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.

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.

Mustard bran in lactating dairy cow diets.

Two trials using lactating Holstein cows were conducted to evaluate effects of a diet containing oriental mustard bran on dry matter intake (DMI), milk production, milk components, and organoleptic properties. In experiment 1, 34 lactating cows (24 multiparous and 10 primiparous; days in milk ≥ 50 d) were used in a switchback design to determine the lactational response and organoleptic quality of milk when the diet contained 8% oriental mustard bran (MB) versus a control diet (CON). Mustard bran replaced a portion of soybean meal and all the beet pulp in the CON diet. Milk yields were greater for cows fed the MB diet; however, no differences were found in DMI, 3.5% fat- (FCM) or solids-corrected milk. Milk components and components production were not affected by treatment. Milk organoleptic qualities were not affected by diet. In experiment 2, 22 lactating cows (16 multiparous and 6 primiparous; days in milk ≥ 21 d) were assigned randomly within parity to receive MB or CON from wk 4 to 19 postpartum in a randomized complete block design. Cows were fed CON wk 1 to 3 postpartum. The MB diet contained the same ingredients as the CON, except sunflower seed and a portion of soybean meal were replaced with mustard bran. Milk and components data were collected during wk 3 postpartum and used as covariates to adjust treatment means. Intake was greater for cows fed the MB diet; however, daily milk, 3.5% FCM, and solids-corrected milk yields were not different between diets. Milk components and component yields were not affected by treatment. Milk urea concentration was less for cows fed the MB diet. Although cows fed the MB diet had greater DMI, this was not translated into a higher milk 3.5% FCM/DMI production efficiency ratio. During experiment 2, many cows fed MB experienced minor to severe hemolysis with bloody urine. This hemolysis believed to be caused by the S-methyl-cysteine sulfoxide contained in mustard bran could have affected milk production efficiency. The increased milk yield observed in experiment 1 was not observed in experiment 2. Adding 8% of MB to lactating cow diets had a mixed effect on DMI and milk production. Milk component yields and milk quality were not affected. Feeding this level of MB presents a hemolytic danger to lactating dairy cows.

Pre-synchronization of ovulation timing and delayed fixed-time artificial insemination increases pregnancy rates when sex-sorted semen is used for insemination of heifers.

This study was conducted to determine effects of pre-synchronization of ovulation timing among heifers and delayed fixed-time artificial insemination (TAI) with sex-sorted semen on proportion of heifers pregnant after TAI (PR/AI). Heifers were assigned to one of eight treatments: 1 and 2), 7-d CO-Synch + CIDR treatment regimen with administration of gonadotropin-releasing hormone and a CIDR insert on Day 0, prostaglandin F2α (PGF) at CIDR removal on Day 7, and TAI occurring 54 h later with conventionally processed (CTRL54-CNV) or sex-sorted semen (CTRL54-SEX); 3 and 4), same as CTRL54 but TAI delayed to 72 h with conventionally processed (CTRL72-CNV) or sex-sorted semen (CTRL72-SEX); 5 and 6), same as CTRL54 but additional administration of PGF on Day -7 and TAI with conventionally processed (PRE54-CNV) or sex-sorted semen (PRE54-SEX); 7 and 8), same as PRE54 treatments but TAI delayed to 72 h with conventionally processed (PRE72-CNV) or sex-sorted semen (PRE72-SEX). Proportion of heifers pregnant after TAI was greater (P ≤  0.02) with conventionally processed semen compared with sex-sorted semen, yet PR/AI did not differ (P =  0.14) between heifers in PRE72-CNV and PRE72-SEX groups. There were greater PR/AI in the PRE72-SEX (P =  0.03) than CTRL54-SEX group (46.1 % and 36.9 %) and there was no difference (P =  0.31) in PR/AI between CTRL54-CNV and PRE72-SEX groups (50.4 % and 46.1 %). In conclusion, pre-synchronization of ovulation timing among heifers combined with delayed TAI resulted in increased PR/AI with sex-sorted semen compared with the 7-d CO-Synch+CIDR treatment regimen.

Postpartum ovarian activity in multiparous Holstein cows treated with bovine somatotropin and fed n-3 fatty acids in early lactation.

Multiparous cows (n = 59) were blocked by expected calving date and previous 305-d mature-equivalent milk yield and assigned randomly to a 2 x 2 factorial design to determine the effects of bovine somatotropin (bST; Posilac, Monsanto Animal Agricultural Group, St. Louis, MO) and dietary fat on ovarian activity during the first 90 d in milk (DIM). Diets that included whole, high-oil sunflower seeds [SS; 10% of dietary dry matter; rich in linoleic acid (18:2)] or a mixture of Alifet-High Energy and Alifet-Repro [AF; Alifet USA, Cincinnati, OH; 3.5 and 1.5% of dietary dry matter, respectively; protected source of linolenic (18:3), eicosapentaenoic, and docosahexaenoic fatty acids] were provided from calving. Diets were isocaloric at equal intakes, but AF provided more net energy for lactation at actual intakes (1.54 vs. 1.66 Mcal/kg of dry matter). Cows received 0 or 500 mg of bST (N, Y) every 10 d from 12 to 70 DIM and at 14-d intervals from 70 to 280 DIM. Breeding was initiated after 90 DIM. Follicular dynamics, luteal growth and development (15 to 90 DIM), and plasma progesterone concentrations (1 to 90 DIM) were evaluated (3 times per week). Days to first ovulation (33.6 +/- 1.4) and incidence of anovulation at 45 or 70 DIM did not differ among treatments. Interovulatory intervals were similar among treatments (22.1 +/- 0.9 d). Incidence of estrous cycles with 2 follicular waves was greater for SSY (71.0%) and AFN (80.0%) than for other groups, but more 3-wave cycles occurred with AFY (83.3%). Growth rate of the ovulatory follicle was greater for AF than SS (1.9 vs. 2.2 +/- 0.11 mm/d) and diameter of ovulatory follicles was larger for AFN than the other treatments (17.9 vs. 15.7 +/- 0.7 mm). Area under the progesterone curve was reduced for SSY (63.2, 48.1, 55.5, and 61.4 +/- 5.1 ng.d/mL for SSN, SSY, AFN, and AFY, respectively). The number of class 1 (3 to 5 mm) follicles was decreased and the number of class 2 (6 to 9 mm) follicles was increased by bST. The number of class 2 follicles was reduced by AF. Initiation of bST administration at 12 DIM and dietary n-3 fatty acids altered ovarian activity during the first 90 DIM and could benefit reproductive performance. Dietary n-3 fatty acids interacted with bST administration in early lactation to increase the incidence of estrous cycles with 3 follicular waves. Although these changes could benefit reproductive performance, evaluation with a larger number of cows is needed to determine if these alterations improve fertility.

Production response of multiparous Holstein cows treated with bovine somatotropin and fed diets enriched with n-3 or n-6 fatty acids.

Multiparous cows (n = 59) were blocked by expected calving date and previous milk yield and assigned randomly to treatments to determine the effects of bovine somatotropin (bST; Posilac, Monsanto Animal Agricultural Group, St. Louis, MO) and source of dietary fat on production responses. Diets were provided from calving and included whole, high-oil sunflower seeds [SS; 10% of dietary dry matter (DM); n-6:n-3 ratio of 4.6] as a source of linoleic acid (18:2) or a mixture of Alifet-High Energy and Alifet-Repro (AF; Alifet USA, Cincinnati, OH; 3.5 and 1.5% of dietary DM, respectively; n-6/n-3 ratio of 2.6) as a source of protected n-3 fatty acids. Diets contained 181 versus 188 g of crude protein and 183 versus 186 g of acid detergent fiber/kg of DM and 1.54 versus 1.66 Mcal of net energy for lactation at the actual DM intake for SS versus AF, respectively. Cows received 0 or 500 mg of bST every 10 d from 12 to 70 d in milk (DIM) and at 14-d intervals through 280 DIM. The 2 x 2 factorial combination of diet (SS or AF) with or without bST administration resulted in treatments designated as SSY, SSN, AFY, and AFN, respectively. Data were analyzed as repeated measures using mixed model procedures to determine the effects of diet, bST, and their interactions. Yield of 3.5% fat-corrected milk was not altered by diet, but was increased by 4.0 +/- 1.9 kg/d from 12 to 70 DIM and by 5.1 +/- 1.2 kg/d from 12 to 280 DIM by bST. Treatment did not affect DM intake or energy balance (EB) nadir. There was an interaction of bST and diet on EB because AF decreased the impact of bST on overall EB and allowed AFY cows to reach a positive EB earlier than SSY cows. Gross feed efficiency adjusted for body weight change was greater for bST-treated cows (1.03 vs. 1.15 +/- 0.03 kg of fat-corrected milk/Mcal of net energy for lactation). Circulating insulin-like growth factor-I concentrations were increased by bST (85 vs. 125 +/- 8 ng/mL). Body weight, body condition score, and backfat thickness were reduced by bST, but differences between treated and nontreated cows did not differ by 280 DIM. Results indicate cows responded to bST administration in early lactation, but the magnitude of the response was greater after 70 DIM. Source of dietary fat had a minimal effect on most production measurements, but relative to SS, AF decreased the impact of bST on overall EB. Results support the premise that bST administration prolongs the delay in postpartum tissue replenishment.

Fatty acid composition of milk from multiparous Holstein cows treated with bovine somatotropin and fed n-3 fatty acids in early lactation.

Multiparous cows (n = 59) were blocked by expected calving date and previous milk yield and assigned randomly to treatments to determine effects of bovine somatotropin (bST; Posilac, Monsanto Animal Agricultural Group, St. Louis, MO) and source of dietary fat on milk fatty acid composition during the first 140 d in milk. Diets were provided from calving and included whole, high-oil sunflower seeds (SS; 10% of dietary dry matter; n-6/n-3 ratio of 4.6) as a source of linoleic acid or a mixture of Alifet-High Energy and Alifet-Repro (AF; Alifet USA, Cincinnati, OH; 3.5 and 1.5% of dietary dry matter, respectively; n-6/n-3 ratio of 2.6) as a source of protected n-3 fatty acids (15.7% 18:3, 1.3% 20:5, and 1.3% 22:6). Treatments were derived from a 2 x 2 combination of supplemental fat source (SS, AF) and with 0 (SSN, AFN) or 500 (SSY, AFY) mg of bST administered every 10 d from 12 to 70 d in milk and at 14-d intervals thereafter. Milk fatty acid composition was determined in samples collected from 32 cows (8 complete blocks) during wk 2, 8, and 20 of lactation. Data were analyzed as repeated measures using mixed model procedures to determine the effects of diet, bST, week of lactation, and their interactions. Proportions of 18:3 (4.02 vs. 3.59 +/- 0.16%), 20:5 (0.52 vs. 0.41 +/- 0.02%), and 22:6 (0.11 vs. 0.02 +/- 0.02%) were greater and the n-6/n-3 fatty acid ratio (7.40 vs. 8.80 +/- 0.30) was reduced in milk from cows fed AF compared with SS. Proportions of de novo-synthesized fatty acids increased and preformed fatty acids decreased as lactation progressed, but bST administration delayed this shift in origin of milk fatty acids. Transfer efficiency of 18:3, 20:5, and 22:6 from AF to milk fat averaged 36.2, 4.9, and 5.2%, respectively. These efficiencies increased as lactation progressed, but were delayed by bST. Apparent mammary Delta(9)-desaturase activity and milk conjugated linoleic acid (cis-9, trans-11 conjugated linoleic acid) content increased through the first 8 wk of lactation. Based on the product-to-substrate ratio of 14:1/14:0 fatty acids in milk, there was an interaction of diet and bST because bST decreased apparent Delta(9)-desaturase activity in SSY cows but increased it in AFY cows (0.10, 0.09, 0.08, and 0.09 +/- 0.01 for SSN, SSY, AFN, and AFY, respectively). Feeding Alifet-Repro increased n-3 fatty acids in milk and bST prolonged the partitioning of dietary fatty acids into milk fat.

Detection of anovulation by heatmount detectors and transrectal ultrasonography before treatment with progesterone in a timed insemination protocol.

Our objective was to determine the accuracy of identifying noncycling lactating dairy cows before the application of a timed artificial insemination (AI) protocol [with or without progesterone supplementation via a controlled internal drug-release (CIDR) insert and 2 different timings of AI] by using heatmount detectors and a single ovarian ultrasound examination. At 6 locations in the Midwest, 1,072 cows were enrolled in a Presynch protocol (2 injections of PGF(2alpha) 14 d apart), with the second injection administered 14 d before initiating the Ovsynch protocol (injection of GnRH 7 d before and 48 h after PGF(2alpha) injection, with timed AI at 0 or 24 h after the second GnRH injection). Heatmount detectors were applied to cows just before the first Presynch injection, assessed 14 d later at the second Presynch injection (replaced when activated or missing), and reassessed at initiation of the Ovsynch protocol. Ovaries were examined for the presence of a corpus luteum (CL) by ultrasound before the initiation of treatment. Treatments were assigned to cows based on the presence or absence of a CL detected by ultrasound: 1) no CL + no CIDR; 2) no CL + CIDR insert for 7 d; and 3) CL present. Further, alternate cows within the 3 treatments were assigned to be inseminated concurrent with the second GnRH injection of Ovsynch (0 h) or 24 h later. Pregnancy was diagnosed at 33 and 61 d after the second GnRH injection. By using low (<1 ng/mL) concentrations of progesterone in serum as the standard for noncycling status, heatmount detectors were activated on a large percentage of noncycling cows (>60%), whereas the single ultrasound examination incorrectly classified noncycling cows only 21% of the time. Conversely, cycling cows (progesterone > or =1 ng/mL) were correctly identified 70 to 78% of the time by heatmount detectors, but 85 to 92% were correctly identified by ultrasound. Overall accuracy of heatmount detectors and ultrasound was 71 and 84%, respectively. Application of progesterone to cows without a CL at the time of the first injection of GnRH reduced the incidence of ovulation but increased the proportions of pregnancies per AI at d 33 or 61 compared with nontreated cows without a CL at the onset of the Ovsynch protocol. Percentages of cows pregnant and pregnancy survival did not differ for cows having a CL before treatment compared with those not having a CL and treated with progesterone. Compared with no response, when a follicle ovulated in response to the first GnRH injection, percentage of cows becoming pregnant after the timed AI increased from 33.3 to 41.6%. Timing of AI at 0 or 24 h after the second GnRH injection did not alter pregnancies per AI, but cows having luteal activity before treatment had improved pregnancies per AI compared with noncycling cows. We conclude that identifying noncycling cows by ultrasound was more accurate than by heatmount detectors. Subsequent progesterone treatment of previously cycling cows not having a CL at the onset of Ovsynch increased the proportion of pregnant cows, equal to that of cows having a CL but not treated with progesterone.

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.

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.

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.

Gonadotropin-releasing hormone increased pregnancy risk in suckled beef cows not detected in estrus and subjected to a split-time artificial insemination program.

We hypothesized that GnRH would increase pregnancy risk (PR) in a split-time AI program for cows in which estrus was not detected. A total of 1,236 suckled beef cows at 12 locations in 3 states (Colorado, Kansas, and North Dakota) were enrolled. Before applying the fixed-time AI program, BCS was assessed. Cows were treated on d -7 with a progesterone insert concurrent with 100 µg GnRH and on d 0 with 25 mg PGF plus removal of the insert. Estrus-detection patches were affixed to cows at insert removal. Estrus was defined to have occurred when an estrus-detection patch was >50% colored (activated). Cows in estrus by 65 h ( = 758; 61.3% of all cows) were randomly allocated to 2 treatments: 1) 100 µg GnRH and early + GnRH (E+G; = 373) or 2) AI only at 65 h (early - no GnRH [E-G]; = 385). The remaining cows were randomly allocated to 2 treatments: 1) 5(L+G; = 252) or 2) AI only at 84 h (late no GnRH [L-G]; = 226). Pregnancy was determined 35 d after AI via transrectal ultrasound. Pregnancy risk did not differ ( = 0.68) between E+G and E-G cows (61.9 vs. 60.4%, respectively). Conversely, for cows inseminated at 84 h, PR was greater ( = 0.01) in cows that received GnRH (L+G) compared with their herd mates not receiving GnRH (L- G; 41.7 vs. 30.8%, respectively). Of those cows not detected in estrus by 65 h, 42.1% were detected by 84 h, for a total expression of estrus by all cows of 77.6%. Administration of GnRH increased ( < 0.01) PR in cows not detected in estrus by 84 h (+GnRH = 33.4% [ = 146] vs. no GnRH = 15.0% [ = 128]) but had no effect in cows expressing estrus by 84 h (+GnRH = 65.3% [ = 103] vs. no GnRH = 61.7% [ = 97]). Neither estrus expression by 65 or 84 h nor PR was influenced by BCS, parity, or days postpartum at AI. Cows had greater PR when they had been detected in estrus before AI, and PR was improved by administration of GnRH at 65 h after insert removal in cows that were not detected in estrus and inseminated at 84 h.

Using estrus detection patches to optimally time insemination improved pregnancy risk in suckled beef cows enrolled in a fixed-time artificial insemination program.

A multilocation study examined pregnancy risk (PR) after delaying AI in suckled beef cows from 60 to 75 h when estrus had not been detected by 60 h in response to a 7-d CO-Synch + progesterone insert (CIDR) timed AI (TAI) program (d -7: CIDR insert concurrent with an injection of GnRH; d 0: PGF injection and removal of CIDR insert; and GnRH injection at TAI [60 or 75 h after CIDR removal]). A total of 1,611 suckled beef cows at 15 locations in 9 states (CO, IL, KS, MN, MS, MT, ND, SD, and VA) were enrolled. Before applying the fixed-time AI program, BCS was assessed, and blood samples were collected. Estrus was defined to have occurred when an estrus detection patch was >50% colored (activated). Pregnancy was determined 35 d after AI via transrectal ultrasound. Cows ( = 746) detected in estrus by 60 h (46.3%) after CIDR removal were inseminated and treated with GnRH at AI (Control). Remaining nonestrous cows were allocated within location to 3 treatments on the basis of parity and days postpartum: 1) GnRH injection and AI at 60 h (early-early = EE; = 292), 2) GnRH injection at 60 h and AI at 75 h (early-delayed = ED; = 282), or 3) GnRH injection and AI at 75 h (delayed-delayed = DD; = 291). Control cows had a greater ( < 0.01) PR (64.2%) than other treatments (EE = 41.7%, ED = 52.8%, DD = 50.0%). Use of estrus detection patches to delay AI in cows not in estrus by 60 h after CIDR insert removal (ED and DD treatments) increased ( < 0.05) PR to TAI when compared with cows in the EE treatment. More ( < 0.001) cows that showed estrus by 60 h conceived to AI at 60 h than those not showing estrus (64.2% vs. 48.1%). Approximately half (49.2%) of the cows not in estrus by 60 h had activated patches by 75 h, resulting in a greater ( < 0.05) PR than their nonestrous herd mates in the EE (46.1% vs. 34.5%), ED (64.2% vs. 39.2%), and DD (64.8% vs. 31.5%) treatments, respectively. Overall, cows showing estrus by 75 h (72.7%) had greater ( < 0.001) PR to AI (61.3% vs. 37.9%) than cows not showing estrus. Use of estrus detection patches to allow for a delayed AI in cows not in estrus by 60 h after removal of the CIDR insert improved PR to TAI by optimizing the timing of the AI in those cows.

Evaluation of implant strategies in Angus-sired steers with high or low genetic potential for marbling and gain.

Sixty-nine Angus-sired steer calves (332.3 kg initial BW) were used to determine the effects of single or double implant strategies on steers of high or low genetic potential (GP) determined by the GeneMax (Zoetis, Florham Park, NJ) genetic profiling test. Steers were assigned to treatments in a 2 × 2 factorial design with factors of 1) composite GP score (high, mean GP score of 86.5 [HI]; low, mean GP score of 25.3[LO]) and 2) implant strategy (single, steers implanted on d 70 [1X], or double, steers implanted d 0 and 70 [2X]). All steers were given the same implant (Revalor-S; Merck Animal Health, Summit, NJ), with the 2X group implanted on d 0 and 70 and the 1X group implanted only on d 70. A diet containing 1.38 Mcal NEg/kg DM was fed ad libitum, once daily. Ultrasound was used to measure body composition characteristics on d 0 and 70. Steers were harvested after 140 d on feed. At both the d-0 and d-70 ultrasound, HI steers had greater ( < 0.001) percent intramuscular fat (IMF) than LO steers, but no differences ( ≥ 0.24) were observed in LM area (LMA), rib fat thickness (RF), or rump fat thickness (RMFT). Steers in the 2X group had larger ( = 0.02) LMA and less ( = 0.03) IMF on d 70 than 1X steers and no differences ( ≥ 0.50) in RF or RMFT were observed. From d 0 to 70, HI steers had ADG, DMI, and G:F ( ≥ 0.60) similar to LO steers; however, 2X steers had greater ( < 0.001) ADG and were more ( < 0.001) feed efficient compared with 1X steers during the same interval. Over the entire 140-d feeding period, there were no differences ( ≥ 0.6) in BW, ADG, DMI, or G:F between GP groups; however, 2X steers had greater ( = 0.03) ADG compared with 1X steers and still had similar ( ≥ 0.12) DMI and G:F. Upon slaughter, marbling score tended to be impacted by a GP × implant interaction (499.9 ± 18.5, 545.6 ± 18.5, 487.1 ± 18.5, and 469.8 ± 18.5 for HI and 2X, HI and 1X, LO and 2X, and LO and 1X, respectively; = 0.06). No differences ( ≥ 0.7) were observed between GP groups for HCW, LMA, RF, KPH, or yield grade (YG). Steers in the 1X group had less ( = 0.003) RF than 2X steers but similar ( ≥ 0.14) HCW, marbling, LMA, KPH, and YG. A greater proportion ( = 0.03) of HI steers had choice carcasses (100 ± 0.0%) compared with LO steers (87.8 ± 3.9%). Results of this study indicate that the GP test used in the current study predicted differences in IMF, carcass marbling, and percent carcasses graded as choice.

Effects of alternate day feeding of dried distiller's grains plus solubles in forage-fed steers on intake, ruminal fermentation and passage rates, and serum nonesterified fatty acid.

Four ruminally and duodenally cannulated Holstein steers (BW = 449 ± 7.3 kg) were used to examine the effects of feeding either dried distiller's grains plus solubles (DG) or grass hay on alternate days (every other day) on intake, ruminal fermentation and passage rates, and serum NEFA in forage-fed steers. Steers were assigned to 1 of 4 dietary treatments in a 4 × 4 Latin square: 1) only hay (CON), 2) hay and 0.4% of BW as DG DM daily (DG7), 3) hay daily and 0.8% BW DG every other day (DG2), and 4) alternate day feeding of hay and 0.8% of BW as DG (DGA). Treatment periods consisted of 13 d of adaptation and 8 d of collecting digesta and blood. Over the entire collection period, DMI was decreased ( = 0.004) for DGA compared with other treatments (13.0 ± 0.8, 12.7 ± 0.8, 13.3 ± 0.8, and 10.9 ± 0.8 kg/d for CON, DG7, DG2, and DGA, respectively). Immediately after feeding on days supplement was fed to DG2 and DGA (supplemented days [SUP]), ruminal pH of DGA was less than other treatments but by the end of the day was greater than other treatments (treatment × time, < 0.001). At feeding time on nonsupplemented days (NSUP), ruminal pH of DGA steers was greater than other treatments but was similar (treatment × time, < 0.001) to DG2 and CON by 5 h after feeding. Total concentrations of VFA were similar ( = 0.09) among treatments on SUP; however, on NSUP, total VFA concentrations were least in DGA from feeding until 4 h after feeding (treatment × time, = 0.02). No differences ( ≥ 0.06) were observed among treatments for apparent ruminal, total intestinal, and total tract DM, OM, or CP digestibility. There were no differences ( = 0.36) in serum NEFA among treatments on SUP; however, on NSUP, steers fed DGA (209.5 ± 12.7 m) had greater ( < 0.01) NEFA compared with other treatments (84.4 ± 12.7, 88.0 ± 12.7, and 77.7 ± 12.7 m for CON, DG7, and DG2, respectively). The DGA feeding strategy influenced DMI and ruminal kinetics and circulating NEFA without impacting total tract digestibility.