Abstract
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.
Keywords: amino acids, early gestation, fetal fluids, heifer, hexose, nutrition
INTRODUCTION
Embryonic mortality due to insufficiencies in maternal–conceptus interactions is a major cause of infertility during the pre-implantation period of pregnancy (Forde et al., 2014). During the first 50 d of gestation, placental circulation is being established, which will support nutrient, respiratory gas, and waste exchange between the maternal and fetal systems. During this time, the conceptus must receive its nutrients through histotroph (Bazer, 1975; Mullen et al., 2012), which is a mixture of growth factors, respiratory gases, and nutrients, including hexoses (Gao et al., 2009) and AAs (Gao et al., 2009; Groebner et al., 2011), secreted into the uterine lumen (Bazer et al., 2012).
Hexoses and AA play vital roles in development of the conceptus (Kwon et al., 2003; Gao et al., 2009) through various roles including: fuels for fetal growth (Bell et al., 1989), osmoregulation of fetal fluids (Wu et al., 2014), cell proliferation (Wang et al., 2016), and fetal programming via epigenetic modifications (Snell and Fell, 1990; Waterland and Jirtle, 2004; Wu et al., 2014).
Both Groebner et al., (2011) and Forde et al., (2014) have determined the concentrations of AA in the histotroph of beef heifers during the pre-implantation and peri-implantation periods of gestation. Additionally, Hugentobler et al., (2008) determined the concentration of glucose and its metabolites in oviductal and uterine fluids throughout the estrous cycle in heifers. Despite the importance of maternal plane of nutrition for the proper development of the conceptus, minimal data are available related to the effects of maternal nutrition on AA and hexose concentrations in utero-placental fluids of beef heifers through organogenesis. Thus, we examined the hypothesis that maternal nutrition and day of gestation would affect the concentrations of AA and hexoses in bovine utero-placental fluids and maternal serum from days 16 to 50 of gestation.
MATERIALS AND METHODS
All animal procedures were approved by the North Dakota State University Institutional Animal Care and Use Committee.
Animals, Housing, and Diet
Treatments are as described by Crouse et al., (2017). Briefly, Crossbred Angus heifers (n = 49) were individually fed (American Calan, Northwood, NH) at the NDSU Animal Nutrition and Physiology Center for 2 wk before the beginning of the trial. All heifers were exposed to the 5-d CO-Synch + CIDR estrus synchronization protocol (Bridges et al., 2008). Six heifers were randomly selected and were not inseminated to serve as nonpregnant (NP) controls, received control diets, and were ovariohysterectomized for tissue collection on day 16 of the synchronized estrous cycle. The remaining heifers (n = 43) were bred by AI and were randomly assigned to one of two dietary intake groups. Control heifers (CON), received 100% of NRC (NRC, 2000) recommended nutritional requirements for 0.45 kg/d of gain (actual ADG = 0.51 kg/d). Restricted heifers (RES) were placed on a 40% global nutrient restriction to maintain BW (i.e., zero net growth), which was accomplished by reducing total diet delivery to 60% of the CON delivery (actual ADG = −0.08 kg/d). The diet was delivered via a total mixed ration consisting of grass hay, corn silage, alfalfa haylage, grain and mineral mix, and dried distillers grains with solubles (53.4% NDF, 31.3% CP) were supplemented to the basal diet to meet individual heifer’s protein requirements. Preliminary data from our laboratory determined that days 16, 34, and 50 were timepoints of peak expression of nutrient transporters (Crouse et al., 2016a), and were therefore selected for analysis in the present study. All pregnant heifers were ovariohysterectomized on either day 16 (CON, n = 7; RES, n = 7), 34 (CON, n = 6; RES, n = 9), or 50 (CON, n = 7; RES, n = 7) of gestation. Thus, experimental design for the dietary intake × days of pregnancy was a completely randomized design with a 2 × 3 + 1 factorial arrangement of treatments.
Sample Collection and Analysis
Ovariohysterectomy procedures were as described by McLean et al. (2016). Serum samples were collected via jugular venipuncture at the time of ovariohysterectomy using 10-mL serum vacutainer tubes (Becton Dickinson HealthCare, Franklin Lakes, NJ), allowed to clot for 20 min at room temperature, and centrifuged at 1,500 × g for 30 min. Serum was separated from blood constituents and stored at −20 °C. Allantoic fluid (ALF) was collected by isolating the embryo within the uterine horn and extracting 10 mL of fluid from the chorioallantois using a 22-gauge needle (Medtronic, Minneapolis, MN) to prevent rupture. AMF was collected using a 22-gauge needle (Medtronic) inserted through the amnion with suction applied via the syringe after the amniotic sac containing the embryo was visualized; 1 mL of fluid was collected from the day 34 embryos and 10 mL from day 50 embryos. Allantoic and amniotic fluids were only collected on days 34 and 50 of gestation. Once collected, all fluids were snap frozen in liquid nitrogen-cooled isopentane and stored at −20 °C. Uterine luminal fluid (histotroph) was collected from the uterine horn ipsilateral to the corpus luteum and containing the embryo. Histotroph was collected by flushing the horn with 20 mL of 10 mM Tris (pH = 7.2). On days 34 and 50, an incision was made 10 cm from the tip of the horn, the chorioallantois, amnion, and embryo were removed, and then the uterine horn was flushed. Recovered histotroph was centrifuged at 1,000 × g for 15 min, and the supernatant was decanted and immediately snap frozen in 1 mL aliquots in liquid nitrogen-cooled isopentane and stored in −20 °C for subsequent AA analysis (Forde et al., 2014).
AA and intermediary metabolite (alanine [Ala], arginine [Arg], asparagine [Asn], aspartate [Asp], citrulline [Cit], cysteine [Cys], glutamate [Glu], glutamine [Gln], glycine [Gly], histidine [His], isoleucine [Ile], leucine [Leu], lysine [Lys], methionine [Met], ornithine [Orn], phenylalanine [Phe], proline [Pro], serine [Ser], threonine [Thr], tryptophan [Trp], tyrosine [Tyr], and valine [Val]) concentrations were determined using the ACQUITY UPLC System (Waters Corporation, Milford, MA). For UPLC, 250 µL of fluid was used for serum, ALF, and AMF. The MassTrac Amino Acid Analysis System from Waters UPLC was used to determine the full profile of AA in these physiological fluids. Derivatization chemistry for physiological samples is a precolumn method and is based on a derivatizing reagent, 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate, which converts both primary and secondary AA to stable chromophores for UPLC detection. Minimum detectable limits for UPLC detection were 5 µmol/L. Therefore, we assigned values at 0.1× of the lower detectable limit. These values were set at 0.5 µmol/L for serum and 10 µmol/L after dilution of 20 mL for histotroph flushing. Therefore, AA concentrations in histotroph <5 µmol/L for serum, ALF, and AMF, as well as 10 µmol/L for histotroph were reported as undetected (Und). Glucose concentrations were determined using Infinity Glucose Hexokinase Liquid Stable Reagent (Fisher Diagnostics, Middletown, VA) and analyzed with a Synergy H1 Microplate Reader (BioTek, Winooski, VT). For glucose determination, 5 µL of fluid was used for serum, ALF, and AMF with 250 µL of reagent (intraplate CV = 3.04; across-plate CV = 7.39; NDSU sheep serum control 2.72 mM ± 0.07 mM). Fructose concentrations were determined using an EnzyChrom Fructose Assay Kit (Bio Assay Systems, Hayward, CA) and analyzed with a Synergy H1 Microplate Reader (BioTek). For fructose determination, 500 µL of serum, histotroph, ALF, and AMF samples were centrifuged before pipetting. After centrifugation, ALF and AMF fluid samples were diluted 1:10 before the assay. For the assay, 20 µL of the diluted sample was mixed with 80 µL of working reagent (intraplate CV = 7.16; across plate CV = 12.92; NDSU sheep serum control 0.079 mM ± 0.018 mM).
Statistical Analysis
Data were analyzed using the GLM procedure of SAS (SAS Inst. Inc.), with day, treatment, and day × treatment in the model, with individual heifer serving as the experimental unit. If no significant interactions were present, main effects of maternal nutrition and day of gestation were reported. Means with a significant protected F-test (P ≤ 0.05) were separated using the LSMEANS procedure of SAS, and P-values ≤ 0.05 were considered significant. For serum and histotroph, if no significant interactions were present, contrasts were conducted. Contrasts included incorporation of the NP heifers and comparing the nutrient concentrations for NP vs. pregnant heifers, as well as pre-attachment (day 16) vs. post-attachment (days 34 and 50), and a postattachment day comparison (day 34 vs. 50) for each nutrient using the GLM procedure of SAS, with P-values ≤ 0.05 considered different. For Und samples, the minimum detectable value of 0.5 µmol/L for serum, ALF, and AMF as well as 10 µmol/L for histotroph was assigned to run statistical comparisons.
RESULTS
Serum
Data for maternal serum concentrations are detailed in Table 1. No nutrients measured in serum were influenced by a day × treatment interaction (P ≥ 0.06), and a main effect of maternal nutritional treatment and day of gestation was only seen in a few instances. Therefore, we describe only significant main effects and contrasts that are not covered in main effects. Glucose concentrations in serum were greater (P = 0.04) on days 16 and 34 of gestation compared with day 50. Additionally, glucose concentrations were greater (P < 0.01) in NP compared with pregnant heifers. Serum fructose concentrations were greater (P = 0.01) in NP compared with pregnant heifers. Arginine concentrations were greater (P = 0.01) on day 50 compared with days 16 and 34. In addition, Arg concentrations were greater (P = 0.03) in pregnant compared with NP heifers. Asparagine was greater (P < 0.01) on day 34 compared with days 16 and 50. Furthermore, Asn in NP heifers was greater (P < 0.01) than pregnant heifers. Citrulline concentrations were greater (P = 0.02) on days 16 and 34 compared with day 50, and was greater in NP compared with pregnant heifers. Cysteine concentrations tended (P = 0.06) to be greater for day 16 CON compared with the other days and nutritional treatments, except day 34 RES. Glutamine was greater (P = 0.03) on day 50 compared with days 16 and 34. Glycine was greater (P = 0.05) for CON compared with RES heifers. Histidine and Lys were greater (P = 0.02) in pregnant compared with NP heifers. Phenylalanine was greater (P < 0.01) on day 50 compared with days 16 and 34. Serine was greater (P = 0.04) in CON compared with RES. Threonine was greater (P = 0.03) on day 34 compared with days 16 and 50; furthermore, CON was greater (P = 0.04) than RES. Threonine was greater (P = 0.02) in NP compared with pregnant heifers. Tyrosine was greater (P < 0.01) on day 50 compared with days 16 and 34. Furthermore, Tyr was greater (P < 0.01) in pregnant compared with NP heifers.
Table 1.
Concentrations of hexoses (mM) and AAs (µmol/L) in heifer serum samples as influenced by dietary treatment in NP heifers, and pregnant heifers from days 16 to 50 of gestation
| Day of gestation1 | P- values2 | |||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Nutrient3 | Nutr.Trt4 | NP | 16 | 34 | 50 | Trt Avg5 | SEM6 | Day | Trt | Day×Trt | NPvs.Preg. | 16vs.34 + 50 | 34vs.50 | |
| Hexoses | ||||||||||||||
| Glc | CON | 4.76 | 4.09 | 4.31 | 3.73 | 4.04 | 0.21 | 0.04 | 0.29 | 0.16 | <0.01 | 0.04 | 0.15 | |
| RES | — | 4.30 | 3.71 | 3.57 | 3.86 | |||||||||
| Day7 | 4.76 | 4.20a | 4.02a | 3.65b | ||||||||||
| Frc | CON | 0.17 | 0.08 | 0.07 | 0.07 | 0.07 | 0.01 | 0.97 | 0.35 | 0.82 | 0.02 | 0.35 | 0.20 | |
| RES | — | 0.08 | 0.09 | 0.09 | 0.09 | |||||||||
| Day | 0.17 | 0.08 | 0.08 | 0.08 | ||||||||||
| AA | ||||||||||||||
| Ala | CON | 252.8 | 216.2 | 237.6 | 219.9 | 224.6 | 13.3 | 0.86 | 0.54 | 0.13 | 0.15 | 0.46 | 0.85 | |
| RES | — | 234.8 | 201.1 | 217.7 | 217.9 | |||||||||
| Day | 252.8 | 225.5 | 219.4 | 218.8 | ||||||||||
| Arg | CON | 144.3 | 166.9 | 178.1 | 207.4 | 184.2 | 13.1 | 0.01 | 0.46 | 0.98 | 0.01 | 0.02 | <0.01 | |
| RES | – | 161.2 | 167.7 | 199.8 | 176.2 | |||||||||
| Day | 144.3 | 164.1a | 172.9a | 203.6b | ||||||||||
| Asn | CON | 45.2 | 22.9 | 34.4 | 20.4 | 25.9 | 3.26 | <0.01 | 0.50 | 0.67 | <0.01 | 0.16 | <0.01 | |
| RES | - | 19.4 | 30.9 | 22.0 | 24.1 | |||||||||
| Day | 45.2 | 21.2a | 32.6b | 21.2a | ||||||||||
| Asp | CON | 10.15 | 11.61 | 10.15 | 9.66 | 10.48 | 0.70 | 0.18 | 0.11 | 0.64 | 0.83 | 0.24 | 0.70 | |
| RES | — | 9.88 | 9.55 | 9.13 | 9.52 | |||||||||
| Day | 10.15 | 10.74 | 9.85 | 9.40 | ||||||||||
| Cit | CON | 74.5 | 70.2 | 63.9 | 53.0 | 62.3 | 4.08 | 0.02 | 0.14 | 0.40 | <0.01 | 0.23 | 0.05 | |
| RES | — | 58.4 | 60.7 | 52.7 | 57.2 | |||||||||
| Day | 74.5 | 64.3a | 62.3a | 52.8b | ||||||||||
| Cys | CON | Und8 | 0.85 | Und | Und | 0.62 | 0.11 | 0.32 | 0.68 | 0.06 | 0.39 | 0.31 | 0.22 | |
| RES | — | Und | 0.73 | Und | 0.58 | |||||||||
| Day | Und | 0.67 | 0.61 | Und | ||||||||||
| Gln | CON | 226.5 | 246.5 | 259.9 | 222.6 | 243.00 | 17.8 | 0.16 | 0.64 | 0.74 | 0.41 | 0.71 | 0.05 | |
| RES | — | 223.9 | 257.3 | 227.3 | 236.2 | |||||||||
| Day | 226.5 | 235.2 | 258.6 | 225.0 | ||||||||||
| Glu | CON | 67.1 | 60.2 | 68.2 | 86.2 | 71.5 | 7.33 | 0.03 | 0.32 | 0.72 | 0.40 | 0.11 | 0.03 | |
| RES | — | 60.0 | 62.5 | 74.0 | 65.5 | |||||||||
| Day | 67.1 | 60.1a | 65.3a | 80.1b | ||||||||||
| Gly | CON | 294.91 | 415.2 | 338.2 | 364.6 | 372.7c | 23.7 | 0.26 | 0.05 | 0.29 | 0.08 | 0.21 | 0.53 | |
| RES | — | 333.1 | 330.8 | 336.8 | 333.6d | |||||||||
| Day | 294.91 | 374.1 | 334.5 | 350.1 | ||||||||||
| His | CON | 30.64 | 47.4 | 45.1 | 46.1 | 46.2 | 5.04 | 0.85 | 0.66 | 0.99 | 0.02 | 0.59 | 0.87 | |
| RES | — | 46.5 | 43.2 | 43.5 | 44.4 | |||||||||
| Day | 30.64 | 47.0 | 44.1 | 44.8 | ||||||||||
| Ile | CON | 70.8 | 71.7 | 74.2 | 84.3 | 76.7 | 5.19 | 0.25 | 0.15 | 0.58 | 0.83 | 0.10 | 0.31 | |
| RES | — | 66.9 | 72.6 | 72.1 | 70.5 | |||||||||
| Day | 70.8 | 69.2 | 73.4 | 78.12 | ||||||||||
| Leu | CON | 105.2 | 108.4 | 118.4 | 127.1 | 118.0 | 6.83 | 0.22 | 0.38 | 0.65 | 0.35 | 0.09 | 0.30 | |
| RES | — | 110.3 | 112.6 | 116.2 | 113.0 | |||||||||
| Day | 105.2 | 109.4 | 115.5 | 121.7 | ||||||||||
| Lys | CON | 60.3 | 71.7 | 79.3 | 73.0 | 74.6 | 5.45 | 0.34 | 0.70 | 0. 94 | 0.03 | 0.22 | 0.36 | |
| RES | — | 68.3 | 77.0 | 73.4 | 72.9 | |||||||||
| Day | 60.3 | 70.0 | 78.1 | 73.2 | ||||||||||
| Met | CON | 22.6 | 19.2 | 22.1 | 19.7 | 20.3 | 1.33 | 0.64 | 0.43 | 0.38 | 0.11 | 0.69 | 0.62 | |
| RES | — | 19.8 | 19.1 | 19.5 | 19.5 | |||||||||
| Day | 22.6 | 19.5 | 20.6 | 19.6 | ||||||||||
| Orn | CON | 38.4 | 38.7 | 40.3 | 36.7 | 38.6 | 1.09 | 0.30 | 0.80 | 0.94 | 0.98 | 0.63 | 0.15 | |
| RES | — | 39.0 | 39.2 | 36.2 | 38.1 | |||||||||
| Day | 38.4 | 38.9 | 39.7 | 36.4 | ||||||||||
| Phe | CON | 45.8 | 47.4 | 50.6 | 71.7 | 56.6 | 3.33 | <0.01 | 0.96 | 0.52 | 0.19 | 0.58 | 0.09 | |
| RES | — | 51.8 | 47.5 | 70.8 | 56.7 | |||||||||
| Day | 45.8 | 49.6a | 49.1a | 71.3b | ||||||||||
| Pro | CON | 76.7 | 68.2 | 75.5 | 71.7 | 71.8 | 3.55 | 0.13 | 0.32 | 0.89 | 0.37 | 0.12 | 0.62 | |
| RES | — | 64.3 | 71.7 | 70.8 | 68.9 | |||||||||
| Day | 76.7 | 66.3 | 73.6 | 71.3 | ||||||||||
| Ser | CON | 92.1 | 96.4 | 84.3 | 85.6 | 88.3c | 5.28 | 0.32 | 0.04 | 0.64 | 0.15 | 0.21 | 0.91 | |
| RES | — | 81.6 | 79.2 | 78.2 | 79.7d | |||||||||
| Day | 92.1 | 89.0 | 81.7 | 81.9 | ||||||||||
| Thr | CON | 57.9 | 55.2 | 75.1 | 57.7 | 62.7c | 4.77 | 0.03 | 0.04 | 0.14 | 0.02 | 0.93 | 0.80 | |
| RES | — | 56.4 | 56.5 | 49.7 | 54.2d | |||||||||
| Day | 57.9 | 55.8a | 65.8b | 53.7a | ||||||||||
| Trp | CON | 28.7 | 32.0 | 33.1 | 33.4 | 32.9 | 3.18 | 0.83 | 0.18 | 0.64 | 0.70 | 0.86 | 0.62 | |
| RES | — | 31.1 | 30.3 | 26.5 | 29.3 | |||||||||
| Day | 28.7 | 31.6 | 31.7 | 30.0 | ||||||||||
| Tyr | CON | Und | 12.3 | 19.0 | 42.0 | 24.5 | 5.55 | <0.01 | 0.65 | 0.97 | <0.01 | <0.01 | <0.01 | |
| RES | — | 8.8 | 18.5 | 39.9 | 22.4 | |||||||||
| Day | Und | 10.6a | 18.7a | 40.9b | ||||||||||
| Val | CON | 156.0 | 161.5 | 175.4 | 184.9 | 173.9 | 9.26 | 0.30 | 0.31 | 0.65 | 0.42 | 0.13 | 0.35 | |
| RES | — | 163.7 | 164.6 | 170.1 | 166.1 | |||||||||
| Day | 156.0 | 162.6 | 170.0 | 177.5 | ||||||||||
Means within row without common superscript differ (P ≤ 0.05).
Means within row without common superscript differ (P ≤ 0.05).
Means within column without common superscript differ (P ≤ 0.05).
Means within column without common superscript differ (P ≤ 0.05).
Day of gestation = number of days after insemination.
Probability values for the effect of day, treatment and day × treatment on the concentrations of hexoses and AAs. Contrast statements comparing pregnant vs. nonpregnant concentration (NP vs. P), day pre-implantation vs. day post-implantation (day 16 vs. 34 + 50) and day post-attachment comparison (34 vs. 50).
Nutrient-hexoses-glucose (Glc) and fructose (Frc). AA-alanine (Ala), arginine (Arg), asparagine (Asn), aspartate (Asp) citrulline (Cit), cysteine (Cys), glutamine (Gln), glutamate (Glu) glycine (Gly), histidine (His), isoleucine (Ile), leucine (Leu), lysine (Lys), methionine (Met), ornithine (Orn), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), tryptophan (Trp), tyrosine (Tyr), and valine (Val).
CON = heifers fed a total mixed ration that meets 100% of NRC requirements to gain 0.45 kg daily. RES = heifers restricted to 60% of CON diet.
Mean hexose and AA concentrations of treatment groups across day of gestation in maternal serum.
Average SEM for day × treatment interaction. Day 16 CON n = 7, day 16 RES n = 7, day 34 CON n = 6, day 34 RES n = 9, day 50 CON n = 7, day 50 RES n = 7.
Mean AA concentration across treatment within day of gestation.
Und-nutrient undetectable. Minimum detectable limits for UPLC detection were 5 µmol/L. Therefore, we assigned values at 0.1× of the lower detectable limit. The concentration is below 0.5 µmol/L.
Histotroph
Data for histotroph are detailed in Table 2. No nutrients in histotroph were influenced by a day × treatment interaction (P ≥ 0.25). Therefore, we will discuss only main effects of day, maternal nutritional treatment, and contrasts when significant. Glucose concentrations were greater (P = 0.03) in histotroph of pregnant compared with NP heifers. Fructose was greater (P < 0.01) on day 50 compared with days 16 and 34 of gestation. Alanine concentrations were greater (P < 0.01) on day 50 compared with day 16. Asparagine was greater (P = 0.01) in pregnant compared with NP heifers. Arginine concentrations were greater (P = 0.05) on day 34 of gestation compared with day 50. Aspartate was greater (P = 0.02) in RES compared with CON heifers. Glutamine concentrations were greater (P < 0.01) on days 34 and 50 compared with day 16 of gestation. Histidine and Leu were greater (P = 0.03) on day 50 compared with day 16. Lysine and Met were greater (P = 0.02 and P = 0.03, respectively) on day 50 compared with day 16. Intermediary metabolite Orn was greater (P < 0.01) on day 50 compared with days 16 and 34. Proline was greater (P < 0.01) on days postattachment compared with preattachment. Valine was greater (P = 0.03) on day 50 compared with day 16.
Table 2.
Concentrations of hexoses (mM) and AAs (µmol/L) in heifer histotroph samples as influenced by dietary treatment in NP heifers, and from days 16 to 50 of gestation
| Day of gestation1 | P-values2 | |||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Nutrient3 | Nutr.Trt4 | NP | 16 | 34 | 50 | Trt Avg5 | SEM6 | Day | Trt | Day×Trt | NPvs.Preg. | 16vs.34 + 50 | 34vs.50 | |
| Hexoses | ||||||||||||||
| Glc | CON | 2.07 | 3.95 | 3.92 | 3.49 | 3.79 | 0.55 | 0.68 | 0.74 | 0.35 | 0.03 | 0.40 | 0.82 | |
| RES | - | 2.93 | 3.87 | 4.10 | 3.64 | |||||||||
| Day7 | 2.07 | 3.44 | 3.90 | 3.80 | ||||||||||
| Frc | CON | 0.17 | 0.12 | 0.58 | 1.03 | 0.57 | 0.31 | <0.01 | 0.27 | 0.65 | 0.09 | <0.01 | 0.02 | |
| RES | - | 0.23 | 0.69 | 1.64 | 0.84 | |||||||||
| Day | 0.17 | 0.18a | 0.63a | 1.34b | ||||||||||
| AA | ||||||||||||||
| Ala | CON | 337.3 | 206.3 | 397.2 | 799.1 | 467.5 | 172.3 | <0.01 | 0.52 | 0.78 | 0.35 | <0.01 | 0.08 | |
| RES | - | 192.9 | 622.7 | 860.7 | 558.7 | |||||||||
| Day | 337.3 | 199.6a | 509.9ab | 829.9b | ||||||||||
| Arg | CON | 113.4 | 151.4 | 192.2 | 27.2 | 123.6 | 47.6 | 0.14 | 0.88 | 0.30 | 0.25 | 0.63 | 0.05 | |
| RES | - | 124.9 | 144.4 | 118.7 | 129.3 | |||||||||
| Day | 113.4 | 138.2 | 168.3 | 72.9 | ||||||||||
| Asn | CON | 119.9 | 489.0 | 394.7 | 364.2 | 416.0 | 85.3 | 0.94 | 0.28 | 0.51 | 0.01 | 0.72 | 0.93 | |
| RES | - | 298.0 | 356.3 | 364.3 | 339.5 | |||||||||
| Day | 119.9 | 393.5 | 375.5 | 364.2 | ||||||||||
| Asp | CON | Und8 | Und | Und | 52.4 | 52.4c | 39.4 | 0.09 | 0.02 | 0.20 | 0.23 | 0.02 | 0.96 | |
| RES | - | Und | 153.6 | 136.0 | 136.0d | |||||||||
| Day | Und | Und | 81.8 | 94.2 | ||||||||||
| Cit | CON | Und | Und | Und | Und | — | — | — | — | — | — | — | — | |
| RES | - | Und | Und | Und | — | |||||||||
| Day | Und | Und | Und | Und | ||||||||||
| Cys | CON | Und | Und | 22.5 | 52.2 | 28.2 | 18.8 | 0.45 | 0.58 | 0.29 | 0.74 | 0.17 | 0.95 | |
| RES | - | Und | 38.6 | Und | 19.5 | |||||||||
| Day | Und | Und | 30.5 | 31.1 | ||||||||||
| Gln | CON | 491.9 | 143.2 | 160.5 | 168.3 | 157.4 | 165.4 | 0.39 | 0.30 | 0.46 | 0.25 | 0.19 | 0.82 | |
| RES | — | 46.1 | 444.2 | 408.8 | 299.7 | |||||||||
| Day | 491.9 | 94.7 | 302.4 | 288.6 | ||||||||||
| Glu | CON | 197.0 | 77.9 | 381.7 | 621.3 | 360.3 | 171.1 | <0.01 | 0.33 | 0.59 | 0.21 | <0.01 | 0.55 | |
| RES | - | 56.7 | 710.6 | 730.4 | 499.2 | |||||||||
| Day | 197.0 | 67.3a | 546.2b | 675.8b | ||||||||||
| Gly | CON | 1,159.9 | 975.0 | 594.2 | 1,141.1 | 903.5 | 311.5 | 0.20 | 0.43 | 0.54 | 0.72 | 0.46 | 0.15 | |
| RES | - | 773.7 | 1,017.5 | 1,529.2 | 1,106.8 | |||||||||
| Day | 1,159.9 | 874.4 | 805.8 | 1,335.2 | ||||||||||
| His | CON | 109.1 | 122.6 | 243.7 | 332.1 | 232.8 | 112.5 | 0.03 | 0.19 | 0.67 | 0.13 | 0.01 | 0.31 | |
| RES | — | 133.0 | 387.2 | 542.7 | 354.3 | |||||||||
| Day | 109.1 | 127.8a | 315.4ab | 437.4b | ||||||||||
| Ile | CON | 19.6 | Und | Und | 41.9 | 20.6 | 24.7 | 0.30 | 0.25 | 0.25 | 0.58 | 0.08 | 0.62 | |
| RES | — | Und | 82.2 | 41.2 | 44.5 | |||||||||
| Day | 19.6 | Und | 46.1 | 41.5 | ||||||||||
| Leu | CON | 28.1 | Und | 52.6 | 128.7 | 63.8 | 58.1 | 0.03 | 0.18 | 0.60 | 0.27 | <0.01 | 0.39 | |
| RES | — | Und | 167.6 | 209.2 | 129.0 | |||||||||
| Day | 28.1 | Unda | 110.1ab | 169.0b | ||||||||||
| Lys | CON | 21.0 | 15.1 | 54.9 | 141.7 | 70.6 | 38.6 | 0.02 | 0.95 | 0.73 | 0.18 | <0.01 | 0.14 | |
| RES | — | 14.9 | 88.6 | 114.5 | 72.7 | |||||||||
| Day | 21.0 | 15.0a | 71.8ab | 128.1b | ||||||||||
| Met | CON | 28.8 | Und | 103.9 | 113.2 | 75.7 | 56.7 | 0.03 | 0.29 | 0.61 | 0.25 | <0.01 | 0.33 | |
| RES | — | 18.8 | 128.2 | 228.1 | 125.1 | |||||||||
| Day | 28.8 | 14.4a | 116.1ab | 170.7b | ||||||||||
| Orn | CON | 29.7 | 13.5 | 36.9 | 111.4 | 53.9 | 22.3 | <0.01 | 0.72 | 0.79 | 0.24 | <0.01 | <0.01 | |
| RES | — | 15.8 | 60.2 | 105.6 | 60.5 | |||||||||
| Day | 29.7 | 14.6a | 48.5a | 108.5b | ||||||||||
| Phe | CON | Und | Und | Und | Und | Und | 69.2 | 0.69 | 0.25 | 0.69 | 0.63 | 0.33 | 0.64 | |
| RES | — | Und | 126.9 | 90.4 | 75.8 | |||||||||
| Day | Und | Und | 68.5 | 50.2 | ||||||||||
| Pro | CON | 72.5 | 75.4 | 215.7 | 310.6 | 200.6 | 76.1 | <0.01 | 0.96 | 0.82 | 0.12 | <0.01 | 0.09 | |
| RES | — | 30.9 | 217.3 | 362.6 | 203.6 | |||||||||
| Day | 72.5 | 53.2a | 216.5b | 336.6b | ||||||||||
| Ser | CON | 524.6 | 550.1 | 244.0 | 278.0 | 357.4 | 141.1 | 0.11 | 0.21 | 0.90 | 0.61 | 0.06 | 0.62 | |
| RES | — | 652.6 | 359.7 | 498.0 | 503.5 | |||||||||
| Day | 524.6 | 601.3 | 301.9 | 388.0 | ||||||||||
| Thr | CON | 100.8 | 117.1 | 164.0 | 220.1 | 167.0 | 90.3 | 0.29 | 0.18 | 0.89 | 0.23 | 0.13 | 0.45 | |
| RES | — | 174.3 | 269.5 | 364.0 | 269.2 | |||||||||
| Day | 100.8 | 145.7 | 216.7 | 292.0 | ||||||||||
| Trp | CON | Und | Und | Und | Und | Und | 19.2 | 0.69 | 0.25 | 0.69 | 0.63 | 0.33 | 0.65 | |
| RES | — | Und | 42.3 | 32.7 | 28.3 | |||||||||
| Day | Und | Und | 26.1 | 21.3 | ||||||||||
| Tyr | CON | Und | Und | Und | Und | Und | — | — | — | — | — | — | — | |
| RES | — | Und | Und | Und | Und | |||||||||
| Day | Und | Und | Und | Und | ||||||||||
| Val | CON | 68.9 | 65.3 | 170.7 | 218.7 | 151.5 | 67.3 | 0.03 | 0.22 | 0.88 | 0.11 | <0.01 | 0.41 | |
| RES | — | 97.3 | 246.0 | 319.0 | 220.8 | |||||||||
| Day | 68.9 | 81.3a | 208.4ab | 268.8b | ||||||||||
Means within row without common superscript differ (P ≤ 0.05).
Means within row without common superscript differ (P ≤ 0.05).
Means within column without common superscript differ (P ≤ 0.05).
Means within column without common superscript differ (P ≤ 0.05).
Day of gestation = number of days after insemination.
Probability values for the effect of day, treatment and day × treatment on the concentrations of hexoses and AAs. Contrast statements comparing pregnant vs. nonpregnant concentration (NP vs. P), day pre-implantation vs. day post-implantation (day 16 vs. 34 + 50) and day post-attachment comparison (34 vs. 50).
Nutrient-hexoses-glucose (Glc) and fructose (Frc). AA-alanine (Ala), arginine (Arg), asparagine (Asn), aspartate (Asp) citrulline (Cit), cysteine (Cys), glutamine (Gln), glutamate (Glu) glycine (Gly), histidine (His), isoleucine (Ile), leucine (Leu), lysine (Lys), methionine (Met), ornithine (Orn), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), tryptophan (Trp), tyrosine (Tyr), and valine (Val).
CON = heifers fed a total mixed ration that meets 100% of NRC requirements to gain 0.45 kg daily. RES = heifers restricted to 60% of CON diet.
Mean hexose and AA concentrations of treatment groups across day of gestation in maternal serum.
Average SEM for day × treatment interaction. Day 16 CON n = 7, day 16 RES n = 7, day 34 CON n = 6, day 34 RES n = 9, day 50 CON n = 7, day 50 RES n = 7.
Mean AA concentration across treatment within day of gestation.
Und-nutrient undetectable. Minimum detectable limits for UPLC detection were 5 µmol/L. Therefore, we assigned values at 0.1× of the lower detectable limit. The concentration is below 10 µmol/L (0.5 µmol/L with 20× dilution factor due to luminal flushing).
Allantoic Fluid
Data for ALF are presented in Table 3. No data are present for day 16 of gestation due to lack of ALF at this time point. Glucose concentrations were greater (P = 0.05) on day 34 of gestation, compared with day 50 and tended (P = 0.07) to be greater in CON vs. RES heifers. Valine was greater (P = 0.05) for day 50 CON compared with all other days and treatments. All other AAs were not affected by a day × treatment interaction (P > 0.05). Thus, for the remainder of the ALF data, we will discuss only significant main effects. Asparagine was greater (P < 0.01) on day 34 compared with day 50. Aspartate was greater (P < 0.01) on day 50 compared with day 34. Furthermore, Asp was greater (P = 0.03) in CON compared with RES heifers. Citrulline and Gly were greater (P < 0.01) on day 50 compared with day 34. Isoleucine and Leu were greater (P < 0.01) on day 34 compared with day 50. Lysine tended (P = 0.06) to be greater for day 34 CON compared with day 34 RES. Methionine concentrations tended (P = 0.07) to be greater in CON compared with RES. Ornithine was greater (P = 0.04) on day 50 compared with day 34. Phenylalanine and Trp concentrations were greater (P = 0.02 and P = 0.03, respectively) on day 34 compared with day 50.
Table 3.
Concentrations of hexoses (mM) and AAs (µmol/L) in heifer allantoic fluid samples as influenced by dietary treatment on days 34 and 50 of gestation
| Day of gestation1 | P-values2 | ||||||||
|---|---|---|---|---|---|---|---|---|---|
| Nutrient3 | Nutr.Trt4 | 34 | 50 | Trt Avg5 | SEM6 | Day | Trt | Day×Trt | |
| Hexoses | |||||||||
| Glc | CON | 1.88 | 1.39 | 1.63 | 0.17 | 0.05 | 0.07 | 0.45 | |
| RES | 1.42 | 1.20 | 1.31 | ||||||
| Day7 | 1.64a | 1.29b | |||||||
| Frc | CON | 5.53 | 5.07 | 5.30 | 0.76 | 0.90 | 0.44 | 0.64 | |
| RES | 4.56 | 4.83 | 4.69 | ||||||
| Day | 5.04 | 4.95 | |||||||
| AA | |||||||||
| Ala | CON | 813.2 | 680.0 | 746.6 | 51.3 | 0.11 | 0.37 | 0.23 | |
| RES | 594.5 | 632.2 | 613.4 | ||||||
| Day | 703.8 | 656.1 | |||||||
| Arg | CON | 620.9 | 456.6 | 538.8 | 90.2 | 0.60 | 0.15 | 0.35 | |
| RES | 498.1 | 475.3 | 486.7 | ||||||
| Day | 559.5 | 466.0 | |||||||
| Asn | CON | 336.1 | 175.4 | 255.8 | 29.3 | <0.01 | 0.55 | 0.15 | |
| RES | 274.6 | 201.9 | 238.2 | ||||||
| Day | 305.3a | 188.6b | |||||||
| Asp | CON | 5.8 | 15.6 | 10.7c | 1.6 | <0.01 | 0.03 | 0.13 | |
| RES | 4.7 | 9.5 | 7.1d | ||||||
| Day | 5.3a | 12.6b | |||||||
| Cit | CON | 34.2 | 50.9 | 42.6 | 8.1 | <0.01 | 0.66 | 0.42 | |
| RES | 23.8 | 53.9 | 38.9 | ||||||
| Day | 29.0a | 52.4b | |||||||
| Cys | CON | 35.1 | 51.0 | 43.0 | 6.1 | 0.07 | 0.11 | 0.49 | |
| RES | 29.1 | 36.4 | 32.7 | ||||||
| Day | 32.1 | 43.7 | |||||||
| Glu | CON | 25.9 | 25.9 | 25.9 | 10.3 | 0.79 | 0.60 | 0.80 | |
| RES | 17.6 | 23.0 | 20.3 | ||||||
| Day | 21.7 | 24.4 | |||||||
| Gln | CON | 1,510.9 | 1,331.9 | 1,421.4 | 145.3 | 0.17 | 0.09 | 0.85 | |
| RES | 1,280.2 | 1,045.7 | 1,163.0 | ||||||
| Day | 1,395.6 | 1,188.8 | |||||||
| Gly | CON | 961.1 | 1,426.9 | 1,194.0 | 103.0 | <0.01 | 0.11 | 0.86 | |
| RES | 809.0 | 1,237.0 | 1,023.0 | ||||||
| Day | 885.0a | 1,331.9b | |||||||
| His | CON | 809.4 | 1,053.6 | 931.5 | 119.5 | 0.46 | 0.08 | 0.20 | |
| RES | 746.7 | 679.9 | 713.3 | ||||||
| Day | 778.0 | 866.7 | |||||||
| Ile | CON | 171.6 | 99.0 | 135.3 | 13.7 | <0.01 | 0.30 | 0.19 | |
| RES | 138.2 | 103.0 | 120.6 | ||||||
| Day | 154.9a | 101.0b | |||||||
| Leu | CON | 404.0 | 254.5 | 329.2 | 31.9 | <0.01 | 0.21 | 0.18 | |
| RES | 318.9 | 257.4 | 288.1 | ||||||
| Day | 361.5a | 255.9b | |||||||
| Lys | CON | 377.2 | 263.1 | 320.1 | 53.7 | 0.90 | 0.27 | 0.06 | |
| RES | 209.4 | 310.0 | 259.7 | ||||||
| Day | 293.3 | 286.5 | |||||||
| Met | CON | 551.2 | 526.7 | 539.0 | 60.3 | 0.94 | 0.07 | 0.64 | |
| RES | 406.9 | 440.2 | 423.6 | ||||||
| Day | 479.1 | 483.4 | |||||||
| Orn | CON | 63.6 | 71.1 | 67.4 | 9.4 | 0.04 | 0.31 | 0.19 | |
| RES | 41.2 | 74.1 | 57.6 | ||||||
| Day | 52.4a | 72.6b | |||||||
| Phe | CON | 439.0 | 288.6 | 363.8 | 49.0 | 0.02 | 0.19 | 0.53 | |
| RES | 341.1 | 252.8 | 297.0 | ||||||
| Day | 390.1a | 270.7b | |||||||
| Pro | CON | 421.3 | 465.7 | 443.5 | 47.0 | 0.17 | 0.14 | 0.64 | |
| RES | 326.8 | 416.3 | 371.6 | ||||||
| Day | 374.1 | 441.0 | |||||||
| Ser | CON | 795.4 | 599.4 | 697.4 | 72.5 | 0.15 | 0.35 | 0.24 | |
| RES | 639.1 | 617.9 | 628.5 | ||||||
| Day | 717.3 | 608.7 | |||||||
| Thr | CON | 292.6 | 138.5 | 215.6 | 92.4 | 0.23 | 0.75 | 0.66 | |
| RES | 222.2 | 149.5 | 185.9 | ||||||
| Day | 257.4 | 144.0 | |||||||
| Trp | CON | 113.6 | 74.5 | 94.1 | 24.4 | 0.03 | 0.81 | 0.52 | |
| RES | 135.8 | 64.5 | 100.2 | ||||||
| Day | 124.7a | 69.5b | |||||||
| Tyr | CON | 200.1 | 89.1 | 144.6 | 60.7 | 0.25 | 0.34 | 0.53 | |
| RES | 101.5 | 68.3 | 84.9 | ||||||
| Day | 150.8 | 78.7 | |||||||
| Val | CON | 559.1e | 357.7f | 458.4 | 49.2 | 0.06 | 0.16 | 0.05 | |
| RES | 385.4f | 389.2f | 387.3 | ||||||
| Day | 472.2 | 373.4 | |||||||
Means within row without common superscript differ (P ≤ 0.05).
Means within row without common superscript differ (P ≤ 0.05).
Means within column without common superscript differ (P ≤ 0.05).
Means within column without common superscript differ (P ≤ 0.05).
Means without common superscript differ (P ≤ 0.05).
Means without common superscript differ (P ≤ 0.05).
Day of gestation = number of days after insemination.
Probability values for effect of day, treatment, and day × treatment on the concentrations of hexoses and AAs in serum. Probability values for the contrast of nutrient concentrations of NP vs. Preg (all days of gestation), day 16 of gestation vs. days 34 and 50 of gestation, and day 34 vs. day 50 of gestation.
Nutrient-hexoses-glucose (Glc) and fructose (Frc). AA-alanine (Ala), arginine (Arg), asparagine (Asn), aspartate (Asp) citrulline (Cit), cysteine (Cys), glutamine (Gln), glutamate (Glu) glycine (Gly), histidine (His), isoleucine (Ile), leucine (Leu), lysine (Lys), methionine (Met), ornithine (Orn), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), tryptophan (Trp), tyrosine (Tyr), and valine (Val).
CON = heifers fed a total mixed ration that meets 100% of NRC requirements to gain 0.45 kg daily. RES = heifers restricted to 60 percent of CON diet.
Mean hexose and AA concentrations of treatment groups across day of gestation in maternal serum.
Average SEM for day × treatment interaction. Day 34 CON n = 6, day 34 RES n = 9, day 50 CON n = 7, day 50 RES n = 7.
Mean AA concentration across treatment within day of gestation.
Amniotic Fluid
Data for AMF are presented in Table 4. No data are presented for day 16 of gestation due to lack of AMF at this time point. Concentrations of glucose in AMF were greater (P = 0.05) in CON compared with RES heifers. Fructose was influenced by a day × treatment interaction, being greater (P = 0.04) for day 34 RES compared with day 50 CON and RES heifers; additionally, day 34 RES and day 50 CON were greater (P ≤ 0.05) compared with day 50 RES. A day × treatment interaction (P = 0.04) was observed for Gln, with day 34 RES being greater (P ≤ 0.05) than all other days and treatments. All other AAs were not influenced by a day × treatment interaction (P > 0.05). Thus, for the remainder of the AMF data, we will discuss only significant main effects. Alanine was greater (P < 0.01) on day 34 compared with day 50. Arginine concentrations were greater (P = 0.04) on day 50 compared with day 34. Asparagine and Asp were greater (P < 0.01) on day 34 compared with day 50. Cysteine was greater (P < 0.01) on day 50 compared with day 34. Glutamate was greater (P < 0.01) on day 34 compared with day 50. Glycine tended (P = 0.07) to be greater for day 34 RES compared with all other days and nutritional treatments; furthermore, Gly was greater (P < 0.01) on day 34 compared with day 50. Isoleucine, Leu, Lys, Met, Orn, Phe, Pro, Ser, Thr, and Val were greater (P ≤ 0.05) on day 34 compared with day 50.
Table 4.
Concentrations of hexoses (mM) and AAs (µmol/L) in heifer amniotic fluid samples as influenced by dietary treatment on days 34 and 50 of gestation
| Day of gestation1 | P-values2 | ||||||||
|---|---|---|---|---|---|---|---|---|---|
| Nutrient3 | Nutr.Trt4 | 34 | 50 | Trt Avg5 | SEM6 | Day | Trt | Day×Trt | |
| Hexoses | |||||||||
| Glc | CON | 1.85 | 1.74 | 1.79a | 0.22 | 0.34 | 0.05 | 0.16 | |
| RES | 1.08 | 1.61 | 1.34b | ||||||
| Day6 | 1.46 | 1.68 | |||||||
| Frc | CON | 3.30cd | 2.57d | 2.93 | 0.29 | <0.01 | 0.21 | 0.04 | |
| RES | 3.56c | 1.55e | 2.55 | ||||||
| Day | 3.43 | 2.06 | |||||||
| AA | |||||||||
| Ala | CON | 852.3 | 424.5 | 638.4 | 59.4 | <0.01 | 0.40 | 0.91 | |
| RES | 794.4 | 380.4 | 587.4 | ||||||
| Day | 823.4f | 402.4g | |||||||
| Arg | CON | 203.6 | 253.6 | 228.6 | 24.2 | 0.04 | 0.63 | 0.88 | |
| RES | 212.1 | 269.4 | 240.7 | ||||||
| Day | 207.8f | 261.5g | |||||||
| Asn | CON | 160.1 | 90. | 125.2 | 23.5 | <0.01 | 0.57 | 0.60 | |
| RES | 186.5 | 91.2 | 138.8 | ||||||
| Day | 173.3f | 90.8g | |||||||
| Asp | CON | 10.2 | Und | 10.2 | 2.8 | <0.01 | 0.29 | 0.29 | |
| RES | 16.3 | Und | 16.3 | ||||||
| Day | 13.3f | Undg | |||||||
| Cit | CON | Und7 | Und | Und | - | - | - | - | |
| RES | Und | Und | Und | ||||||
| Day | Und | Und | |||||||
| Cys | CON | 5.0 | 25.5 | 15.3 | 5.13 | <0.01 | 0.51 | 0.53 | |
| RES | 11.8 | 25.7 | 18.7 | ||||||
| Day | 8.4f | 25.6g | |||||||
| Glu | CON | 42.13 | 0.50 | 21.32 | 6.10 | <0.01 | 0.40 | 0.60 | |
| RES | 50.67 | 2.53 | 26.60 | ||||||
| Day | 46.40f | 1.52g | |||||||
| Gln | CON | 956.2c | 543.4e | 749.8 | 53.8 | <0.01 | 0.04 | 0.04 | |
| RES | 1,191.7e | 543.1e | 867.4 | ||||||
| Day | 1,073.9 | 543.3 | |||||||
| Gly | CON | 615.4 | 460.7 | 538.1 | 53.5 | <0.01 | 0.13 | 0.07 | |
| RES | 804.4 | 443.4 | 623.9 | ||||||
| Day | 709.9f | 452.1g | |||||||
| His | CON | 243.9 | 184.7 | 214.3 | 60.2 | 0.05 | 0.21 | 0.29 | |
| RES | 385.2 | 196.7 | 291.0 | ||||||
| Day | 314.6 | 190.7 | |||||||
| Ile | CON | 47.3 | 26.2 | 36.7 | 19.5 | 0.01 | 0.28 | 0.22 | |
| RES | 79.5 | 24.0 | 51.7 | ||||||
| Day | 63.4f | 25.1g | |||||||
| Leu | CON | 131.1 | 75.4 | 103.2 | 30.2 | 0.01 | 0.26 | 0.23 | |
| RES | 204.3 | 72.7 | 138.5 | ||||||
| Day | 167.7f | 74.0g | |||||||
| Lys | CON | 103.1 | 47.5 | 75.3 | 53.7 | <0.01 | 0.30 | 0.17 | |
| RES | 137.4 | 39.8 | 98.6 | ||||||
| Day | 130.2f | 43.7g | |||||||
| Met | CON | 246.9 | 161.8 | 204.4 | 28.6 | <0.01 | 0.38 | 0.31 | |
| RES | 302.8 | 158.0 | 230.4 | ||||||
| Day | 274.9f | 159.9g | |||||||
| Orn | CON | 37.4 | 14.3 | 25.9 | 5.56 | <0.01 | 0.99 | 0.60 | |
| RES | 40.3 | 11.3 | 25.8 | ||||||
| Day | 38.9f | 12.8g | |||||||
| Phe | CON | 137.1 | 108.0 | 122.6 | 41.5 | 0.05 | 0.33 | 0.18 | |
| RES | 237.0 | 92.4 | 164.7 | ||||||
| Day | 187.0f | 100.2g | |||||||
| Pro | CON | 285.7 | 128.4 | 207.4 | 11.5 | <0.01 | 0.39 | 0.18 | |
| RES | 312.3 | 122.3 | 217.3 | ||||||
| Day | 299.0f | 125.3g | |||||||
| Ser | CON | 459.8 | 180.9 | 320.4 | 39.9 | <0.01 | 0.18 | 0.11 | |
| RES | 582.3 | 169.9 | 376.1 | ||||||
| Day | 521.0f | 175.4g | |||||||
| Thr | CON | 392.6 | 189.5 | 291.0 | 39.4 | <0.01 | 0.71 | 0.36 | |
| RES | 444.9 | 167.2 | 306.0 | ||||||
| Day | 418.7f | 178.3g | |||||||
| Trp | CON | 41.1 | 34.1 | 37.6 | 10.4 | 0.11 | 0.40 | 0.32 | |
| RES | 60.9 | 32.5 | 46.7 | ||||||
| Day | 51.0 | 33.3 | |||||||
| Tyr | CON | Und | Und | Und | - | - | - | - | |
| RES | Und | Und | Und | ||||||
| Day | Und | Und | |||||||
| Val | CON | 163.5 | 111.9 | 137.7 | 43.8 | 0.04 | 0.26 | 0.32 | |
| RES | 260.6 | 118.3 | 189.4 | ||||||
| Day | 212.0f | 115.1g | |||||||
Means within column without a common superscript differ in day × treatment (P ≤ 0.05).
Means within column without a common superscript differ in day × treatment (P ≤ 0.05).
Means without a common superscript differ in main effect of day (P ≤ 0.05).
Means without a common superscript differ in main effect of day (P ≤ 0.05).
Means within row without a common superscript differ in main effect of treatment (P ≤ 0.05).
Day of gestation = number of days after insemination. NP = heifers who were not bred but received ovariohysterectomy on day 16 of their synchronized estrous cycle.
Probability values for effect of day, treatment, and day × treatment on the concentrations of hexoses and AAs in serum. Probability values for the contrast of nutrient concentrations of NP vs. Preg (all days of gestation), day 16 of gestation vs. days 34 and 50 of gestation, and day 34 vs. day 50 of gestation.
Nutrient-hexoses-glucose (Glc) and fructose (Frc). AA-alanine (Ala), arginine (Arg), asparagine (Asn), aspartate (Asp) citrulline (Cit), cysteine (Cys), glutamine (Gln), glutamate (Glu) glycine (Gly), histidine (His), isoleucine (Ile), leucine (Leu), lysine (Lys), methionine (Met), ornithine (Orn), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), tryptophan (Trp), tyrosine (Tyr), and valine (Val).
CON = heifers fed a total mixed ration that meets 100% of NRC requirements to gain 0.45 kg daily. RES = heifers restricted to 60% of CON diet.
Mean hexose and AA concentration of treatment group across day of gestation within tissue and nutrient of interest.
Average SEM for the day × treatment interaction was used within gene. NP n = 6, d 16 CON n = 7, d 16 RES n = 7, d 34 CON n = 6, d 34 RES n = 9, d 50 CON n = 7, d 50 RES n = 7.
Mean nutrient concentration across treatment within day and gene of interest.
Und-nutrient undetectable. Minimum detectable limits for UPLC detection were 5 µmol/L. Therefore, we assigned values at 0.1× of the lower detectable limit. The concentration is below 0.5 µmol/L.
DISCUSSION
Proper nutrient supply to the conceptus throughout early gestation is critical to the establishment and maintenance of pregnancy in all species. For ruminants, successful establishment of pregnancy requires conceptus elongation and secretion of sufficient interferon-tau for the maintenance of pregnancy (Bazer et al., 1997; Bazer et al., 2011b). If the pregnancy is maintained, significant cellular and tissue differentiation, as well as fetal organogenesis continue through day 50 of gestation (Winters et al., 1942) after which time the majority of fetal organs are present (Winters et al., 1942; Larsen, 2001). During this time frame, a proper milieu of nutrients from histotroph is required by the conceptus for growth and development until the establishment of transplacental exchange. Histotroph is made up of cytokines, enzymes, hormones, growth factors, vitamins, AA, hexoses, and other substances that are secreted into the uterine lumen for subsequent transport into the fetal-placental vascular system (Bazer, 1975; Spencer and Bazer, 2004; Bazer et al., 2011a). Gray et al. (2001) demonstrated the necessity of histotroph via a uterine gland knockout model in sheep, which resulted in the loss of conceptus viability. Therefore, it is no surprise that histotrophic nutrition deficiencies, prior to hemotrophic nutrition establishment, can lead to reduced embryonic and placental growth, resulting in IUGR, or even failed pregnancies (Bazer et al., 2011b).
We hypothesized that both maternal nutrition and day of gestation would affect the concentrations of AA and hexoses in bovine utero-placental fluids and maternal serum from days 16 to 50 of gestation. The results of this study support our hypothesis in that Val, Gln, and fructose were influenced by a day × treatment interaction in ALF, AMF, and AMF, respectively. Furthermore, maternal nutritional treatment affected the concentrations of Asp, Gly, Ser, Thr, and glucose in the various fluids investigated, whereas day of gestation affected the concentrations of all 17 AA as well as glucose and fructose in at least one of the fluids investigated. It is important to note that these data complement our previously determined mRNA data where the expression of five neutral and acidic AA transporters, as well as six glucose, fructose, and cationic AA transporters in utero-placental tissues were determined relative to NP endometrial samples from beef heifers (Crouse et al., 2016b, 2017; Greseth et al., 2017). Concentrations of nutrients in maternal and fetal fluids analyzed in this study were collected from the same animals and tissues used in the previously published studies.
When comparing days 34 and 50 of gestation, 9 of 20 AA (Asn, Asp, Cys, Gly, Ile, Leu, Phe, Trp, and Val) as well as Orn, Cit, and glucose differed in ALF, or tended to differ due to day of gestation. The same day of gestation effect was observed for 18 of 20 AA (excluding: Trp and Tyr) in AMF as well as intermediary metabolites Cit and Orn. Of the AA investigated in histotroph, 7 of 20 had a greater concentration on days postattachment (days 34 and 50) compared with day pre-attachment (day 16). Past research has suggested that the abundance of specific AA (Steeves and Gardner, 1999) and hexoses (Suga et al., 1973; Suga, 1975) changes depending on developmental stage of the conceptus. Recent research by Forde et al. (2014) and Groebner et al. (2011) further support this, as both groups found multiple main effects of day on AA concentrations in the histotroph of heifers during the pre-implantation and peri-implantation periods of gestation. Additionally, concentrations of hexoses in histotroph varied by developmental stage in both the bovine (Suga et al., 1973) and the ovine (Gao et al., 2009). Around day 16 of gestation in the bovine, maternal recognition of pregnancy occurs (Thatcher et al., 1994), along with rapid placental growth and elongation, and around day 34 of gestation embryonic organogenesis is still taking place (Winters et al., 1942). Furthermore, by day 50 of gestation, the conceptus has developed all of its organ systems and is deemed a fetus. Moreover, the uterus undergoes dramatic structural and functional changes to prepare for gestation and implantation. Due to the key events mentioned and the fact that the majority of embryonic loss occurs before day 40 of gestation (Thatcher et al., 1994), data regarding concentrations of these AA and hexoses that are vital to successful pregnancy establishment during the first 50 d of gestation are important.
Concentrations of glucose in ovine histotroph were greater in pregnant vs. cyclic ewes and increased between days 10 and 16 of gestation (Gao et al., 2009). Our data are similar in that concentrations of glucose were greater in pregnant vs. NP; however, concentrations remained similar from days 16 to 50 of gestation. It was expected that histotroph glucose concentrations would be greater than those of fructose due to glucose being the main fuel source for the fetus and placenta (Hay, 1995). However, the placenta is also the site of conversion of glucose to fructose (Alexander et al., 1955a, b; White et al., 1979), thus the increase in fructose from histotroph to ALF and AMF was to be expected. The role of placental conversion of glucose to fructose and subsequent transport of fructose to the conceptus can be further explained when the current glucose and fructose data are coupled with the greater expression of fructose transporters reported in intercotyledonary placenta (Crouse et al., 2017) compared with the cotyledonary placenta and amnion. Glucose concentrations in AMF differed by maternal nutritional treatment and concentrations of fructose differed by day of gestation and nutritional treatment on day 50 of gestation. Differences seen in both hexoses may be due to either reduced transport due to reduced availability from the maternal system or differential metabolism of glucose and fructose by the placenta and fetus. Both glucose and fructose independently stimulate cell proliferation through activation of the mechanistic target of rapamycin signaling pathway (Kim et al., 2012). Changes in the concentrations of these nutrients due to treatment and day of gestation may culminate in differences in cell cycle regulation and thus cell growth, differentiation, and proliferation of the conceptus. Fructose is involved in nucleotide synthesis via the pentose-phosphate pathway and increases in fructose concentration for day 34 RES and CON AMF compared with day 50 RES and CON may suggest an increase in nucleotide synthesis on day 34, which may be due to fetal organogenesis and an increased need of nucleotides compared with day 50.
When examining the histotroph of beef heifers, Forde et al., (2014) found that neutral AA had the largest concentrations during the pre-implantation stage. Data from the present study support those findings in that eight of 15 neutral AA had greater concentrations than Glu, Asp, His, Arg, or Lys in histotroph on day 16 of gestation. Furthermore, the present study indicates that glycine was the AA with the greatest concentration in histotroph during the pre-implantation period, which supports similar findings in beef heifer histotroph (Forde et al., 2014) and sheep histotroph (Gao et al., 2009). Arginine concentrations were greater on day 34 compared with day 50, which could be due to Arg’s association with secreted phosphoprotein 1 and elongation of the placenta during early pregnancy similar to what has been previously shown in sheep (Bazer et al., 2011b). Additionally, Crouse et al., (2017) reported the expression of CAT-1, a high-affinity Arg transporter (Closs et al., 1997) was greater in intercaruncular endometrium on days 34 and 50 compared with day 16.
On day 34 of gestation, Gln was the most abundant AA in both ALF and AMF. This abundance highlights Gln’s function as a fuel for rapidly proliferating cells. In addition, on day 34, maternal nutrition affected the concentration of Gln in AMF, where RES was greater than CON. Due to Gln’s function as a major fuel for fetal growth, these results may suggest the action of a compensatory mechanism during maternal nutrient deprivation. Despite receiving fewer nutrients, the dam may utilize more nutrient reserves, in this case Gln specifically, to provide a sufficient amount of the AA to the conceptus. Additionally, with such an abundance of this AA in the fetal fluids, it is possible that the maternal and/or fetal system may synthesize Gln. Valine is known to serve as a substrate for Gln synthesis (Wu, 2009). In fact, when the porcine placenta is treated with branched-chain AA (Leu, Ile, and Val), Val loss is exhibited that corresponds with an increase in Gln synthesis (Self et al., 2004). In the present study, Val concentrations in ALF were affected by maternal nutrition status on day 34 of gestation, where CON was greater than RES. This restriction could be linked to synthesis of Gln. Unfortunately, the present study did not examine that possibility. However, a future study of similar design with isotope labeling of nutrients is warranted to allow for the determination of flux in nutrient supply between the maternal circulation, uterine luminal fluids (i.e., histotroph), ALF, AMF, and the fetal circulation. Such a study would further delineate the basis for the differences in AA concentrations between treatment groups on day 34. It is also important to note that Greseth et al. (2017) determined that in intercaruncular tissue the relative mRNA expression of SLC38A7 (Gln transporter; Hagguland et al., 2011) was greater for RES compared with the CON group on day 16 of gestation; however, this difference was absent on days 34 and 50 of gestation.
Data in the present study show that the concentration of AA involved in one-carbon metabolism including Ser, Gly, and Met was greater in AMF on day 34 compared with day 50 of gestation. There are many events during early gestation that require one-carbon metabolites to ensure proper development and differentiation, such as global DNA demethylation at fertilization followed by subsequent de novo DNA methylation (Santos et al., 2002); however, around day 34 of gestation dorsal portions of the body are differentiating, vertebrae are becoming more organized, and limbs are undergoing regional specialization (Winters et al., 1942). In addition, coordinated DNA methylation events at myogenic loci as well as genes such as Myf5, MyoD, and MYOG must take place for proper muscle development and myogenic commitment to take place (Palacios and Puri, 2006). As previously mentioned, Ser, Gly, and Met have roles in one-carbon metabolism and thus in providing methyl donors for DNA methylation. These events involve the use of Ser, Gly, and Met to maintain the methionine-folate cycle and thus synthesis of S-adenosyl methionine (Pogribna et al., 2001), which serves as the primary methyl donor for methylation of CpG in genes involved in the myogenic differentiation pathway (Palacios and Puri, 2006). Expression of fetal myogenic genes collected from our current study on day 50 of gestation is upregulated in fetuses from RES compared with CON heifers (Ward et al., 2017). Accompanied with decreased Met in AMF of RES compared with CON heifers, myogenic regulation may be altered by maternal nutrient restriction and the supply of one-carbon metabolites. Additionally, our data on homocysteine concentrations (not reported herein) showed increased serum homocysteine in RES heifers. Therefore, the greater concentrations of Ser, Gly, and Met, as well as altered concentrations of Met and homocysteine due to nutritional treatment may indicate a deficiency in the one-carbon metabolite pathway, which would affect the methylation of homocysteine to methionine across the maternal–fetal interface (Castro et al., 2006). Thus, these changes may affect programming of feto-placental gene expression due to alterations in DNA methylation and thereby may alter the differentiation and commitment of muscle precursor cells to myoblast development that is occurring at this point of gestation.
In conclusion, continued moderate maternal nutrient restriction, resulting in −0.08 kg/d gain vs. 0.51 kg/d gain in control-fed dams, may further exacerbate differences observed in AA and hexose concentrations due to limited nutrient availability. Whereas the transporters of these nutrients are critical for conceptus development, determination of the concentrations of AA and hexoses in maternal circulation and fetal fluids is important in nutrient-restricted beef heifers during early pregnancy. Data presented in this study, as well as previously reported mRNA expression data (Crouse et al., 2016b, 2017; Greseth et al., 2017) in combination with immunolocalization of the transporters to determine location and abundance, will provide a more complete AA and hexose transport profile in the utero-placental tissues of beef heifers during the first 50 d of gestation.
Footnotes
This project was supported by Agriculture and Food Research Initiative Competitive (grant no. 2016-67016-24946) from the USDA National Institute of Food and Agriculture.
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