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.

Effect of ewe birth litter size and estimation of genetic parameters on ewe reproductive life traits.

Ewe lifetime productivity has economic implications for producers because shorter lifetime productivity results in less profit. Productive years of ewes from extensive, range-based systems of the United States West are generally less than ewes from more temperate regions of the United States. Accordingly, ewes from range-based systems, especially those employing shed-lambing strategies, have been selected for increased litter size to offset decreased lifetime productivity. However, the relationship of the ewe's birth litter size (ELSB) has not been considered a potential contributor to lifetime productivity. Longevity (number of productive years, n = 1 per population) and stayability (probability to survive to the next age; ages 2-7 years, n = 6 per population) were investigated to understand ELSB effects on productive life. Columbia, Polypay, Rambouillet, and Targhee breeds were used in this study. Across-breed (n = 11 550) and within-breed (Columbia, n = 4 398; Polypay, n = 4 534; Rambouillet, n = 5 922; Targhee, n = 6 482) analyses were used. Depending on the population, records spanned from 1950 to 2008, where ewe's birth year was included as a fixed effect in the animal model using restricted maximum likelihood estimation procedures. Fixed effects investigated included ELSB (single-, twin-, or triplet-born) and ewe breed (across-breed analyses only). Regardless of trait or population used, heritability ranged from 0.06 ± 0.02 to 0.34 ± 0.03, where stayability at younger ages had the highest estimates. The breed effect was significant in all across-breed analyses (0.0001 ≤ P ≤ 0.038; n = 7), where Polypay, a breed selected for accelerated lambing and increased fertility, averaged shorter productive life or had a lower probability of survival to the next age compared with other breeds (longevity: 0.009 ≤ P ≤ 0.223; stayability: 0.000 ≤ P ≤ 0.842). The ELSB was significant in 60% (n = 5) and 37% (n = 30) of longevity and stayability analyses, respectively. Except for Targhee, all analyses showed ewes born in smaller litter sizes were associated with longer productive lives or higher probability of surviving to the next age, particularly in across-breed analyses (e.g., longevity: single- vs twin-born ewes, P = 0.004; vs triplet-born ewes, P = 0.003). This study provides evidence that increasing prolificacy in ewes from extensive, range-based production systems may impact productive life. Due to the low heritability of these traits, additional investigation into modeling these traits with dominance effects and litter size needs to be conducted.

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 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.

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.

Gap junctional connexin messenger RNA expression in the ovine uterus and placenta: effects of estradiol-17ß-treatment, early pregnancy stages, and embryo origin.

Gap junctions play a major role in direct, contact-dependent cell-cell communication, and they have been implicated in the regulation of cellular metabolism and the coordination of cellular functions during growth and differentiation of organs and tissues. Gap junctional channels, composed of connexin (Cx) proteins, have been detected and shown to be influenced by hormones (eg, estrogen and progesterone) in uterine and placental tissues in several species. We hypothesized that (1) the messenger RNA (mRNA) for Cx26, Cx32, Cx37, and Cx43 is expressed in the uterus of ovariectomized sheep treated with estradiol-17ß (E2) and in ovine placenta during early pregnancy, (2) E2-treatment of ovariectomized ewes would cause time-specific changes in Cx26, Cx32, Cx37, and Cx43 mRNA expression (experiment 1), and (3) expression of these 4 Cx would vary across the days of early pregnancy (experiment 2) and will be affected by embryo origin (ie, after application of assisted reproductive technologies [ARTs]; experiment 3). Thus, we collected uterine tissues at 0 to 24 h after E2 treatments (experiment 1), and placental tissues during days 14 to 30 of early pregnancy after natural (NAT) breeding (experiment 2) and on day 22 of early pregnancy established after transfer of embryos generated through natural breeding (NAT-ET), in vitro fertilization (IVF), or in vitro activation (IVA, parthenotes; experiment 3). In experiment 1, the expression of Cx26, Cx37, and Cx43 mRNA increased (P < 0.05) and Cx32 mRNA decreased (P < 0.06) in both caruncular and intercaruncular tissues after E2 treatment. In experiment 2, during early pregnancy, there were significant changes (P < 0.01) across days in the expression of Cx26, Cx37, and Cx43 mRNA in the maternal placenta, accompanied by changes (P < 0.001) in Cx37 and Cx43 mRNA in the fetal placenta. In experiment 3, in maternal placenta, Cx32 mRNA expression was decreased (P < 0.001) in NAT-ET, IVF, and IVA groups compared to the NAT group; but in fetal placenta, Cx32 mRNA expression was increased (P < 0.05) in NAT-ET, IVF and IVF groups, and Cx26 mRNA expression was increased (P < 0.05) in IVA compared to NAT group. These data suggest that Cx26, Cx32, Cx37, and Cx43 play specific roles in E2-regulated uterine function and in placental development during early gestation both after natural mating and with application of ART.

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.

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.

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.

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.

Impacts of supplemental arginine on the reproductive performance of fall lambing ewes.

In sheep, embryonic and fetal death during pregnancy can account for 25% to 50% of the total number of corpora lutea (and thus potential embryos). The objective of this study was to determine the effects of injectable and oral Arg supplementation provided for 14 d postbreeding on the reproductive performance of naturally stimulated fall lambing ewes. Rambouillet ewes ( = 210) were exposed to rams equipped with marking harnesses to induce cyclicity in April 2012. Upon estrus detection (d 0) ewes were randomly assigned, in a completely random design, to 1 of 6 treatments for a 14-d treatment period: injectable saline (CON; = 25), injectable Ala (IVALA; = 20), injectable Arg (IVARG; = 23), oral rumen-protected Arg (RPARG; = 20), oral fish meal (FM; = 24), or oral soybean meal (SBM; = 23). Daily treatments, except CON, IVALA, and SBM, were formulated to provide supplemental Arg at 30 mg·kg BW·d and were provided at 0800 h daily. Ewes receiving injectable treatments were provided 454 g corn/d postinjection, whereas ewes receiving oral supplements were provided a ground ration of their respective treatments with corn individually at 0800 h daily. Plasma and serum samples were collected on d 0, 2, 4, 6, 8, 10, 12, and 14 from 12 ewes per treatment to evaluate plasma progesterone and serum AA concentrations. At lambing, birth weight, birth type, and sex were recorded. Weaning weights were recorded when the average age of lambs was 85 d. No differences ( ≥ 0.39) were detected for pregnancy, prolificacy, and lambing rates or lamb birth weights among treatments. However, litter weaning weight tended to be greater ( = 0.06) and weaning rates were greater ( = 0.05) in Arg-injected ewes (1.09, 0.95, 1.29, 0.72, 1.00, and 0.86, respectively). Plasma progesterone and serum Arg concentrations showed a treatment and day effect ( < 0.001), but no treatment × day interaction ( ≥ 0.99) was observed. In contrast to previous research, supplemental Arg during the first 14 d of pregnancy did not improve pregnancy or lambing rates; however, IVARG did positively impact weaning rates.

TRIENNIAL REPRODUCTION SYMPOSIUM: Developmental programming of fertility.

The 2015 Triennial Reproduction Symposium focused on developmental programming of fertility. The topics covered during the morning session included the role of the placenta in programming of fetal growth and development, effects of feeding system and level of feeding during pregnancy on the annual production cycle and lifetime productivity of heifer offspring, effects of litter size and level of socialization postnatally on reproductive performance of pigs, effects of postnatal dietary intake on maturation of the hypothalamic-pituitary-gonadal axis and onset of puberty in heifers, effects of housing systems on growth performance and reproductive efficiency of gilts, and effects of energy balance on sexual differentiation in rodent models. The morning session concluded with presentation of the American Society of Animal Science L. E. Casida Award for Excellence in Graduate Education to Dr. Michael Smith from the University of Missouri, Columbia, who shared his philosophy of graduate education. The afternoon session included talks on the role of epigenetic modifications in developmental programming and transgenerational inheritance of reproductive dysfunction, effects of endocrine disrupting compounds on fetal development and long-term physiology of the individual, and potential consequences of real-life exposure to environmental contaminants on reproductive health. The symposium concluded with a summary talk and the posing of 2 questions to the audience. From an evolutionary standpoint, programming and epigenetic events must be adaptive; when do they become maladaptive? If there are so many environmental factors that induce developmental programming, are we doomed, and if not, what is or are the solution or solutions?

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.

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.

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.

Placental development during early pregnancy in sheep: estrogen and progesterone receptor messenger RNA expression in pregnancies derived from in vivo-produced and in vitro-produced embryos.

Sex steroids are important regulators of angiogenesis and growth in reproductive tissues, including the placenta. In experiment (exp.) 1, to examine the expression of a suite of sex steroid receptors throughout early pregnancy, maternal (caruncular [CAR]) and fetal (fetal membranes [FM]) placental tissues were collected on days 14 to 30 after mating and on day 10 after estrus (nonpregnant controls). In exp. 2, to examine the hypothesis that assisted reproductive technology would affect the expression of the same suite of sex steroid receptors, pregnancies were achieved through natural mating (NAT) or transfer of embryos from natural mating (NAT-ET), in vitro fertilization (IVF), or in vitro activation (IVA), and CAR and FM were collected on day 22. In exp. 1, for CAR messenger RNA (mRNA) expression of estrogen receptors (ESR) 1 and 2, nuclear (n) progesterone receptors (PGR) and membrane (m) PGRα, ß, and γ were affected (P < 0.02) by pregnancy stage, as were ESR1, nPGR, and mPGRα, ß, and γ for FM (P < 0.03). In exp. 2, for CAR, mRNA expression of ESR1 and nPGR was decreased (P < 0.001) in NAT-ET, IVF, and IVA groups compared with NAT. For FM, mRNA expression of ESR1 tended to be greater (P = 0.10) in the IVA group compared with NAT and NAT-ET, and GPER1 was greater (P < 0.05) in NAT-ET and IVF compared with NAT. These data establish the normal pattern of sex steroid receptor mRNA expression in maternal and fetal placenta during early pregnancy in sheep, and in addition, suggest that altered expression of placental sex steroid receptors may be an early event leading to poor placental vascularization and growth after assisted reproductive technology.

Activation of the CXCL12/CXCR4 signaling axis may drive vascularization of the ovine placenta.

Early pregnancy, when most embryonic losses occur, is a critical period in which vital placental vascularization is established. Vascular endothelial growth factor (VEGF) is a potent inducer of angiogenesis, and factors that regulate VEGF function, expression, or both may ultimately affect vascularization. Activation of the C-X-C chemokine receptor type 4 (CXCR4) by its cognate ligand, C-X-C chemokine ligand 12 (CXCL12), increases VEGF synthesis and secretion, which in turn stimulates CXCL12 and CXCR4 production and this synergistic regulation may influence placental vascularization. We hypothesized that expression of CXCL12, CXCR4, select angiogenic factors, and their receptors would increase in placental tissues during early pregnancy and that treatment of ovine trophectoderm cells with CXCL12 would increase production of angiogenic factors. To test this hypothesis, maternal caruncle (CAR) and fetal extraembryonic membrane (FM) tissues were collected on days 18, 20, 22, 25, 26, and 30 of pregnancy and on day 10 of the estrous cycle (control, NP) to determine relative mRNA or protein expression of CXCL12 and CXCR4 and selected angiogenic factors. In CAR, expression of mRNA for CXCR4 increased on day 18, 20, 22, and 25 and CXCL12 increased on day 18 and 20 compared with NP ewes. CXCL12 protein followed a similar pattern in CAR tissue, with greater levels on day 20 than in NP tissue. Greater levels of fibroblast growth factor 2 (FGF2) mRNA was observed in CAR on day 20 of gestation than on day 30. In FM, CXCL12, CXCR4, angiopoietin 1, VEGF, and VEGF receptor 1 were enhanced with advancing pregnancy, whereas FGF2 and kinase insert domain receptor (or VEGF receptor 2) peaked on day 25. An increase in protein levels occurred on day 25 compared with day 20 in FM for CXCL12 and CXCR4, as well as a similar tendency for FGF2 protein. Both CXCL12 and CXCR4 are specifically localized to trophoblast cells and to the uterine luminal and glandular epithelium. Treatment of ovine trophectoderm cells with CXCL12 increased mRNA expression for VEGF and FGF2. The relationship between VEGF, FGF2, and the CXCL12/CXCR4 signaling underscores the potential role for this chemokine axis in driving placentation.