DOHaD: A MENAGERIE OF ADAPTATIONS AND PERSPECTIVES: Large animal models of developmental programming: sustenance, stress, and sex matter.

In brief: Developmental programming refers to the long-term programming of gene expression during fetal and postnatal development, resulting in altered organ function even into adulthood. This review describes how maternal and paternal sustenance and stress, as well as fetal sex, all matter in large animal models and affect developmental programming of the offspring. Abstract: Developmental programming is the concept that certain health outcomes throughout life can be linked to early fetal or postnatal development. Progress in understanding concepts and mechanisms surrounding developmental programming is heavily leveraged by the use of large animal models. Numerous large animal models have been developed that apply a host of different maternal stressors and, more recently, paternal stressors. Maternal nutrition is the most researched maternal stressor applied during gestation and includes both global nutrient supply and models that target specific macro- or micro- nutrients. The focus of this review is to provide an overview of the many large animal models of developmental programming and to discuss the importance of sex effects (including paternal contributions) in study design and data interpretation.

Effects of realimentation after nutrient restriction during mid- to late gestation on pancreatic digestive enzymes, serum insulin and glucose levels, and insulin-containing cell cluster morphology.

This study examined effects of stage of gestation and nutrient restriction with subsequent realimentation on maternal and foetal bovine pancreatic function. Dietary treatments were assigned on day 30 of pregnancy and included: control (CON; 100% requirements; n = 18) and restricted (R; 60% requirements; n = 30). On day 85, cows were slaughtered (CON, n = 6; R, n = 6), remained on control (CC; n = 12) and restricted (RR; n = 12), or realimented to control (RC; n = 11). On day 140, cows were slaughtered (CC, n = 6; RR, n = 6; RC, n = 5), remained on control (CCC, n = 6; RCC, n = 5) or realimented to control (RRC, n = 6). On day 254, the remaining cows were slaughtered and serum samples were collected from the maternal jugular vein and umbilical cord to determine insulin and glucose concentrations. Pancreases from cows and foetuses were removed, weighed, and subsampled for enzyme and histological analysis. As gestation progressed, maternal pancreatic α-amylase activity decreased and serum insulin concentrations increased (p ≤ 0.03). Foetal pancreatic trypsin activity increased (p < 0.001) with advancing gestation. Foetal pancreases subjected to realimentation (CCC vs. RCC and RRC) had increased protein and α-amylase activity at day 254 (p ≤ 0.02), while trypsin (U/g protein; p = 0.02) demonstrated the opposite effect. No treatment effects were observed for maternal or foetal pancreatic insulin-containing cell clusters. Foetal serum insulin and glucose levels were reduced with advancing gestation (p ≤ 0.03). The largest maternal insulin-containing cell cluster was not influenced by advancing gestation, while foetal clusters grew throughout (p = 0.01). These effects indicate that maternal digestive enzymes are influenced by nutrient restriction and there is a potential for programming of increased foetal digestive enzyme production resulting from previous maternal nutrient restriction.

Identifying the immunoglobulin G transporter in equine tissues: A look at the neonatal Fc receptor.

The neonatal Fc receptor (FcRn) is the receptor responsible for bidirectional transport of immunoglobulin G (IgG) across cells, maintenance of IgG levels in serum, and assisting with antigen presentation. Unfortunately, little is known about FcRn in horses. Therefore, the objective of this study was to provide fundamental information regarding the location of FcRn in equine tissues. Tissues were collected from six horses of mixed breed, age, and sex immediately following euthanasia. Sampling locations included the respiratory tract, gastrointestinal tract (GIT), other visceral organs, cornea, and synovial membrane of the stifle and carpal joints. Tissues for histological analysis were fixed, cross sectioned, and stained for FcRn. Areas of interest were captured and analyzed with data represented as relative fluorescence (RF) to indicate FcRn abundance. Tissues for qPCR analysis were placed in RNAlater and relative quantification (RQ) of FcRn transcripts (FCGRT) was calculated using the 2-ΔΔCT method, normalized to the geometric mean of three reference genes (ACTB, GADPH, HPRT1). Data were analyzed using the general linear model procedure of SAS. Abundance of FcRn differed between tissue types by immunofluorescence and qPCR analysis (P < 0.01). Joint synovium and respiratory tract tissues had the highest RF, GIT tissues expressed moderate RF, and other visceral organs had the lowest RF. Conversely, liver and kidney tissues had the highest RQ while the stomach and cornea had the lowest RQ. These data lay the foundation for future studies regarding FcRn and IgG in horses and their roles in disease prevention and treatment.

Role of the placenta in developmental programming: Observations from models using large animals.

Developmental programming, which proposes that "insults" or "stressors" during intrauterine or postnatal development can have not only immediate but also long-term consequences for healthy and productivity, has emerged as a major biological principle, and based on studies in many animal species also seems to be a universal phenomenon. In eutherians, the placenta appears to be programmed during its development, which has consequences for fetal growth and development throughout pregnancy, and likewise has long-term consequences for postnatal development, leading to programming of organ function of the offspring even into adulthood. This review summarizes our current understanding of the placenta's role in developmental programming, the mechanisms involved, and the challenges remaining.

Supranutritional selenium increases mammary gland vascularity in postpartum ewe lambs.

Objectives were to determine the effects of maternal dietary supranutritional Se and nutritional plane during gestation on capillary surface density, capillary area density, and angiogenic factor expression in the developing mammary gland of primiparous ewes. Selenium treatments were initiated at breeding [adequate Se (ASe; 9.5 µg/kg of body weight) vs. high Se (HSe; 81.8 µg/kg of body weight)] and nutritional planes at d 50 of gestation [Low, 60%; moderate (Mod), 100%; and High, 140% of requirements). Mammary glands were collected within 24h postpartum. Vascular development was assessed in the glandular portion of the mammary gland. Vascularity was determined for mammary tissue with the following measurements taken: the cross-sectional capillary area density (total capillary area as a proportion of tissue area) and capillary surface density (CSD; total capillary circumference per unit of tissue area). High-Se ewes had greater capillary surface and area densities compared with ASe ewes. A tendency existed for an Se × plane of nutrition interaction for CSD with maternal diet not affecting CSD in HSe ewes, but Low ewes had a decreased CSD compared with Mod ewes, with High being intermediate in ASe ewes. Moreover, HSe-Low and HSe-High ewes had increased CSD compared with ASe-Low and ASe-High, respectively. Although Se status did not influence angiogenic factor mRNA expression, mammary glands from Low ewes tended to have increased VEGF and FLT1 mRNA expression compared with High ewes, with Mod being intermediate. Maternal plane of nutrition did not affect mammary gland glutathione peroxidase activity, but it was increased in HSe compared with ASe ewes. Increased mammary capillary nutrient exchange area may contribute to previously observed changes in colostrum quality.

Effects of fetal and maternal genotype on placentome morphology in sheep.

Both maternal and fetal genotypes contribute to conceptus development. The objective was to determine how placentome number, size, and type and fetal weight was influenced after reciprocal embryo transfer in Columbia and Romanov sheep. Reciprocal embryo transfer was conducted between Columbia and Romanov ewes where a single embryo was transferred into Romanov and Columbia recipients [Romanov embryo in a Romanov uterus (RinR, n = 9); Romanov embryo in a Columbia uterus (RinC, n = 7); Columbia embryo in a Columbia uterus (CinC, n = 8); Columbia embryo in a Romanov uterus (CinR, n = 4)]. On day 130 of gestation, fetuses were weighed and placentomes were morphologically typed, weighed, and measured. Regardless of maternal genotype, Romanov fetuses were smaller (P < 0.05) compared to Columbia fetuses. Moreover, CinC fetuses were larger (P < 0.05) than CinR fetuses. There was a tendency (P = 0.12) for a fetal by maternal genotype interaction on total placentome weight, but main effects were significant for fetal genotype (P = 0.04) and maternal genotype (P < 0.01). The number of Type A placentomes was greater than any other types. Type A placentomes had a greater (P < 0.05) contribution to total placentome weight within the Romanov uterus, or when associated with a Romanov fetus, than within the Columbia breed, in which placentome type was evenly distributed. The hypothesis that the Romanov uterus would limit the growth of a Columbia conceptus is accepted; however, the Romanov conceptus did not experience augmented growth when transferred into a Columbia uterus as predicted.

Effects of nutrition and genotype on prion protein (PrPC) gene expression in the fetal and maternal sheep placenta.

For placental transmission of scrapie to occur, the normal cellular prion protein (PrPC) must be converted to an abnormal infectious form known as PrPSc. PrPC genotype influences susceptibility to contracting scrapie, but we still do not understand whether genotype or expression levels of PrPC are important in transmission of scrapie. Some evidence exists that nutrition affects expression levels of PrPC. Thus, we evaluated the effects of genotype and nutrition on PrPC mRNA and protein expression in adolescent ewes fed at control (100% of National Research Council [NRC] requirements) or restricted (60% of NRC) levels of diet intake during two periods of pregnancy (days 50-90 and days 90-130)]. Gravid uteri (n=50) from singleton pregnancies were collected at day 130, and placentomes were either separated into caruncular (CAR; maternal) or cotyledonary (COT; fetal) placenta and snap-frozen for PrPC mRNA expression or perfusion fixed for PrPC protein expression. PrPC genotypes were determined (codons 136 and 171) using SNP assay. There were no genotype effects on PrPC mRNA expression in CAR or on PrPC protein expression in either CAR or COT, but PrPC mRNA expression in COT was greater (P<0.02) when codon 136 was homozygous for alanine. Some PrPC protein-positive cells were found in the epithelium of CAR, but most were found in trophoblast binucleate and mononucleate cells of COT. In CAR, from days 90 to 130, PrPC protein abundance was greater (P=0.003) in diet-restricted ewes than in control ewes, but was less uniformly distributed (P<0.007). Additionally, in COT, from days 90 to 130, PrPC protein was less uniformly distributed (P<0.01) in diet-restricted ewes. The localized increase in PrPC protein expression, found in ewes diet-restricted late in pregnancy, may suggest a protective role for PrPC in placental biology. Further study is needed to evaluate whether nutrition, PrPC genotype, and PrPC expression levels influence placental transmission of scrapie.

The impact of diet and arginine supplementation on pancreatic mass, digestive enzyme activity, and insulin-containing cell cluster morphology during the estrous cycle in sheep.

To determine the effect of feed intake and arginine treatment during different stages of the estrous cycle on pancreatic mass, digestive enzyme activity, and histological measurements, ewes (n = 120) were randomly allocated to 1 of 3 dietary groups; control (CON; 2.14-Mcal metabolizable energy/kg), underfed (UF; 0.6 × CON), or overfed (OF; 2 × CON) over 2 yr. Estrus was synchronized using a controlled internal drug release device for 14 d. At controlled internal drug release withdrawal, ewes from each dietary group were assigned to 1 of 2 treatments; Arg (L-Arg HCl, 155-µmol/kg BW) or Sal (approximately 10-mL saline). Treatments were administered 3 times daily via jugular catheter and continued until slaughter on d (day) 5 and 10 of the second estrus cycle (early luteal phase, n = 41 and mid-luteal phase, n = 39; yr 1) and d 15 of the first estrus cycle (late luteal phase, n = 40; yr 2). A blood sample collected from jugular catheters for serum insulin analysis before slaughter. The pancreas was then removed, trimmed of mesentery and fat, weighed, and a sample snap-frozen until enzyme analysis. Additional pancreatic samples were fixed in 10% formalin solution for histological examination of size and distribution of insulin-containing cell clusters. Data were analyzed as a completely randomized design with a factorial arrangement of treatments. Diet, treatment, and diet × treatment were blocked by yr and included in the model with initial BW used as a covariate. Day of the estrous cycle was initially included in the model but later removed as no effects (P > 0.10) were observed for any pancreatic variables tested. Overfed ewes had the greatest (P < 0.001) change in BW, final BW, change in BCS, and final BCS. A diet × treatment interaction was observed for change in BW and final BW (P ≤ 0.004). Overfed and CON had increased (P < 0.001) pancreas weight (g) compared with UF ewes. Protein concentration (g/pancreas) was the lowest (P < 0.001) in UF ewes, whereas protein content (mg/kg BW) was greater (P = 0.03) in UF than OF ewes. Activity of α-amylase (U/g, kU/pancreas, U/kg of BW, and U/g protein) and trypsin (U/pancreas) was greater (P ≤ 0.003) in OF than UF ewes. Serum insulin was the greatest (P < 0.001) in OF ewes. No effects were observed for pancreatic insulin-containing cell clusters. This study demonstrated that plane of nutrition affected several measurements of pancreatic function; however, the dosage of Arg used did not influence pancreatic function.

The effects of maternal nutrition on the messenger ribonucleic acid expression of neutral and acidic amino acid transporters in bovine uteroplacental tissues from day sixteen to fifty of gestation.

We hypothesized that both day of gestation and maternal nutrition would alter the relative mRNA expression of neutral and acid AA transporters , , , , and . Crossbred Angus heifers ( = 49) were synchronized, bred via AI, assigned to nutritional treatment (100% of NRC requirements for 0.45 kg/d gain [control heifers {CON}] and 60% of CON [restricted heifers {RES}]), and ovariohysterectomized on d 16, 34, or 50 of gestation ( = 6 to 9/d). Nonbred, nonpregnant (NB-NP) controls were ovariohysterectomized on d 16 of the estrous cycle ( = 6) after synchronization. The resulting arrangement was a 2 × 3 factorial + 1 (CON vs. RES × d 16, 34, or 50 + NB-NP controls). Tissues collected included caruncular endometrium (CAR), intercaruncular endometrium (ICAR), fetal membranes (FM; chorioallantois; d 16 and 34), cotyledonary placenta (COT; d 50 only), intercotyledonary placenta (ICOT; d 50 only), and amnion (AMN; d 50 only]). Relative expression of , , , , and was determined for each tissue using NB-NP CAR and NB-NP ICAR tissues for the baseline; for FM, endometrium from NB-NP controls served as the baseline. In CAR, no day × treatment interaction was observed ( > 0.05). However, day of gestation affected relative expression of , where expression on d 16 was greater ( < 0.01) than expression on d 34 and 50. Additionally, relative expression of and was greater ( ≤ 0.05) in pregnant heifers compared with NB-NP heifers. For ICAR, was influenced by a day × treatment interaction ( < 0.01), where expression in d 16 RES was greater ( ≤ 0.05) than that of any other day or nutritional treatment. Furthermore, expression in d 16 CON was greater ( ≤ 0.05) than that in d 50 RES, with those in d 34 CON and RES and d 50 CON being intermediate. In addition, was affected by day of gestation, where expression on d 16 was greater ( < 0.01) than that on d 34 and 50. A day × treatment interaction was not observed ( > 0.05) in FM; however, expression on d 34 was greater ( = 0.02) than on d 50, with that on d 16 being intermediate. Day of gestation also affected expression of , where expression on d 34 and 50 was greater ( < 0.01) than that on d 16. These data support our hypothesis in that both day of gestation and maternal nutrition affected the relative mRNA expression of AA transporter in ICAR, whereas day of gestation has a greater effect on the relative mRNA expression of other neutral and acidic AA transporters in the various tissues studied.

Impacts of maternal nutrition on uterine and placental vascularity and mRNA expression of angiogenic factors during the establishment of pregnancy in beef heifers.

We hypothesized that maternal nutrient restriction starting at the time of breeding would influence placental vascular development and gene expression of angiogenic factors during the first 50 d of gestation in beef heifers. Commercial Angus crossbred heifers (n = 49) were maintained on a total mixed ration and supplemented with dried distillers grains with solubles. All heifers were subject to 5-d CO-Synch + CIDR estrous synchronization protocol, AI to a single Angus sire, and randomly assigned to dietary treatments. One half were assigned to control diet (CON) targeted to gain 0.45 kg/d and the remaining half were assigned to restricted diet (RES), which received 60% of CON. Heifers were subjected to ovariohysterectomy on d 16, 34, or 50 of gestation. Utero-placental tissues were obtained from the uterine horns ipsilateral and contralateral to the corpus luteum and separated into maternal caruncle (CAR); maternal endometrium, inter-caruncle (ICAR), and fetal membranes (FM). After collection, all tissues were snap frozen and stored at -80°C. There were no treatment × stage of gestation interactions (P >0.13) on the mRNA expression of vascular endothelial growth factor (VEGF) or endothelial nitric oxide synthase (eNOS). Heifers on CON treatment had greater (P = 0.03) expression of VEGF compared with RES heifers in NP-ICAR. On d 50 expression of eNOS was increased (P = 0.05) compared with d 16 in P-CAR. Expression of eNOS mRNA was decreased (P = 0.04) on d 16 compared with d 34 and 50 in CON heifer. Gene expression of eNOS was increased (P < 0.001) in the pregnant uterine horn compared with the NP uterine horn on d 34 and 50. Expression of eNOS was also increased (P < 0.003) on d 34 and 50 in the pregnant uterine horn compared with FM. There was a maternal nutritional plane × stage of gestation interaction (P = 0.01) on the vascular ratio (vascular volume/tissue volume) in maternal tissues. The RES heifers had a greater vascular ratio on d 16 compared with d 34 and 50; whereas, CON heifers had a greater vascular ratio on d 34 compared with d 16 and 50. In the NP uterine horn, there was also an increase (P = 0.02) in vascular volume of FM from CON heifers compared with FM from RES heifers. We conclude that maternal nutrient restriction did alter both vascularity and mRNA expression of angiogenic factor in utero-placental tissues during the establishment of pregnancy in first parity beef heifers.

Endogenous retroviral gene elements (syncytin-Rum1 and BERV-K1), interferon-τ, and pregnancy associated glycoprotein-1 are differentially expressed in maternal and fetal tissues during the first 50 days of gestation in beef heifers.

We hypothesized that the endogenous retroviruses [ERV: syncytin-Rum1 and (BERV-K1)], and pregnancy hormones [interferon-τ (IFN-τ), and pregnancy associated glycoprotein-1 (PAG-1)] would be differentially expressed whereas progesterone and insulin concentrations in maternal blood would remain steady during early gestation. To test this hypothesis Angus crossbred heifers (n = 46; ∼15 mo of age; BW = 363 ± 35 kg) were fed native grass hay, supplemented with cracked corn to gain 0.3 kg/d, and given ad libitum access to water. All heifers were subjected to a 5-d CO-Synch + CIDR estrous synchronization protocol and AI (breeding = d 0). Ovariohysterectomies were performed on d 16, 22, 28, 34, 40, and 50 of gestation and at d 16 of the estrous cycle for non-pregnant (NP) controls. Utero-placental tissues [maternal caruncle (CAR); maternal intercaruncular endometrium (ICAR); and fetal membranes, (FM, chorion on d 16, chorioallantois on d 22 to 50)] were collected from the uterine horn ipsilateral to the corpus luteum (CL). Tissues were flash frozen and stored at -80°C. Expression of mRNA was evaluated using qPCR. In CAR, syncytin-Rum1 expression was greater (P < 0.01) on d 50 (81.5-fold) compared with NP controls or any other day of early pregnancy. In contrast, syncytin-Rum1 expression in I-CAR only tended (P = 0.09) to change across days of early pregnancy and did not differ (P = 0.27) in FM tissues. In CAR, the expression of BERV-K1 was not different (P > 0.79) at d 16 and 22, was intermediate at d 28, 34, and 40, and was greatest on d 50 (108-fold increase compared with NP). Expression of BERV-K1 in FM was increased (P < 0.01) on d 28, 34, and 50 compared with NP controls, but at d 40 did not differ from NP controls. The mRNA expression of IFN-τ in FM at d 22 was greater (P < 0.01) than all other days of gestation. In CAR, expression of PAG-1 increased (P < 0.001) dramatically on d 40 (20,000-fold) and d 50 (86,000-fold) compared with NP heifers (P < 0.01). In ICAR, expression of PAG-1 was greater (P < 0.05) on d 28 and 40 (fold increases of 113 and 102, respectively, compared with NP). Insulin concentrations were not different (P = 0.53) but progesterone was greater (P < 0.01) on d 16, 22, 28, 34, and 40 compared with d 50 of gestation. These data confirm differential ERV, IFN-τ, and PAG-1 gene expression during critical time points of early gestation in utero-placental tissues.

Maternal nutrition and stage of early pregnancy in beef heifers: Impacts on expression of glucose, fructose, and cationic amino acid transporters in utero-placental tissues.

We hypothesized that maternal nutrition and day of gestation would impact utero-placental mRNA expression of the nutrient transporters , , , , and in beef heifers. Crossbred Angus heifers (n = 49) were estrous synchronized, bred via AI, assigned to nutritional treatment (CON = 100% of NRC requirements for 0.45 kg/d gain and RES = 60% of CON) and ovariohysterectomized on d 16, 34, or 50 of gestation (n = 6 to 9/d); Non-bred, non-pregnant (NB-NP) controls were fed the CON diet, not bred, and were ovariohysterectomized on d 16 of the synchronized estrous cycle = 6). The resulting arrangement of treatments was a 2 × 3 factorial + 1 (CON vs. RES × d 16, 34, or 50 + NB-NP controls). Caruncle (CAR), intercaruncular endometrium (ICAR), and fetal membranes (FM [chorioallantois]), were obtained from the pregnant uterine horn (the uterine horn containing the conceptus) immediately after ovariohysterectomy. On d 50 cotyledons (COT), intercotyledonary placenta (ICOT) and amnion (AMN) were also collected. Relative expression of nutrient transporters was determined for each tissue utilizing NB-NP-CAR and NB-NP-ICAR tissues as the baseline. For FM, NB-NP endometrium served as the baseline. There was no interaction of day × treatment ( ≥ 0.20) for any genes in CAR. However, CAR expression of was greater ( < 0.01) on d 16 compared with d 34 and 50, and , , and were greater ( ≤ 0.05) on d 34 compared with d 16 and 50. In ICAR, was the only gene to be influenced by the day × treatment interaction ( = 0.01), being greater in d 50 CON compared with d 34 CON and d 16 and 50 RES. In ICAR, expression of was greater ( < 0.01) on d 16 compared with d 34, and expression of was greater ( < 0.01) on d 34 and 50 compared with d 16. In FM, expression of was greater ( = 0.04) on d 16 compared with d 50 of gestation, and expression of was greater ( < 0.01) on d 34 and 50 compared with d 16. On d 50, expression of , , and expression were all greater ( < 0.05) in AMN compared with COT and ICOT, and expression of was greater ( < 0.01) in ICOT compared with COT and AMN. These data indicate that day was a more influential factor for mRNA expression of utero-placental glucose and cationic AA transporters than maternal nutritional status in heifers during early pregnancy.

Placental development during early pregnancy: Effects of embryo origin on expression of chemokine ligand twelve (CXCL12).

The aim was to localize chemokine ligand twelve (CXCL12) in sheep placental tissues during early gestation and after assisted reproductive technologies (ART). Uteri were collected from naturally (NAT) mated ewes and ewes receiving embryo transfer (ET), in vitro fertilization (IVF) or in vitro activation (IVA). CXCL12 was immunolocalized to endometrial stroma, glands, and trophoblast. Greater CXCL12 immunoreactivity was present in trophoblast on day 22 and 24 and in NAT ewes compared to IVF and IVA. Increased CXCL12 expression suggests CXCL12 promotes implantation and placentation. Decreased CXCL12 in IVF and IVA embryos, may compromise pregnancy establishment when utilizing ART methods.

Technical note: A new surgical technique for ovariohysterectomy during early pregnancy in beef heifers.

We hypothesized that a standing flank ovariohysterectomy procedure could be developed in beef heifers that would provide high quality tissues for addressing critical questions during early pregnancy, while concomitantly keeping livestock stewardship a high priority. To test the hypothesis, we: 1) developed a standing flank ovariohysterectomy procedure for use in beef heifers, and 2) implemented this procedure in a cohort of heifers up to d 50 of pregnancy for tissue collections, documentation of post-surgical recovery, and assessment of feedlot finishing performance. Ovariectomy and cesarean section protocols are well established in research and veterinary medicine and were used as starting points for procedural development. Crossbred Angus heifers ( = 46; ∼ 15 mo of age; BW = 362.3 ± 34.7 kg) were used to develop this new surgical tissue collection technique. Heifers were subjected to the 5-d CO-Synch + CIDR estrous synchronization protocol so ovariohysterectomy occurred at d 16, 22, 28, 34, 40, and 50 of gestation. Key aspects of the standing flank ovariohysterectomy technique included 1) use of local anesthetic for a standing flank incision, 2) locate the uterine and ovarian arteries via blind palpation and ligate them through the broad ligament via an improved clinch knot, 3) cut the ovaries and uterus free from the broad ligament, 4) ligate the cervix and uterine branch of the vaginal artery, and 5) cut through the cervix and remove the reproductive tract. Surgical times, from skin incision to placement of the last suture, were influenced ( = 0.04) by stage of gestation. In pregnant heifers, time decreased from d 22 (120.0 ± 12.0 min) of gestation to d 40 (79.5 ± 12.0 min) of gestation; then increased at d 50 (90.5 ± 14.7 min) of gestation. Using this procedure, we obtained uterine, placental, and embryo/fetal tissues that had experienced limited hypoxia, little or no trauma, and thus were excellent quality for scientific study. All heifers recovered from surgery quickly and were moved to a finishing period. During the finishing period, ovariohysterectomized heifers had a DMI of 13.8 kg, gained 1.99 ± 0.35 kg/d, and had a G:F of 0.145 over 132-d. The standing flank ovariohysterectomy technique represents a new and viable model to economically obtain high quality tissues for investigating critical biological mechanisms during early pregnancy in beef heifers.

Maternal environment and placental vascularization in small ruminants.

Uteroplacental development is a crucial step facilitating conceptus growth. Normal placental development comprises extensive placental angiogenesis to support fetoplacental transport, meeting the metabolic demands of the fetus. Compromised pregnancies due to maternal stressors such as over or undernutrition, maternal age or parity, altered body mass index, or genetic background result in altered vascular development of the placenta. This negatively affects placental growth and placental function and ultimately results in poor pregnancy outcomes. Nonetheless, the placenta acts as a sensor to the maternal stressors and undergoes modifications, which some have termed placental programming, to ensure healthy development of the conceptus. Sex steroid hormones such as estradiol-17ß and progesterone, chemokines such as chemokine ligand 12, and angiogenic/vasoactive factors such as vascular endothelial growth factors, placental growth factor, angiopoietins, and nitric oxide regulate uteroplacental development and hence are often used as therapeutic targets to rescue compromised pregnancies. Interestingly, the presence of sex steroid receptors has been identified in the fetal membranes (developing fetal placenta). Environmental steroid mimetics known as endocrine disrupting compounds disrupt conceptus development and lead to transgenerational impairments by epigenetic modification of placental gene expression, which is another area deserving intense research efforts. This review attempts to summarize current knowledge concerning intrinsic and extrinsic factors affecting selected reproductive functions with the emphasis on placental development.

Influence of nutrient restriction and melatonin supplementation of pregnant ewes on maternal and fetal pancreatic digestive enzymes and insulin-containing clusters.

Primiparous ewes (n=32) were assigned to dietary treatments in a 2×2 factorial arrangement to determine effects of nutrient restriction and melatonin supplementation on maternal and fetal pancreatic weight, digestive enzyme activity, concentration of insulin-containing clusters and plasma insulin concentrations. Treatments consisted of nutrient intake with 60% (RES) or 100% (ADQ) of requirements and melatonin supplementation at 0 (CON) or 5 mg/day (MEL). Treatments began on day 50 of gestation and continued until day 130. On day 130, blood was collected under general anesthesia from the uterine artery, uterine vein, umbilical artery and umbilical vein for plasma insulin analysis. Ewes were then euthanized and the pancreas removed from the ewe and fetus, trimmed of mesentery and fat, weighed and snap-frozen until enzyme analysis. In addition, samples of pancreatic tissue were fixed in 10% formalin solution for histological examination including quantitative characterization of size and distribution of insulin-containing cell clusters. Nutrient restriction decreased (P⩽0.001) maternal pancreatic mass (g) and α-amylase activity (U/g, kU/pancreas, U/kg BW). Ewes supplemented with melatonin had increased pancreatic mass (P=0.03) and α-amylase content (kU/pancreas and U/kg BW). Melatonin supplementation decreased (P=0.002) maternal pancreatic insulin-positive tissue area (relative to section of tissue), and size of the largest insulin-containing cell cluster (P=0.04). Nutrient restriction decreased pancreatic insulin-positive tissue area (P=0.03) and percent of large (32 001 to 512 000 µm2) and giant (⩾512 001 µm2) insulin-containing cell clusters (P=0.04) in the fetus. Insulin concentrations in plasma from the uterine vein, umbilical artery and umbilical vein were greater (P⩽0.01) in animals receiving 100% requirements. When comparing ewes to fetuses, ewes had a greater percentage of medium insulin-containing cell clusters (2001 to 32 000 µm2) while fetuses had more (P<0.001) pancreatic insulin-positive area (relative to section of tissue) and a greater percent of small, large and giant insulin-containing cell clusters (P⩽0.02). Larger insulin-containing clusters were observed in fetuses (P<0.001) compared with ewes. In summary, the maternal pancreas responded to nutrient restriction by decreasing pancreatic weight and activity of digestive enzymes while melatonin supplementation increased α-amylase content. Nutrient restriction decreased the number of pancreatic insulin-containing clusters in fetuses while melatonin supplementation did not influence insulin concentration. This indicated using melatonin as a therapeutic agent to mitigate reduced pancreatic function in the fetus due to maternal nutrient restriction may not be beneficial.

RAPID COMMUNICATION: Isolation of glucose transporters and in bovine uteroplacental tissues from days 16 to 50 of gestation.

Glucose transporter solute carrier family 2 member 14 () is a duplicon of glucose transporter solute carrier family 2 member 3 () with a 95% shared homology to and has not previously been isolated in ruminant uteroplacental tissues. The transporter has been previously isolated in Holstein heifer uterine epithelium but not in ovine epithelium. We hypothesized that and its duplicon would be found in bovine uteroplacental tissues and that maternal nutrition and day of gestation would impact mRNA expression of and . Crossbred Angus heifers ( = 49) were estrus synchronized, bred via AI, and assigned to nutritional treatment (CON = 100% of requirements to gain 0.45 kg/d; RES = 60% of CON) at breeding. Ovariohysterectomy was performed on d 16, 34, or 50 of gestation ( = 6 to 9/d); nonpregnant (NP) controls were not bred and ovariohysterectomized on d 16 of the synchronized estrous cycle ( = 6). The resulting treatment arrangement was a 2 × 3 factorial + 1. Uteroplacental tissues (caruncle, CAR; intercaruncular endometrium, ICAR; and fetal membrane [chorioallantois], FM) were obtained from the pregnant uterine horn immediately after ovariohysterectomy. For NP controls, only CAR and ICAR were obtained. There were no day × treatment interactions for or gene expression in CAR, ICAR, or FM. Expression of in CAR was greater ( = 0.03) on d 50 compared with d 16. In ICAR, was greatest ( = 0.02) on d 50 compared with d 16 and 34 of gestation. In FM, was greater ( = 0.04) on d 16 compared with d 50. Expression of was greater ( = 0.05) in pregnant compared with nonpregnant heifers. Additionally, expression of was greater ( = 0.01) on d 34 and 50 compared with d 16. Expression of in CAR was greater ( = 0.03) on d 50 compared to d 16 and 34. In CAR, tended ( = 0.07) to be greater on d 34 and 50 than on d 16 and was greater ( = 0.02) on d 50 than on d 34. There was no effect of treatment for either or in CAR, ICAR, or FM. These data demonstrate that glucose transporters and are expressed in beef heifer uteroplacental tissues and that they are expressed differentially by day of gestation in bovine uteroplacental tissues.

Nutrient transporters in bovine uteroplacental tissues on days sixteen to fifty of gestation.

During early gestation, nutrients are transported to the developing embryo via transporters in the uterine endometrium and chorioallantois. In the present study, we examined glucose transporters and and the cationic AA transporters , , and to test the hypotheses that 1) relative mRNA expression of transporters would be different among uteroplacental tissue type as gestation progresses and 2) concentrations of glucose and cationic AA would be different among target sites (placental compartments, serum, and histotrophic) and days of gestation. To test these hypotheses, crossbred Angus heifers ( = 46) were synchronized, bred via AI, and then ovariohysterectomized on d 16, 22, 28, 34, 40, or 50 of gestation (5 to 9/d) or not bred and ovariohysterectomized on d 16 of the synchronized estrous cycle ( = 7) to serve as nonpregnant (NP) controls. Uteroplacental tissues (maternal caruncle [CAR], intercaruncular endometrium [ICAR], and fetal membranes [FM; chorioallantois, d 22 and later]) were collected from the uterine horn ipsilateral to the corpus luteum immediately following ovariohysterectomy. Relative mRNA expression of the glucose transporters and cationic AA transporters was determined for each tissue from d 16 to 50 of gestation and from NP controls. Chorioallantoic, amniotic, and plasma fluids were collected from heifers on d 40 and 50 of gestation to determine concentrations of glucose and cationic AA. Expression of and showed a tendency ( < 0.10) toward being greater in d 16 ICAR and d 34 ICAR, respectively. Day × tissue interactions ( < 0.05) were present for , , and . Expression of was greater in d 50 CAR, expression of was greater on d 34 in ICAR, and expression of was greater in CAR tissue on d 34 compared with all other tissues and days of gestation. Glucose concentrations tended ( = 0.10) to be impacted by a day × fluid interaction. A day × fluid interaction ( = 0.01) for arginine concentration was observed, with greater concentrations in allantoic fluid on d 40 compared with all other days and fluid types. These data support our hypothesis that glucose and cationic AA transporters differ in their level of mRNA expression due to day of gestation and uteroplacental tissue type. In addition, concentrations of nutrients were differentially impacted by day, target site, and/or their respective interaction.

RAPID COMMUNICATION: Expression of an endogenous retroviral element, during early gestation in beef heifers.

Endogenous retroviral gene elements have been implicated in development and formation of the feto-maternal interface. A variant of the syncytin endogenous retroviral envelope gene family, , was recently found in ruminants. We hypothesized that mRNA would be differentially expressed in utero-placental tissues and would fluctuate during key time points of early gestation in beef heifers. Commercial Angus crossbred heifers ( = 46; ∼15 mo of age; BW = 362.3 ± 34.7kg) housed in 6-animal pens were fed daily with native grass hay and supplemented with cracked corn to gain 0.3 kg/d. The heifers were estrus synchronized, artificially inseminated, (d of breeding= d 0) and ovariohysterectomized on d 16, 22, 28, 34, 40, and 50 ( = 9, 6, 6, 7, 6, and 5, respectively) of gestation and at d 16 of the estrous cycle for non-bred, non-pregnant controls (NP; = 7). Harvested tissues were separated into maternal caruncle (CAR), intercarunclar endometrium (ICAR), and fetal membranes, (FM; chorioallantois, d 22 and later). All tissues were obtained from the ipsilateral uterine horn to the CL. Statistical analyses were conducted via the GLM procedure of SAS. Maternal CAR expression of was greater ( = 0.003) on d 50 by 81.5-fold compared to NP controls. At d 50 expression of in CAR was 190.3-fold greater than ( < 0.0001) ICAR. Fetal membranes had greater ( < 0.002) expression of from d 22 until d 50 of gestation compared to maternal ICAR (d 16 not analyzed). Expression of in FM was greater ( < 0.004) than in CAR until d 40 of gestation. Therefore, we conclude that is differentially expressed in utero-placental tissues and may be involved in the establishment of pregnancy. The expression of in maternal tissues is completely novel and indicates unique functions of syncytin in ruminant pregnancy.

Animal models of placental angiogenesis.

The study of the development of the fetal membranes is an ancient one, and the importance of placental vascular development to placental function has long been recognized. Animal models have been important in these studies, as they allow for controlled experiments and analysis of multiple time-points during pregnancy. Since the demonstration nearly 20 years ago that the placenta produces angiogenic factors, the major factors regulating placental angiogenesis have been identified. These major factors include vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), the angiopoietins (ANG), and their receptors. Recently, sophisticated computerized image analysis methods have been developed to establish the pattern of placental vascular development in sheep. The maternal placental capillary bed develops primarily by increased size of capillaries, with only small increases in capillary number or surface densities. In contrast, the microvasculature of the fetal placenta develops primarily by increased branching, resulting in a large increase in capillary number and surface densities. These observations help to explain the relatively large increase in umbilical blood flow and nutrient delivery to the fetus that occurs during the last half of gestation. In addition, expression of mRNAs for VEGF, bFGF, ANG, and their receptors have recently been correlated with normal placental vascular development in sheep, and further refinement of these mathematical models is warranted. Lastly, the recent development of animal models of compromised pregnancies, including those resulting from maternal nutrition (both restriction and excess), multiple fetuses, environmental stress (heat stress and high altitude), and fetal and maternal breed effects, has already indicated that reductions in placental vascular development and expression of angiogenic factors are probably a root cause of fetal growth restriction. With these methods and models now in place, we should soon be able to establish the mechanisms involved in both normal and abnormal placental angiogenesis.