Conceptus Growth Research Articles

3DMOUSEneST: a volumetric label-free imaging method evaluating embryo-uterine interaction and decidualization efficacy.

Effective interplay between the uterus and the embryo is essential for pregnancy establishment; however, convenient methods to screen embryo implantation success and maternal uterine response in experimental mouse models are currently lacking. Here, we report 3DMOUSEneST, a groundbreaking method for analyzing mouse implantation sites based on label-free higher harmonic generation microscopy, providing unprecedented insights into the embryo-uterine dynamics during early pregnancy. The 3DMOUSEneST method incorporates second-harmonic generation microscopy to image the three-dimensional structure formed by decidual fibrillar collagen, named 'decidual nest', and third-harmonic generation microscopy to evaluate early conceptus (defined as the embryo and extra-embryonic tissues) growth. We demonstrate that decidual nest volume is a measurable indicator of decidualization efficacy and correlates with the probability of early pregnancy progression based on a logistic regression analysis using Smad1/5 and Smad2/3 conditional knockout mice with known implantation defects. 3DMOUSEneST has great potential to become a principal method for studying decidual fibrillar collagen and characterizing mouse models associated with early embryonic lethality and fertility issues.

A critical review on garbhopghatkarabhava related to dietetics

Garbhopaghatakara Bhavas deals with concerns that may cause Upaghata (damage) to the growing fetus (Garbha). It includesAhara, viharaj and mansikfactors that are dangerous during pregnancy. Maternal Ahara has a crucial role in regulating maternal health and fetal growth. The word "Ambu" in Garbhasambhavasamagri refers to a nutrient-dense portion for good conceptus growth. Any defect in "Ambubhava" has a negative impact on pregnancy by preventing the growing fetus from receiving adequate nutrients. This paper discuss the Garbhopghatakarabhava related to Aharawith rationality. Scientific knowledge of these factors can improve birth outcomes by removing hazardous variables.

A review of progesterone roles in implantation

Implantation failure constitutes one of the major causes of pregnancy loss in all mammalian species. The complexity of implantation mechanism involves series of hormones, macromolecules, cytokines, chemokines, growth factors and repertoire of genes, however, progesterone receptor through which physiological responses to progesterone are initiated is the most essential for implantation across all mammalian species. Progesterone is also known as hormone of pregnancy because of its crucial roles to establish and sustain pregnancy and implantation. Progesterone facilitates blastocyst implantation to the maternal endometrium by promoting uterine secretion for conceptus growth and development, initiating window of receptivity, inducing quiescence and non-contractility of endometrium to avoid abortion and protection of embryo against maternal immune system. The aforementioned roles of progesterone and their mechanisms are the subject matter of this review.

A review of calcium and phosphorus requirement estimates for gestating and lactating sows

Abstract Calcium (Ca) and phosphorus (P) are minerals involved in biological functions and essential structural components of the skeleton. The body tightly regulates Ca and P to maintain homeostasis. Maternal needs for Ca and P increase during gestation and lactation to support conceptus growth and milk synthesis. Litter size and litter average daily gain (ADG) have a large effect on Ca and P requirements for sows because as they increase, the requirements increase due to a greater need from the sow. The objective of this review was to summarize published literature on Ca and P requirements in gestating and lactating sows derived from empirical data and factorial models. A total of nine empirical studies and seven factorial models were reviewed for determining the Ca and P requirements in gestation. For lactation, there were six empirical studies and seven factorial models reviewed. Empirical studies determined requirements based on the observed effect of Ca and P on bone mineralization, sow and litter performance, and milk characteristics. Factorial models generated equations to estimate Ca and P requirements using the main components of maintenance, fetal and placental growth, and maternal retention in gestation. The main components for factorial equations in lactation include maintenance and milk production. In gestation, the standardized total tract digestible phosphorus (STTD P) requirement estimates from empirical studies range from 5.4 to 9.5 g/d with total Ca ranging from 12.9 to 18.6 g/d to maximize bone measurements or performance criteria. According to the factorial models, the requirements increase throughout gestation to meet the needs of the growing fetuses and range from 7.6 to 10.6 g/d and 18.4 to 38.2 g/d of STTD P and total Ca, respectively, on day 114 of gestation for parity 1 sows. During lactation, STTD P requirement estimates from empirical studies ranged from 8.5 to 22.1 g/d and total Ca ranged from 21.2 to 50.4 g/d. For the lactation factorial models, STTD P requirements ranged from 14.2 to 25.1 g/d for STTD P and 28.4 to 55.6 g/d for total Ca for parity 1 sows with a litter size of 15 pigs. The large variation in requirement estimates makes it difficult to define Ca and P requirements; however, a minimum level of 6.0 and 22.1 g/d of STTD P during gestation and lactation, respectively, appears to be adequate to meet basal requirements. The limited data and high variation indicate a need for future research evaluating Ca and P requirements for gestating and lactating sows.

Low progesterone concentration in early pregnancy is detrimental to conceptus development and pregnancy outcome in horses

High progesterone concentrations in the early luteal phase support pregnancy, whereas subphysiological progesterone concentrations delay embryonic development at least until placentation. In this study, fetal growth and development of pregnancy was investigated in pregnancies with prostaglandin F2α-induced low progesterone concentrations (PGF) in the early luteal phase and control pregnancies (CON) in the same mares (n = 12). Mares were inseminated and in PGF pregnancies received the prostaglandin F2α analogue cloprostenol (62.5 µg) on days 0–3 after ovulation to induce subphysiological progesterone concentrations; CON pregnancies remained untreated. Mares were assigned to PGF or CON treatments in alternating order and received the opposite treatment in the following year. Blood was collected and conceptus size determined repeatedly by transrectal (≤day 101) and transabdominal (>day 101) ultrasonography. After birth, foals were weighed, measured and submitted to a clinical examination. Treatment PGF resulted in fewer pregnancies than CON treatment. All foals born from CON pregnancies were healthy and mature, whereas 4/7 PGF pregnancies were either lost (one embryonic death, one abortion) or resulted in the birth of compromised foals (P = 0.018). Size of the conceptus (e.g., diameter day 49: PGF 6.6 ± 0.7, CON 7.7 ± 0.7 cm, P = 0.006) and embryo proper (e.g., crown rump length day 54; PGF 4.4 ± 0.8, CON 5.8 ± 0.6 cm, P = 0.015) differed between treatments. These size differences decreased over time and at birth PGF foals did not differ significantly from CON foals. In conclusion, reduced progesterone concentration in the early luteal phase leads to delayed conceptus growth beyond placentation and increased pregnancy loss.

Induced endometrial inflammation compromises conceptus development in dairy cattle†.

Endometrial inflammation is associated with reduced pregnancy per artificial insemination (AI) and increased pregnancy loss in cows. It was hypothesized that induced endometritis alters histotroph composition and induces inflammatory signatures on conceptus that compromise development. In Experiment 1, lactating cows were assigned to control (CON; n = 23) or to an intrauterine infusion of Escherichia coli and Trueperella pyogenes (ENDO; n = 34) to induce endometritis. Cows received AI 26days after treatment, and the uterine fluid and conceptuses were collected on day 16 after AI. In Experiment 2, Holstein heifers were assigned to CON (n = 14) or ENDO (n = 14). An embryo was transferred on day 7 of the estrous cycle, and uterine fluid and conceptuses were recovered on day 16. Composition of histotroph and trophoblast and embryonic disc gene expression were assessed. Bacterial-induced endometritis in lactating cows altered histotroph composition and pathways linked to phospholipid synthesis, cellular energy production, and the Warburg effect. Also, ENDO reduced conceptus length in cows and altered expression of genes involved in pathogen recognition, nutrient uptake, cell growth, choline metabolism, and conceptus signaling needed for maternal recognition of pregnancy. The impact of ENDO was lesser on conceptuses from heifers receiving embryo transfer; however, the affected genes and associated pathways involved restricted growth and increased immune response similar to the observed responses to ENDO in conceptuses from lactating cows. Bacterial-induced endometrial inflammation altered histotroph composition, reduced conceptus growth, and caused embryonic cells to activate survival rather than anabolic pathways that could compromise development.

Effects of sub-physiological plasma progesterone concentration in the early luteal phase on embryonic and fetal development, pregnancy outcome and foal characteristics

High progesterone concentration in the early luteal phase supports pregnancy and postovulatory progestin treatment stimulates conceptus growth (Willmann et al., Theriogenology 2011;75,421) whereas reduced progesterone concentration changes histotroph composition (Beyer et al., Theriogenology 2019;125:236). Embryos obtained on day 34 from mares with reduced progesterone concentration were less developed than embryos from control pregnancies (Okada et al., Reprod Fert Develop 2020;32:1156). Sub-physiological progesterone concentrations in early pregnancy thus delay embryonic development at least until placentation. In this study, subsequent fetal growth, pregnancy loss and foal characteristics were investigated in pregnancies with reduced progesterone concentrations in the early luteal phase (PGF; n=7) and control pregnancies (CON; n=7) in the same mares. Estrous mares were inseminated every 48h until ovulation (day 0). Group PGF received 62.5µg of the prostaglandin F2α analogue cloprostenol daily on days 0-3 to induce sub-physiological progesterone concentrations whereas CON pregnancies remained untreated. Mares were assigned to PGF and CON treatments in alternating order and received the opposite treatment in the following year. Blood was obtained and conceptus size determined by transrectal (≤day 100) and transabdominal (>day 100) ultrasonography at regular intervals. IgG concentration was determined in colostrum at foaling and in foal plasma at 18-22 hrs after birth. Foals were weighed,measured, submitted to a clinical examination and the neutrophil / lymphocyte (N / L) ratio was determined as an indicator of maturity. Time from birth to first standing and first suckling was recorded. Data were analyzed with the SPSS statistics software, using two-way repeated measures ANOVA and χ2 analysis. Progesterone concentration was reduced in the first two weeks of PGF pregnancies (p<0.001; day 7 CON 35±3, PGF 11±2 ng/ml). All foals from CON pregnancies were healthy and mature, whereas 3/7 PGF pregnancies were lost (one embryonic death, one abortion, one non-viable foal, p=0.05). Size of the conceptus (day 49: PGF 6.6±07, CON 7.7±07 cm, p=0.006), embryo proper (day 54; PGF 4.4±0.8, CON 5.8±0.6 cm, p=0.015) and fetal orbita diameter (day 73 PGF 0.7±0.1, CON 1.0±0.1 cm, p=0.007) were smaller in PGF than in CON pregnancies. Size differences between PGF and CON pregnancies decreased over time and were no longer present at foaling. The viable PGF foals also did not differ in N / L ratio and time to standing and first suckling from CON foals In conclusion, reduced plasma progesterone concentration in the early luteal phase delays fetal growth beyond placentation and increases pregnancy loss. When viable foals are born, however, they are not compromised. Results indicate that pregnancies with sub-physiological progesterone concentrations will benefit from early progestogen substitution.

46 Adrenomedullin: A Novel Peptide Hormone for Uterine Receptivity and Conceptus Elongation in Pigs?

Abstract Adrenomedullin (ADM) is an evolutionarily conserved multi-functional peptide hormone in mammalian species. The implantation sites of conceptuses (embryo/fetus and its extra-embryonic membranes) in pregnant Adm+/− female mice are abnormally spaced leading to fetal crowding, intrauterine growth restriction (IUGR) and increases in pregnancy loss. As litter-bearing species, pigs exhibit a high incidence of early embryonic death (30-40%) and naturally occurring IUGR (15-25%), and significant numbers of stillborn piglets (3-9%). Therefore, it is imperative that the roles of ADM in regulation of uterine receptivity, as well as growth and development of conceptus in pigs be established. This study determined abundances of ADM in uterine luminal fluid, and the patterns of expression of ADM and its receptor components in uteri from cyclic and pregnant gilts, as well as conceptuses during the peri-implantation period of pregnancy. ADM receptor components include: calcitonin receptor-like receptor (CALCRL; G protein-coupled receptor bound by ADM), receptor activity modifying protein (RAMP2; dimerized with CALCRL to form ADM1 receptor) and RAMP3 (dimerized with CALCRL to form ADM2 receptor with lower specificity to ADM) and atypical chemokine receptor 3 (ACKR3; a decoy receptor that serves as a cell-autonomous molecular rheostat to dampen ADM signaling). Total recoverable ADM was greater in the uterine fluid of pregnant compared with cyclic gilts between days 10 and 16 post-estrus and was from uterine luminal epithelial (LE) and conceptus trophectoderm (Tr) cells. Uterine expression of CALCRL, RAMP2, and ACKR3 were affected by day (P &amp;lt; 0.05), pregnant status (P &amp;lt; 0.01) and/or day x status (P &amp;lt; 0.05). Within porcine conceptuses, expression of CALCRL, RAMP2 and ACKR3 increased between days 10 and 16 of pregnancy. Using an established porcine trophectoderm (pTr1) cell line isolated from elongating porcine conceptuses recovered on day 2 post-breeding, it was determined that 10-7 M ADM stimulated proliferation of pTr1 cells (P &amp;lt; 0.05) at 48 h, and increased phosphorylated mechanistic target of rapamycin (p-MTOR) and 4E binding protein (p-4EBP1) by 6.1- and 4.9-fold (P &amp;lt; 0.0001), respectively. These novel results indicate a significant role for ADM in uterine receptivity for implantation and conceptus growth and development in pigs. They also provide a framework for future studies of ADM signaling to affect proliferation and migration of Tr cells, spacing of blastocysts, implantation and placentation in pigs. This research was supported by Agriculture and Food Research Initiative Competitive Grant no. 2022-67015-36491 from the USDA National Institute of Food and Agriculture.

Heritability of beef cow metabolizable energy for maintenance.

Most of the metabolizable energy that a cow uses during a production year is for maintenance; however, less is known about the heritability of maintenance compared to other traits that can be measured directly. Feed intake is a heritable trait in the mature cow and most of the feed consumed is used for maintenance. We hypothesized that maintenance energy was a heritable trait. Individual feed intake was measured for 84 or 85 d on 5 yr old pregnant cows (N = 887) from a pedigreed population of cattle that represent prominent breeds in the United States. Phenotypic mean (± SD) values were 654 ± 68 kg for cow body weight, 0.21 ± 0.24 kg/d for average daily gain, and 175 ± 17 d for midpoint fetal age. Dry matter intake averaged (± SD) 10.84 ± 1.41 kg/d. Metabolizable energy for maintenance was estimated by subtracting the metabolizable energy used for conceptus growth and tissue accretion from metabolizable energy intake. Metabolizable energy for maintenance averaged (± SD) 139 ± 18 ME kcal/d/BW kg0.75 and had a heritability of 0.31 ± 0.11. Cows have a moderate heritability for maintenance suggesting an opportunity for selection.

Selenium supplementation and pregnancy outcomes

In vertebrates and invertebrates, selenium (Se) is an essential micronutrient, and Se deficiency or excess is associated with gonadal insufficiency and gamete dysfunction in both males and females, leading to implantation failure, altered embryonic development and, ultimately, infertility. During pregnancy, Se excess or deficiency is associated with miscarriage, pre-eclampsia (hypertension of pregnancy), gestational diabetes, fetal growth restriction and preterm birth. None of this is surprising, as Se is present in high concentrations in the ovary and testes, and work in animal models has shown that addition of Se to culture media improves embryo development and survival in vitro in association with reduced reactive oxygen species and less DNA damage. Selenium also affects uterine function and conceptus growth and gene expression, again in association with its antioxidant properties. Similarly, Se improves testicular function including sperm count, morphology and motility, and fertility. In animal models, supplementation of Se in the maternal diet during early pregnancy improves fetal substrate supply and alters fetal somatic and organ growth. Supplementation of Se throughout pregnancy in cows and sheep that are receiving an inadequate or excess dietary intake affected maternal whole-body and organ growth and vascular development, and also affected expression of angiogenic factors in maternal and fetal organs. Supplemental Se throughout pregnancy also affected placental growth, which may partly explain its effects on fetal growth and development, and also affected mammary gland development, colostrum yield and composition as well as postnatal development of the offspring. In conclusion, Se supplementation in nutritionally compromised pregnancies can potentially improve fertility and pregnancy outcomes, and thereby improve postnatal growth and development. Future research efforts should examine in more detail and more species the potential benefits of Se supplementation to reproductive processes in mammals.

Maternal and Fetal PI3K-p110α Deficiency Induces Sex-Specific Changes in Conceptus Growth and Placental Mitochondrial Bioenergetic Reserve in Mice.

Simple SummaryThe placenta is responsible for materno-fetal resource allocation. Placental insufficiency may disrupt fetal growth trajectory, with negative consequences for maternal and fetal health. By manipulating a growth-controlling protein named PI3K-p110α, this work, performed on mice, shows the relevance of the maternal environment and fetal genotype in determining the conceptus growth and mitochondria bioenergetic reserve in the placental transport zone. Interestingly, the disruption of maternal and/or fetal PI3K-p110α exerted these effects in a sex-specific manner, with male fetuses more affected than females. These data highlight the importance of the sex of the fetus and placental mitochondrial function in the regulation of fetal growth.Fetal growth is reliant on placental formation and function, which, in turn, requires the energy produced by the mitochondria. Prior work has shown that both mother and fetus operate via the phosphoinositol 3-kinase (PI3K)-p110α signalling pathway to modify placental development, function, and fetal growth outcomes. This study in mice used genetic inactivation of PI3K-p110α (α/+) in mothers and fetuses and high resolution respirometry to investigate the influence of maternal and fetal PI3K-p110α deficiency on fetal and placental growth, in relation to placental mitochondrial bioenergetics, for each fetal sex. The effect of PI3K-p110α deficiency on maternal body composition was also determined to understand more about the maternal-driven changes in feto-placental development. These data show that male fetuses were more sensitive than females to fetal PI3K-p110α deficiency, as they had greater reductions in fetal and placental weight, when compared to their WT littermates. Placental weight was also altered in males only of α/+ dams. In addition, α/+ male, but not female, fetuses showed an increase in mitochondrial reserve capacity, when compared to their WT littermates in α/+ dams. Finally, α/+ dams exhibited reduced adipose depot masses, compared to wild-type dams. These findings, thus, demonstrate that maternal nutrient reserves and ability to apportion nutrients to the fetus are reduced in α/+ dams. Moreover, maternal and fetal PI3K-p110α deficiency impacts conceptus growth and placental mitochondrial bioenergetic function, in a manner dependent on fetal sex.

Insulin-like growth factor system components expressed at the conceptus-maternal interface during the establishment of equine pregnancy.

In many species, the insulin-like growth factors (IGF1 and IGF2), their receptors and IGF binding proteins play important roles in preparing the endometrium for implantation, and regulating conceptus growth and development. To determine whether the IGF system may contribute to conceptus-maternal interaction during equine pre-implantation development, we evaluated mRNA expression for IGF system components in conceptuses, and endometrium recovered from pregnant and cycling mares, on days 7, 14, 21 and 28 after ovulation. We also investigated expression of IGF1, IGF2 and their receptors 6 and 11 days after transfer of day 8 embryos to synchronous (day 8) or asynchronous (day 3) recipient mares. Expression of IGF1 and IGF2, IGF1R, IGF2R, INSR and IGFBPs 1, 2, 4 and 5 was evident in endometrium and conceptus membranes during days 7–28. Endometrial IGF2, INSR, IGFBP1 and IGFBP2 expression increased between days 7 and 28 of pregnancy. In conceptus membranes, expression of all IGF system components increased with developmental stage. Immunohistochemistry revealed strong expression of IGF1, IGF2 and IGF1R in both endometrium and conceptus membranes, whereas INSR was highly expressed in endometrium but barely detectable in the conceptus. Finally, a negatively asynchronous uterine environment retarded IGF1, IGF2 and INSR expression in the conceptus, whereas in the endometrium only INSR expression was altered by asynchrony. The presence of IGFs, their receptors and IGFBPs in the endometrium and conceptus during early equine pregnancy, and down-regulation in the conceptus following asynchronous embryo transfer, suggest a role in conceptus-maternal communication during the preparation for implantation.

Effects of Mid-Gestation Nutrient Restriction, Realimentation, and Parity on the Umbilical Hemodynamics of the Pregnant Ewe

Previous studies have reported that nutritional restriction from days 50 to 130 applied in young nulliparous ewes reduces umbilical blood flow (UBF). We hypothesized that during restriction, UBF and fetal and placentome dimensional measurements would decrease compared to adequately fed ewes, but upon realimentation, ewes would have similar UBF as ewes that were not restricted. We also hypothesized that multiparous ewes would be more resilient to nutrient restriction compared to nulliparous ewes. In experiment 1, second-parity Dorset ewes carrying singletons were assigned to an adequate nutrition group (CON, n = 7) or a restricted (60% of CON) group (RES, n = 8), from days 50 to 90 of gestation. In experiment 2, on day 50 of gestation, adult (15-month) nulliparous (NUL; n = 12) and multiparous (MUL; n = 16) Dorset ewes carrying singletons were randomly assigned to receive 100% of NRC recommendations (CON) or 60% of CON (RES). On day 90, all ewes were fed 100% of nutritional recommendations according to body weight. Ewe body weight and conceptus measurements via ultrasonography were recorded every 10 days from days 50 to 130 of gestation. We measured 10 random placentomes, fetal biparietal and abdominal length, and kidney length and width. Doppler mode was used to obtain UBF, pulsatility index (PI), and resistance index (RI). Lamb weight and parturition problems were recorded. In experiment 1, on day 80, UBF decreased (P ≤ 0.05 means separation of unprotected F test), placentome size tended to decrease (P ≤ 0.10), and PI and RI tended to increase in RES vs. CON ewes (P ≤ 0.10). In experiment 2, there were no three-way interactions or main effects of treatments on UBF, PI, RI, and placentome size (P ≥ 0.57). There was a parity-by-day interaction (P &amp;lt; 0.05) for RI, but UBF was not affected by parity or diet. After realimentation, there was no effect of treatment on ultrasound measurements in both experiments. At birth, lambs and placental measurements were not different (P ≥ 0.43). Restriction from days 50 to 90 does not seem to influence umbilical hemodynamics or conceptus growth in adult white face sheep, regardless of parity.

Variability of bovine conceptus-related volumes in early pregnancy measured with transrectal 3-dimensional ultrasonography

Up until now, bovine fetometry has been entirely based on 2-dimensional ultrasonography. Fetal size is estimated by several linear measurements such as crown-rump length (CRL). However, the advent of 3-dimensional ultrasonography (3D-US) provides in vivo access to the volumes of the fetus and its amniotic sac. The objective of this preliminary observational study was to determine the variability of conceptus-related volumes using transrectal 3D-US in dairy cows and to identify factors affecting them. Furthermore, relationships between the gained measurements and calf birth weight were investigated. In total, 315 Simmental and Holstein-Friesian dairy cows were transrectally examined at d 42 after breeding using a portable ultrasound device (Voluson I, GE Healthcare). Gestational volumes including fetal volume (FV) and amniotic sac volume (ASV) were determined with the software tool VOCAL (Virtual Organ Computer-Aided Analysis, GE Healthcare), whereas amniotic fluid volume (AFV) values were derived from the subtraction of FV from ASV. The CRL was determined by means of 3-dimensional data. The mean values and standard deviations for FV, ASV, AFV, and CRL were 1.47 ± 0.25 cm3, 5.86 ± 1.22 cm3, 4.38 ± 1.02 cm3, and 2.38 ± 0.18 cm, respectively. All gestational volumes and CRL values were affected by breed. In Simmental cattle, larger concepti were observed compared with pregnancies derived from Holstein-Friesian animals. Parity affected only ASV and AFV, with heifers showing greater values than lactating cows. The CRL was positively associated with milk protein content. It was not possible to predict calf weight at birth by using FV, ASV, or AFV; however, tendencies were found for ASV and AFV. The present study was the first to adopt 3D-US volumetry to assess early pregnancy development in dairy cattle. Our results showed that this method could be used successfully to identify minor variations in conceptus growth.

Early Pregnancy in Jennies in the Caribbean: Corpus Luteum Development and Progesterone Production, Uterine and Embryo Dynamics, Conceptus Growth and Maturation.

Simple SummaryAn understanding of the basic mechanisms of reproduction in donkeys is essential, for several reasons. Some donkey breeds are threatened or endangered, and efforts to save these species depend on improved knowledge of reproductive processes. In some parts of the world, donkeys continue to be valued for purposes of work, recreation, or even meat or milk production, as well as the breeding of mules, and reproduction is essential to maintain suitable populations. In others, donkey populations have become feral and represent a nuisance or even a danger to human populations, and improved contraceptive methods are required. Whether for enhancing or inhibiting reproduction, species-specific information is valuable. While the mare has been extensively studied, few studies have explored early pregnancy in jennies. Therefore, this study characterized early embryo development and differences in progesterone profile and changes in the corpus luteum between pregnant and non-pregnant jennies.We aimed to characterize early embryo development and changes in corpus luteum (CL) development and progesterone profile in pregnant vs. non-pregnant jennies. Eight jennies were enrolled in the study. In the first two cycles, the jennies were monitored by transrectal ultrasonography and had blood harvested for hormone profile assay. In the third cycle, jennies were bred by a jack of proven fertility. Jennies were then monitored and sampled for up to 30 days of pregnancy. Data were evaluated by random-effects multiple linear regression, and correlations were expressed as Pearson’s correlation coefficient. Progesterone concentration rose rapidly from ovulation (D0) until D7, plateaued until D12–14, then precipitously declined between D14 and 15, remaining low until the next ovulation in non-pregnant cycles. In the pregnant jennies, the progesterone concentration rose to maximal concentrations on D7–11, being higher at this stage than in non-pregnant cycles, then declined gradually up to D30. In all cycles, the volume of the CL increased steadily until D6, when it plateaued in pregnant jennies. For non-pregnant jennies, CL volume decreased slowly from D6 to D11 and then had a faster drop. Uterine tone increased following ovulation, becoming turgid around the day of embryo fixation (D15.0 ± 0.9). An embryonic vesicle (EV) was first detected on D9.3 ± 0.5 (2.4 ± 0.5 mm). The EV remained spherical until D18.6 ± 1.4. The embryo proper was first detected ventrally in the vesicle on D20.8 ± 1.1 and the embryonic heartbeat by D22.0 ± 0.9. The allantoic sac was identified at D24.0 ± 0.9, and at D30, the allantoic sac filled the ventral half of the EV. This study provides evidence that higher cumulative concentrations of progesterone are correlated to size of the EV, and there were changes in the luteal dynamics and progesterone profiles in pregnant vs. non-pregnant jennies.

Temporal and spatial expression of adrenomedullin and its receptors in the porcine uterus and peri-implantation conceptuses.

Adrenomedullin (ADM) is an evolutionarily conserved multifunctional peptide hormone that regulates implantation, embryo spacing, and placentation in humans and rodents. However, the potential roles of ADM in implantation and placentation in pigs, as a litter-bearing species, are not known. This study determined abundances of ADM in uterine luminal fluid, and the patterns of expression of ADM and its receptor components (CALCRL, RAMP2, RAMP3, and ACKR3) in uteri from cyclic and pregnant gilts, as well as conceptuses (embryonic/fetus and its extra-embryonic membranes) during the peri-implantation period of pregnancy. Total recoverable ADM was greater in the uterine fluid of pregnant compared with cyclic gilts between Days 10 and 16 post-estrus and was from uterine luminal epithelial (LE) and conceptus trophectoderm (Tr) cells. Uterine expression of CALCRL, RAMP2, and ACKR3 were affected by day (P < 0.05), pregnant status (P < 0.01) and/or day x status (P < 0.05). Within porcine conceptuses, the expression of CALCRL, RAMP2, and ACKR3 increased between Days 10 and 16 of pregnancy. Using an established porcine trophectoderm (pTr1) cell line, it was determined that 10-7M ADM stimulated proliferation of pTr1 cells (P < 0.05) at 48h, and increased phosphorylated mechanistic target of rapamycin (p-MTOR) and 4E binding protein 1 (p-4EBP1) by 6.1- and 4.9-fold (P < 0.0001), respectively. These novel results indicate a significant role for ADM in uterine receptivity for implantation and conceptus growth and development in pigs. They also provide a framework for future studies of ADM signaling to affect proliferation and migration of Tr cells, spacing of blastocysts, implantation, and placentation in pigs.

141 Mineral Requirements of Dairy Cattle: Emphasis on organic minerals

Abstract Requirements for most minerals are expressed on an absorbed basis. The true absorption of minerals can vary widely because of source, presence of antagonists, dietary concentrations, and animal mineral status; therefore, expressing mineral requirements on an absorbed basis is theoretically sound. Unfortunately measuring true absorption of minerals is exceedingly difficult; available data is limited which means that often we are using constants. Requirements for lactation, growth, and conceptus growth are known with reasonable certainty; however establishing maintenance requirements is plagued with methodological difficulties, and the classical definition of maintenance (i.e., replenishment of inevitable fecal and urinary losses) ignores effects on water balance, acid-base balance in the rumen, and the ruminal and intestinal microbiome. For Na, Cl- and K, absorption is essentially 100% regardless of source. Lab methods are available to estimate absorption of P. Source of Ca accounts for the majority of the variation in Ca absorption and constants are available for feedstuffs and supplements. We have good data on absorption of Mg from basal diets and are able to estimate antagonism of Mg absorption caused by K. We are less able to estimate absorption of the various Mg supplements, some of which are highly variable. Except for the electrolytes (which affect water balance), requirements are known with reasonable certainty for macrominerals. Absorption coefficients for trace minerals (TM) are known with much less certainty. Source of supplemental TM (e.g., organic vs sulfates) can affect absorption but the effect depends on the mineral TM (e.g., source affects absorption of Cu more than that of Mn) and on interactions with basal diet. Maintenance requirements are generally poorly defined for TM and because of ‘non-factorial’ requirements such as effects on microbiome, source of TM likely affects requirements. This means that for some TM, the factorial approach to requirements may not be adequate.

Hemodynamics of the umbilical artery and conceptus-related measurements in singleton and twin pregnancies in ewes

In sheep, adequate physiological maternal adaptations to twin fetuses are key for their prenatal development. We hypothesized that there is a relationship between maternal circulating steroid hormone concentration, hematocrit, and patterns of conceptus growth and umbilical artery hemodynamics in twin vs singleton pregnancies. Ewes carrying singletons (n = 6) or twins (n = 7) were selected. Hematocrit, progesterone (P4) and estradiol-17β (E2) concentrations were analyzed. Blood flow of the umbilical artery (UBF) was assessed through Doppler ultrasonography. Ewes carrying twins had a decreased (p = 0.03) hematocrit. There was a tendency (p = 0.08) for ewes carrying twins to have increased P4 from day 20 to 40. From day 50 to 70, P4 tended to be increased (p = 0.07) and E2 was increased (p = 0.01) in ewes carrying twins. From day 90 to 120, ewes carrying twins had greater (p ≤ 0.04) P4. Ewes carrying twins had increased (p < 0.01) P4 and tended to have (p = 0.06) increased E2. From day 60 until 110, placentomes from twins were larger (p ≤ 0.05). On day 50, UBF was greater (p = 0.04; 27.38 vs. 20.95 ± 1.99 ml/min) in twins. It has been suggested that physiological adaptations during early pregnancy can significantly impact fetal development in multiple offspring carrying ewes. Our findings suggest that increased umbilical artery blood flow early in gestation, associated with increased maternal concentrations of P4 and E2, and decreased hematocrit (related to plasma volume) could contribute to those adaptations.

Evidence of metabolic compartmentation in the bovine placenta and significance for the regulation of placental function and fetal growth in pregnancies bearing in vivo- or in vitro-produced embryos

The concept of metabolic zonation in the liver proposes that opposing or complementary metabolic pathways are carried out within distinct regions of the liver lobule to maintain optimal metabolic homeostasis. As bovine placentomes differ in mass and shapes within the reproductive tract, which is even more pronounced in cloned-derived concepti, the aims of this study were to investigate the occurrence of metabolic zonation in the bovine placenta by placentome shape and size, and to compare placental functions between control and cloned concepti, which may relate to the abnormal conceptus growth after cloning. Bovine pregnancies established after superovulation/AI (n=4), in vitro fertilization (n=4) or cloning by nuclear transfer (n=6) were slaughtered on Day 225 of gestation. Placentomes were excised, weighed, measured, and classified by shape as types A, B, C, or D; cDNA from each placentome type/group was used for RT-qPCR analyses of 24 target gene products related to carbohydrate and lipid metabolism, placental function, IGF system and apoptosis. No differences in gene expression were detected between clones and controls. However, the gene expression pattern revealed differences in metabolic profile only between placentome types, with an increase in expression in type C in relation to type A placentomes, in special for IGF2 (5.8-fold) and IGF2r (1.6-fold), glucose transporters (1.2-fold), and fructogenesis (2.0- to 2.3-fold), suggesting a potential metabolic zonation in the bovine placenta. Morphological changes in the placenta of clones may influence placental function and metabolic compartmentalization, likely disturbing placental control on fetal growth and programming towards the end of pregnancy.

Characterization and regulation of extracellular vesicles in the lumen of the ovine uterus†

Secretions of the endometrium are vital for peri-implantation growth and development of the sheep conceptus. Extracellular vesicles (EVs) are present in the uterine lumen, emanate from both the endometrial epithelia of the uterus and trophectoderm of the conceptus, and hypothesized to mediate communication between those cell types during pregnancy establishment in sheep. Size-exclusion chromatography and nanoparticle tracking analysis determined that total EV number in the uterine lumen increased from days 10 to 14 of the cycle but was lower on days 12 and 14 of pregnancy in sheep. Intrauterine infusions of interferon tau (IFNT) did not affect total EV number in the uterine lumen. Quantitative mass spectrometric analyses defined proteins and lipids in EVs isolated from the uterine lumen of day 14 cyclic and pregnant sheep. In vitro analyses found that EVs decreased ovine trophectoderm cell proliferation and increased IFNT production without effects on gene expression as determined by RNA-seq. Collective results support the idea EVs impact conceptus growth during pregnancy establishment via effects on trophectoderm cell growth.