body condition score (BCS), and BHBA of 117 ewes from three flocks were determined weekly during the four weeks before parturition. During the last three weeks of gestation >20% of ewes were identified with moderate risk to develop ketosis. During the last four weeks of gestation, ewes carrying triplets had reduced BCS (P = 0.0002) and increased BHBA concentrations (P < 0.0001) compared with singleton and twin pregnancies. Ewe BHBA did not correlate with lamb birth weight (R2 = 0.003; P = 0.41). In conclusion, the Nova Vet Meter is suitable for sheep-side BHBA monitoring between 0.8 and 1.5 mmol/L, but further testing is necessary to evaluate BHBA readings ≥1.6 mmol/L.

Effects of maternal nutrition on the expression of genomic imprinted genes in ovine fetuses

ABSTRACT Genomic imprinting is an epigenetic phenomenon of differential allelic expression based on parental origin. To date, 263 imprinted genes have been identified among all investigated mammalian species. However, only 21 have been described in sheep, of which 11 are annotated in the current ovine genome. Here, we aim to i) use DNA/RNA high throughput sequencing to identify new monoallelically expressed and imprinted genes in day 135 ovine fetuses and ii) determine whether maternal diet (100%, 60%, or 140% of National Research Council Total Digestible Nutrients) influences expression of imprinted genes. We also reported strategies to solve technical challenges in the data analysis pipeline. We identified 80 monoallelically expressed, 13 new putative imprinted genes, and five known imprinted genes in sheep using the 263 genes stated above as a guide. Sanger sequencing confirmed allelic expression of seven genes, CASD1, COPG2, DIRAS3, INPP5F, PLAGL1, PPP1R9A, and SLC22A18. Among the 13 putative imprinted genes, five were localized in the known sheep imprinting domains of MEST on chromosome 4, DLK1/GTL2 on chromosome 18 and KCNQ1 on chromosome 21, and three were in a novel sheep imprinted cluster on chromosome 4, known in other species as PEG10/SGCE. The expression of DIRAS3, IGF2, PHLDA2, and SLC22A18 was altered by maternal diet, albeit without allelic expression reversal. Together, our results expanded the list of sheep imprinted genes to 34 and demonstrated that while the expression levels of four imprinted genes were changed by maternal diet, the allelic expression patterns were un-changed for all imprinted genes studied.