We examined the hypothesis that maternal nutrition and day of gestation would affect the concentrations of AAs and hexoses in bovine utero-placental fluids and maternal serum from days 16 to 50 of gestation. Forty-nine cross-bred Angus heifers were bred via artificial insemination and fed a control diet (CON = 100% of requirements for growth) or a restricted diet (RES = 60% of CON) and ovariohysterectomized on days 16, 34, or 50 of gestation; nonpregnant controls were not bred and ovariohysterectomized on day 16 of the synchronized estrous cycle. The resulting design was a completely randomized design with a 2 × 3 factorial + 1 arrangement of treatments. Maternal serum, histotroph, allantoic fluid, and amniotic fluid were collected at time of ovariohysterectomy. Samples were then analyzed for concentrations of AAs and intermediary metabolites: alanine (Ala), arginine, asparagine (Asn), aspartate (Asp), citrulline, cysteine, glutamine, glutamate (Glu), glycine (Gly), histidine, isoleucine, leucine (Leu), lysine, methionine (Met), ornithine, phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), tryptophan, tyrosine (Tyr), and valine (Val). The concentrations of Gly, Ser, and Thr in maternal serum were greater (P ≤ 0.05) in CON compared with RES. Furthermore, day of gestation affected (P ≤ 0.05) concentrations of Asn, Glu, Phe, Thr, and Tyr in maternal serum. Status of maternal nutrition affected the Asp concentration of histotroph where RES was greater (P = 0.02) than CON. In histotroph, Ala, Leu, Met, and Val concentrations were greater (P ≤ 0.05) on day 50 compared with day 16. Additionally, Glu and Pro concentrations in histotroph were greater (P < 0.01) on days 34 and 50 compared with day 16. A day × treatment interaction was observed for the concentration of Val in allantoic fluid where day 34 CON was greater (P = 0.05) than all other days and nutritional treatments. In addition, the concentration of Gln in amniotic fluid experienced a day × treatment interaction where day 34 RES was greater (P ≤ 0.05) than day 34 CON, which was greater (P ≤ 0.05) than day 50 CON and RES. These data support our hypothesis that day of gestation and maternal nutrition affect the concentrations of various neutral and acidic AA in beef heifer utero-placental fluids and maternal serum from days 16 to 50 of gestation.

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.

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.

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.

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 &gt;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 &lt; 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 &lt; 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.