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Journal of Animal Science - Animal Physiology

Supplementation of corn dried distillers’ grains plus solubles to gestating beef cows fed low-quality forage: II. Impacts on uterine blood flow, circulating estradiol-17β and progesterone, and hepatic steroid metabolizing enzyme activity1


This article in JAS

  1. Vol. 94 No. 11, p. 4619-4628
    Received: Feb 22, 2016
    Accepted: July 31, 2016
    Published: October 27, 2016

    2 Corresponding author(s):

  1. V. C. Kennedy*,
  2. B. R. Mordhorst*,
  3. J. J. Gaspers*,
  4. M. L. Bauer*,
  5. K. C. Swanson*,
  6. C. O. Lemley and
  7. K. A. Vonnahme 2*
  1. * Department of Animal Sciences, North Dakota State University, Fargo 58108
     Department of Animal and Dairy Sciences, Mississippi State University, Mississippi State 39762


The objective of this study was to investigate the effects of supplementing dried distillers’ grains plus solubles (DDGS) during late gestation on uterine blood flow (BF), circulating steroid hormones and hepatic steroid metabolizing enzymes, and calf and placental weights. Multiparous beef cows were randomly divided into a control group (CON; n = 15) consuming a diet containing 90% corn stover and 10% corn silage (DM basis) for ad libitum intake and a treatment group (SUP; n = 12) consuming the same diet and DDGS (0.3% of BW). Corn silage inclusion was increased to 30% as gestation progressed to meet increasing caloric requirements. Ipsilateral and contralateral uterine BF and cross-sectional area (CSA) of each uterine artery were measured by Doppler ultrasonography on d 180, 216, and 246 of pregnancy. Contralateral BF and CSA increased (P < 0.01) as gestation advanced. Ipsilateral BF and CSA was affected by a treatment × day of gestation interaction (P < 0.05). A main effect of treatment (P = 0.02) and day (P < 0.01) was observed for total BF; BF increased over time and SUP cows had greater BF than CON cows. Circulating concentrations of both progesterone (P4) and estradiol-17β (E2) were affected by an interaction of treatment and day (P < 0.01). Concentrations of circulating E2 steadily increased throughout the study and were greater in CON cows than in SUP cows by d 242. Concentrations of P4 also increased over time; P4 of CON cows was greater than that of SUP cows by d 242. Uridine 5′-diphospho-glucuronosyltransferase (UGT) and cytochrome P450 1A (CYP1A) activity increased with advancing gestation (P < 0.01). There was greater UGT activity (P < 0.05) and a trend for greater CYP1A activity (P = 0.06) in SUP cows than in CON cows. Activity of cytochrome P450 3A was greater (P < 0.01) in SUP cows and decreased (P < 0.05) with advancing gestation. Supplementing DDGS to cows fed low-quality forage during late gestation increased uterine BF but decreased circulating E2 and P4 concentrations and altered hepatic steroid metabolizing enzyme activity. It was anticipated that enzyme activity would reflect circulating hormone levels; however, our data suggests the observed increases in BF are not driven by alterations in hormone concentration. Therefore, further research is warranted to elucidate the underlying mechanisms.


Maternal nutrition is essential to gravid uterine development, which influences the lifetime performance of the calf (Funston et al., 2010). One way to quantify nutrient delivery to the fetus is to measure uterine arterial blood flow (BF; Ferrell, 1991). Maternal feed intake, as well as specific dietary components, can alter uterine BF (Reynolds et al., 2006; Lemley et al., 2012). Besides providing vasoactive factors, the maternal diet may directly influence circulating vasoactive steroids, specifically estradiol-17β (E2), a known vasodilator (Resnik et al., 1974), and progesterone (P4; Lemley et al., 2014). The amount and ratio of E2 and P4 are thought to influence uterine BF (Rupnow et al., 2001).

Recently, we demonstrated that dried distillers’ grains plus solubles (DDGS) supplementation to cows during late gestation fed low-quality forage reduced uterine BF, which disagreed with our hypothesis (Mordhorst et al., 2016). It is plausible that DDGS enhanced steroid catabolism by the liver, as P4 concentrations tended to decrease in supplemented cows (Mordhorst et al., 2016). How maternal diet impacts circulating steroids is not understood; however, in sheep, dietary protein causes an increase in hepatic P450 enzyme activity, which oxidizes steroids (Thomford and Dziuk, 1988). Our laboratory has previously demonstrated protein-deficient ewes have decreased maternal hepatic steroid catabolizing enzyme activity (Lekatz et al., 2015).

We recently indicated that DDGS supplementation can positively influence voluntary intake, eating behavior, and maintenance of BW and BCS (Kennedy et al., 2016). In dairy cows, there was a positive association between intake and metabolic clearance rate of P4 (Sangsritavong et al., 2002). We hypothesized that supplementation of DDGS would increase hepatic metabolism of steroids, namely E2 and P4, and thus reduce uterine BF. The objective of this study was to investigate the effects of supplementing DDGS to cows fed a cornstalk-based diet during late gestation on uterine BF, circulating concentrations of E2 and P4, and their corresponding metabolic enzymes in the liver.


Experimental Design, Cows, and Dietary Treatments

All procedures were approved by the North Dakota State University Animal Care and Use Committee (A14007). Treatments applied to animals have been previously described (Kennedy et al., 2016). Briefly, 27 multiparous beef cows (Angus or Angus-based cross; 674 ± 17 kg and 6 ± 5 yr) were stratified by weight and age of the cow and then randomly divided into a control group (CON; n = 15) and a treatment group (SUP; n = 12). Following a 3-wk acclimation period, intake was monitored and controlled via Insentec roughage feeders (Insentec, B.V., Marknesse, the Netherlands) beginning on d 201 of gestation for 10 wk. Use of Insentec feeders allows for individual animal feed intake monitoring. A basal diet of 90% corn stover and 10% corn silage (5.0% CP [DM basis], marginally NE deficient, and RDP deficient) was fed for ad libitum intake to both treatment groups, with the SUP group supplemented DDGS at 0.3% of BW (DM basis). Corn silage inclusion was increased to 20% on d 246 of gestation (gestational diet 2) to meet increased nutritional demands during pregnancy, but supplementation regimes remained the same. Both pens had free access to water and trace-mineralized salt blocks (95.5 to 98.5% NaCl, 3,500 mg of Zn/kg, 2,000 mg of Fe/kg, 1,800 mg of Mn/kg, 280 to 420 mg of Cu/kg, 100 mg of I/kg, and 60 mg of Co/kg). Following parturition, gestation length was calculated for each cow.

Body Weights, BCS, and Blood Collection

During gestation, cows were weighed and body condition was scored every 2 wk from initiation of the project until d 242 of gestation and effects of diet on weight and BCS have been previously reported (Kennedy et al., 2016). On each weigh day, blood was sampled via jugular venipuncture. Samples were centrifuged for 20 min at 1,380 × g at 4 degrees C to separate serum, which was then stored at −20°C until P4 and E2 analysis.

Uterine Hemodynamic Measurements

To measure uterine hemodynamics, color Doppler ultrasonography was used (Camacho et al., 2014a). Ipsilateral and contralateral uterine BF, cross-sectional area (CSA), and pulsatility indices (PI) were measured by Doppler ultrasonography on d 180, 216, and 246 (±5 d) of pregnancy. A 7.5 MHz finger probe was inserted into the rectum and the bifurcation of the internal and external iliac arteries was identified. By following the former, the uterine artery was identified descending toward the uterus. This was confirmed by its Doppler coloration and maneuverability relative to the iliac artery or caudal aorta. The probe was placed just below the branch point to ensure measurements were taken at the same location in each cow on each ultrasound day. At data collection, the average of 3 separate cardiac cycle waveforms was measured from 2 to 3 separate ultrasound evaluations (i.e., 6 to 9 measurements per artery per cow). Resistance index (RI), PI, peak systolic velocity, end diastolic velocity, flow time, maternal heart rate (HR), mean velocity, BF volume, CSA, and cross-sectional diameter were all recorded. The Doppler software was preprogrammed to calculate PI = (peak systolic velocity − end diastolic velocity)/mean velocity; RI = (peak systolic velocity − end diastolic velocity)/peak systolic velocity; and BF (mL/min) = mean velocity (cm/s) × (π/4) × CSA (cm2) × 60 s. The ipsilateral horn was identified as the side of the animal with greatest BF. Finally, total BF was calculated as the sum of ipsilateral and contralateral uterine artery BF late for statistical analyses.

Liver Biopsy Procedure and Sample Processing

Liver biopsies were performed on d 187 (±1) and d 221 or 222 of gestation, which will be referred to as “pre-” and “post-treatment” biopsies, respectively. Biopsies were performed in a hydraulic squeeze chute following the methods of Voelz et al. (2015). For reference, an imaginary line was drawn from the point of the tuber coxae to the olecranon and the liver biopsy was taken on this plane between the 10th and 11th rib on the right side of the animal. Hair was removed from the surgical site using clippers. The liver was scanned using Doppler ultrasonography to confirm its location and ensure the point of biopsy needle insertion was devoid of major hepatic vessels. The skin was then scrubbed twice with betadine. A local anesthetic (10 mL of 2% lidocaine hydrochloride) was administered at the 10th intercostal space and the skin was punctured using a scalpel. A biopsy tool, machined at Mississippi State University’s Department of Agricultural and Biological Engineering (Mississippi State, MS) according to specifications of Swanson et al. (2000), was inserted until it passed the liver capsule, the biopsy needle was deployed, and the biopsy tool was withdrawn. The biopsy needle collected 0.5 to 1.0 g of liver tissue, which was then placed in a cryogenic vial and snap frozen in liquid nitrogen. Immediately following liver sample collection, the skin was stapled and treated with antiseptic spray (Blu-Kote; H.W. Naylor Co. Inc., Morris, NY). Finally, feed intake and behavior (i.e., decreased feed intake, time, and duration of feeding) were monitored following surgery, and no complications were experienced. Skin staples were removed 7 to 10 d after surgery.


During calving, cows were allowed to remain in their pens with the group until signs of labor were observed. If it was possible to move the cow inside the barn without causing undue stress, she was brought inside and put in an individual pen for calving; otherwise, she was allowed to calve outside with the herd and the pair was immediately brought inside using a sled for the newborn calf. Time of birth was recorded. Calf BW, sex, crown–rump length, and heart girth were measured. Cows and calves were returned together to their individual pen where they were monitored for general calf health. Cow–calf pairs remained in individual pens for 24 h before returning outside to the group.

Time to placenta expulsion was recorded for each cow and placentas immediately were collected. Each placenta was weighed, cotyledons and intercotyledonary tissue was dissected and weighed, and the largest and smallest cotyledons were weighed. The number of cotyledons was counted. Finally, gestation length was calculated for each cow.

Preparation of Liver Samples and Enzyme Activity Assays

Liver samples were prepared according to Hart et al. (2014). Briefly, liver tissue was homogenized and centrifuged at 10,000 × g for 10 min at 4°C. The resulting supernatant was used for all enzymatic procedures. The cytochrome P450 1A (CYP1A), cytochrome P450 2C (CYP2C), cytochrome P450 3A (CYP3A), and uridine 5′-diphospho-glucuronosyltransferase (UGT) assay kits and Nicotinamide adenine dinucleotide phosphate (NADPH) regeneration system were purchased from Promega Corporation (Madison, WI). Enzymatic activity assays were performed according to Hart et al. (2014). Briefly, these assays use a luciferin substrate, which was detected using a plate reader using luminescence detection mode in relative light units (RLU; PPromega Multi+ Microplate Reader using luminescence detection mode; Promega Corporation). Enzyme activity was expressed relative to maternal liver protein (RLU · min-1 · mg-1 of protein), relative to grams of hepatic tissue (RLU · min-1 · g-1), and relative to maternal BW (RLU · min-1 · g-1 of BW). Activity of aldo-keto reductase 1C (AKR1C) was determined using the procedures of Lemley and Wilson (2010). In brief, concentrations of AKR1C in the fractions were determined using the specific substrate 1-acenapthenol (Pfaltz & Bauer Inc., Waterbury, CT). Reactions contained 150 μg of cytoplasmic protein, 250 μM 1-acenapthenol, and 500 μM Nicotinamide adenine dinucleotide phosphate (NADP). Reduction of NADP by 1-acenapthenol was standardized by the amount of cytoplasmic protein used. Reduction of NADP was measured by quantifying absorbance at 340 nm for 10 min using a plate reader (SpectraMax Plus; Molecular Devices, LLC, Sunnyvale, CA). To calculate the rate of reduced NADP produced, the molar absorption coefficient of NADPH was used (6,220 L/mol × cm). Activity of AKR1C is reported in picomoles per minute per milligram of cytoplasmic protein.

Estradiol-17β and Progesterone Analysis

Serum E2 concentrations were evaluated according to manufacturer’s instructions for solid-phase 125I radioimmunoassay (Siemens Healthcare Diagnostics Inc., Los Angeles, CA), except 500 μL of serum from samples was used instead of the recommended 100 μL. Standards and unknowns were pipetted in duplicate to E2 antibody-coated tubes. Next, 1 mL of E2 tracer (iodinated synthetic E2) was added to all tubes, which were then covered with parafilm, vortexed, and incubated at room temperature for 3 h. After incubation, all liquid in all tubes was aspirated and discarded. Finally, tubes were read in a Packard gamma counter (Packard Instrument Company, Inc, Meriden, CT). The average intra-assay CV for the E2 assay was 5.00%.

Progesterone serum concentrations were evaluated according to manufacturer’s instructions for solid-phase 125I radioimmunoassay (Siemens Healthcare Diagnostics Inc.). Standards and unknowns were pipetted in duplicate to P4 antibody-coated tubes. Next, 1 mL of P4 tracer (iodinated P4) was added to all tubes, which were then covered with parafilm, vortexed, and incubated at room temperature for 3 h. After incubation, all liquid in all tubes was aspirated and discarded. Finally, tubes were then read in a Packard gamma counter. The average intra-assay CV for the P4 assay was 3.17%.

Statistical Analysis

Data analyses for E2, P4, PI, RI, BF, CSA, and HR were performed using generalized least squares (mixed procedure; SAS Inst. Inc., Cary, NC) with class statement including cow, treatment, calf sex, and day of gestation. The model statement included day of gestation, treatment (SUP vs. CON), calf sex, and all interactions. Calf sex was removed from all analyses as it had minor contributions (P > 0.20). The repeated measures statement included day of gestation with cow nested within treatment as the subject. Covariance structures, based on information criteria, were compound symmetry for contralateral and ipsilateral PI, contralateral and ipsilateral RI, contralateral and ipsilateral BF, contralateral and ipsilateral CSA, E2, P4, and HR and unstructured for total BF.

Data analysis for liver enzyme activities used generalized least squares (mixed procedure). Class statements included cow, calf sex, treatment, and day of biopsy. Model statements included treatment, day of biopsy, calf sex, and all interactions. Calf sex was removed from all analyses as it had minor contributions (P > 0.20). To further elucidate changes in hormone concentration and enzyme activity, percentage change was calculated as before except without day and its interaction. Data analysis for calf and placental measurements were performed using the ordinary least squares (GLM procedure) with class and model statements including the main effects of treatment and calf sex. Calf sex was retained in the model for cotyledonary number and calf weights as it contributed to those measures (P ≥ 0.11). Calf sex was removed from the analyses for all other measures as it had minor contributions (P > 0.20). Differences between least squares means for all analyses were determined using the LSD method.


As previously reported (Kennedy et al., 2016), there was an effect of diet on maintenance of maternal BW and BCS; SUP cows gained (P < 0.01) weight at an average rate of 1.27 ± 0.15 kg/d and CON cows tended to lose weight (0.25 ± 0.13 kg/d; P = 0.06) while losing (P < 0.01) BCS.

Uterine Blood Flow and Maternal Heart Rate

A treatment × day of gestation interaction (P = 0.05) was observed for both CSA and uterine BF for the ipsilateral uterine artery. Cross-sectional area spanned 0.5 to 1.1 (±0.07) cm2 and was not different (P = 0.30) between treatments on d 181 but was greater (P < 0.01) in SUP than in CON cows on d 216 and 246 (Fig. 1A). Ipsilateral uterine BF was also similar (P > 0.01) on d 181 in SUP cows compared with CON cows but was greater (P < 0.01) on d 216 and 246.

Figure 1.
Figure 1.

Ipsilateral uterine artery cross-sectional area (CSA; A) and blood flow (BF; B) and contralateral uterine artery CSA (C) and BF (D) of beef cows fed the control (control group [CON]) or the control plus supplement (treatment group [SUP]) from d 201 to 270 of gestation. a–eLeast squares means ± SEM with different superscripts differ at P ≤ 0.05. Trt = treatment.


There was no interaction of treatment and day (P > 0.31) for CSA or BF on the contralateral side (Fig. 1C and 1D). A main effect of day (P < 0.01) was observed for CSA and BF on the contralateral side. Contralateral CSA increased from 0.24 to 0.47 (±0.05) cm2 and BF increased from 2.5 to 7.7 (±1.0) L/min.

When ipsilateral and contralateral BF were amalgamated, there were main effects of treatment (P = 0.02) and day (P < 0.01) for total uterine BF. Uterine BF increased as gestation advanced, and SUP cows had greater BF than CON cows (Fig. 2A). There was a treatment × day interaction (P < 0.01) for maternal heart rate. Although HR was similar (P = 0.37) between treatments on d 181, CON cows experienced a reduction in HR on d 221 whereas SUP cows did not. The HR of CON cows returned to d 181 rates by d 241, whereas SUP cows’ HR increased (P < 0.01; Fig. 2B).

Figure 2.
Figure 2.

Total uterine blood flow (A) and maternal heart rate (B) of beef cows fed the control (control group [CON]) or the control plus supplement (treatment group [SUP]) from d 201 to 270 of gestation. a–cLeast squares means ± SEM with different superscripts differ at P ≤ 0.05. Trt = treatment; bpm = beats per minute.


In addition to uterine BF, hemodynamic indices were altered as result of advancing gestation. On both the ipsilateral and contralateral sides, there was an effect of day on PI (P < 0.01), which decreased with advancing gestation (Fig. 3). No effects of day or treatment (P ≥ 0.21) were observed for RI on either side (data not shown).

Figure 3.
Figure 3.

Ipsilateral and contralateral uterine artery pulsatility index of beef cows fed the control or the control plus supplement from d 201 to 270 of gestation. a–c, x–zLeast squares means ± SEM within artery with different superscripts differ at P ≤ 0.05. Trt = treatment.


Circulating Estradiol-17β and Progesterone

There was a treatment × day interaction (P < 0.01) for circulating E2 and P4. Concentrations of E2 were similar on d 181 in both groups. Although E2 concentration remained relatively steady throughout the experiment in SUP cows, in CON cows, E2 concentration increased from d 188 to d 200 through 244. By d 246, the greatest concentrations were observed within the CON cows (Fig. 4A). Concentrations of P4 were similar in both groups until d 246, when CON cows had greater P4 concentration compared with SUP cows (Fig. 4B).

Figure 4.
Figure 4.

Circulating estradiol-17β (A) and progesterone (B) concentrations of beef cows fed the control (control group [CON]) or the control plus supplement (treatment group [SUP]) from d 201 to 270 of gestation. a–eLeast squares means ± SEM with different superscripts differ at P ≤ 0.05. Trt = treatment.


Enzyme Activity

An interaction of treatment and day was observed for aldo-keto reductase activity as expressed per milligram of protein (P < 0.01) and per gram of liver (P = 0.01); all measures of aldo-keto reductase activity decreased after initiation of treatment, with CON cows having higher pretreatment values than SUP cows but no significant difference after treatment (Fig. 5). No interaction of maternal dietary treatment and day of gestation was observed for the remaining steroid metabolizing enzyme activities, but main effects of day and treatment were observed (Table 1).

Figure 5.
Figure 5.

Hepatic aldo-keto reductase (AKR) 1C enzyme activity in beef cows fed the control (control group [CON]) or the control plus supplement (treatment group [SUP]) from d 201 to 270 of gestation. a,b, x,yDifferent superscripts within a measure differ at P ≤ 0.05. RLU = relative light units; Trt = treatment.


View Full Table | Close Full ViewTable 1.

Hepatic steroid metabolizing enzyme activity in cows before and after initiation1 of dietary treatments

Dietary treatment2
Dependent variable CON SUP SEM Before After SEM Treatment Day Treatment × day
    (RLU4 ∙ min-1 ∙ mg-1 protein)×104 79.8 90.1 6.4 65.4 104.4 6.4 0.26 <0.01 0.27
    (RLU ∙ min-1 ∙ g-1)×106 48.4 66.5 4.3 43.6 71.3 4.3 <0.01 <0.01 0.06
    RLU ∙ min-1 ∙ mg-1 protein 3.0 3.0 0.2 2.8 3.2 0.2 0.96 0.15 0.38
    (RLU ∙ min-1 ∙ g-1)×104 18.5 21.8 1.4 18.3 22.0 1.4 0.11 0.07 0.83
    RLU ∙ min-1 ∙ mg-1 protein 569.0 685.1 39.2 690.3 563.8 39.2 0.04 0.03 0.64
    (RLU ∙ min-1 ∙ g-1)×106 35.8 50.6 3.2 46.8 39.7 3.2 <0.01 0.13 0.68
    (RLU ∙ min-1 ∙ mg-1 protein)×105 10.7 13.2 1.4 8.8 15.2 1.4 <0.01 0.20 0.84
    (RLU ∙ min-1 ∙ g-1)×106 68.2 95.8 9.4 57.8 106.2 9.3 <0.01 0.04 0.85
1Initiation of dietary treatments was d 201 of gestation: before = d 187 (±1 d); after = d 221 to 222.
2Maternal diets: CON = control group (consuming basal diet; n = 15); SUP = treatment group (consuming basal diet + dried distillers’ grains plus solubles at 3 g/kg of BW; n = 12).
3CYP1A = cytochrome P450 1A.
4RLU = relative light units.
5CYP2C = cytochrome P450 2C.
6CYP3A = cytochrome P450 3A.
7UGT = uridine 5′-diphospho-glucuronosyltransferase.

A main effect of day (P < 0.01) was also observed for CYP1A activity, as expressed per milligram of protein, which increased with advanced gestation. A tendency for an interaction of treatment and day was observed for CYP1A activity per gram of liver tissue (P = 0.06), with CON cows and SUP cows having similar activities at the first biopsy but SUP cows having greater activities by the second biopsy. Day or treatment did not influence (P ≥ 0.11) CYP2C enzyme activity, although activity expressed per gram of liver tissue tended to be greater (P = 0.07) by the second biopsy. Activity of CYP3A, as expressed per milligram of protein, was influenced by treatment and day (P ≤ 0.04), where concentrations decreased by the second biopsy and supplemented cows had greater average activity. When evaluated per gram of liver sample, a main effect of treatment was observed (P < 0.01), with supplemented cows having greater concentration of activity. A main effect of day was observed for UGT enzyme activity when expressed per milligram of protein (P < 0.01), where enzyme activity was increased by the second liver biopsy. When expressed per gram of liver, an effect of day (P < 0.01) and treatment (P = 0.04) was observed, with increased enzyme activity by the second liver biopsy, and SUP cows had greater activity than CON cows (Table 1).


Gestation length was similar (P = 0.43) between treatments. Calves born to supplemented cows were heavier (P = 0.02) at birth and 24 h later but were similar (P ≥ 0.22) in heart girth and length (Table 2). There was no effect of treatment on dystocia score, placental expulsion time, or any placental measurements (Table 2).

View Full Table | Close Full ViewTable 2.

Calving and placental measurements from beef cows fed the control or the control plus supplementation from d 201 to 270 of gestation

Dietary treatment1
Variable CON SUP SEM P-value
Gestation length, d 277.0 275.8 1.0 0.43
Calf birth wt, kg 39.8 43.2 1.0 0.02
Calving ease2 1.87 1.44 0.36 0.39
Heart girth,3 cm 82.1 84.1 1.2 0.22
Crown rump length,4 cm 84.7 83.1 2.0 0.57
Calf 24 h wt, kg 40.4 44.0 1.1 0.02
Placental expulsion, min 226.8 286.3 54.5 0.45
Cotyledonary number 90.5 85.5 9.1 0.68
Total placenta wt, kg 5.4 4.7 0.4 0.22
Intercotyledonary wt,5 kg 2.7 2.2 0.2 0.13
Cotyledonary wt,6 kg 2.2 2.2 0.2 0.94
Largest, g 90.1 91.7 9.0 0.90
Smallest, g 1.0 0.5 0.2 0.18
1Maternal diets: CON = control group (consuming basal diet; n = 15); SUP = treatment group (consuming basal diet + dried distillers’ grains plus solubles at 0.3% of BW; n = 12).
2Calving ease score (1 = no assistance and 5 = caesarian section).
3Heart girth was measured around calf’s chest at withers.
4Crown rump measured from the point of the skull to the base of the tail head.
5Intercotyledonary weight includes all tissues except cotyledons.
6Cotyledonary weight includes all dissected cotyledons.


Supplementation of DDGS, a source of much higher protein than the basal diet (as reported in Kennedy et al., 2016), increased uterine BF during late gestation. This increase in supplemental protein was also reflected in faster HR and larger CSA in SUP cows than in CON cows. A decrease in RI with advancing gestation was also to be expected to accommodate the increases in total BF needed during pregnancy (Gómez et al., 2006) as cows neared the end of term. Similarly, the increased CSA of both uterine arteries, particularly on the ipsilateral side, where nearly a 4-fold increase was observed, will obviously result in dramatic changes in BF.

In agreement with our hypothesis, circulating concentrations of E2 and P4 were decreased and metabolizing enzymes were increased in the SUP group. Metabolic enzyme activity coincided with the circulating hormone concentrations observed. Lower E2 concentrations were present with greater enzyme activity in the SUP group. In a previous study performed by our laboratory (Mordhorst et al., 2016), supplementation of DDGS (1.7 g/kg of BW) during late gestation to cows fed low-quality hay (20 g/kg of BW) decreased uterine BF and produced a tendency for decreased P4 concentrations. Results from that study led to speculation that decreased uterine BF resulted from increased steroid metabolism and, therefore, decreased E2 levels. In that same experiment, no differences between calf birth weights or weaning weights were observed, whereas the current study resulted in heavier calves at birth and ultimately heavier calves at weaning (this study; Kennedy et al., 2015), despite placental weights not being impacted by supplementation in either experiment. Radunz et al. (2010) reported a greater birth weight to DDGS-supplemented cows during late gestation compared with hay-fed controls, with no difference in dystocia, similar to our study. Placental characteristics were not reported (Radunz et al., 2010). Radunz et al. (2010) reported that DDGS supplementation may increase P4 on d 210 of gestation (approximately 53 d after initiation of DDGS feeding), even though P4 was not different on d 189 or 231 of gestation. Perhaps the mechanisms behind the increased uterine BF in the current study were simply more influential than the potential alterations in circulating concentrations of steroids or their metabolism, thus producing greater BF in SUP cows as well as differences in calf BW. Regardless, both studies further underline the notion that placental function (and potential efficiency) cannot by assessed simply by placental weight alone, as placental size was similar between treatments in each experiment (Mordhorst et al., 2016; this study).

The CYP1A and CYP3A enzymes catalyze the conversion of E2 to 2-hydroxyestradiol and 4-hydroxyestradiol (Tsuchiya et al., 2005). In sheep, evidence implicates these E2 metabolites in partially regulating uterine vasoactivity independent of estrogen receptors (Jobe et al., 2013). Therefore, an increase in catechol estradiol metabolites could explain the increase in uterine artery BF even with a concomitant decrease in peripheral concentrations of E2. Increased metabolic clearance of P4 in the liver of ovariectomized (OVX) sheep has also been observed as a result of increased intake (Parr et al., 1993), creating an inverse relationship between nutrient intake and circulating P4, although feeding isoenergetic and isonitrogenous diets to Holstein cows has been shown to decrease hepatic CYP2C, CYP3A, and AKR1C activity and increase the half-life of P4 when liver BF was equal between dietary treatments (Lemley et al., 2010). Therefore, modulation of enzyme activity, independent of liver BF can alter the metabolic clearance rate of P4. The enzymes AKR1C and UGT are involved in phase I and phase II of P4 inactivation, respectively. The AKR1C enzymes are involved in the reduction of steroids containing aldehyde or ketone groups, which typically convert P4 to 3α-hydroxyprogesterone or 20α-hydroxyprogesterone metabolites (Penning et al., 2000). In the current study, AKR1C was increased before treatment in CON cows and then decreased after treatment; therefore, if P4 synthesis remained steady in the CON group, peripheral concentrations of P4 could be increasing due to the drop in AKR1C activity. The UGT enzymes conjugate hydroxysteroid metabolites with gucuronic acid, generating a more hydrophilic metabolite (Bowalgaha et al., 2007). This is an important step in steroid elimination, as a decrease in UGT activity could cause a buildup of hydroxysteroid metabolites, thereby decreasing phase I metabolism. Similar to our own observations, an increase in UGT activity in SUP cows would be expected to decrease both peripheral E2 and P4 concentrations.

The decreased concentration of E2 and P4 could also be a result of an increase in liver size. Although we did not measure total liver mass in the current experiment, liver mass in response to altered nutritional planes has been reported in pregnant beef cattle (Camacho et al., 2014b), where restricted animals had significantly lower liver masses than those kept on an adequate plane of nutrition. Level of feed intake has also been shown to increase the relative proportion of visceral organs to body mass in sheep, where changes in liver weight in response to level of nutrition were of a greater degree than any other organ (Burrin et al., 1990). The same study also found that changes in organ size were associated with altered whole-body metabolic rate. It has also been shown in dairy cows that a continuous high plane of nutrition may chronically elevate liver BF and metabolic clearance rate of P4 and E2 (Sangsritavong et al., 2002).

It is also possible that the observed results are more understandable within the larger picture of how the altered nutrition is affecting the whole cow. With this in mind, dietary effects from a nutrient intake, feeding behavior, and maternal performance standpoint were analyzed as well. An increase of voluntary DMI in SUP cows was coupled with maintenance of body condition compared to CON cows, who tended to lose weight and condition during the treatment period (Kennedy et al., 2016). In comparison to our previous study (Mordhorst et al., 2016), limited intake of a better quality basal diet (hay was fed at 20 g/kg of BW), was consumed at similar rates between treatment groups (P = 0.41) and provided less DDGS (1.7 g/kg of BW). This difference in nutritional plane between studies as well as similar hay intake between treatments in the first study could help to explain the different effects observed on uterine BF. In Radunz et al. (2010), DDGS was supplemented as a higher-quality hay than studies performed in our laboratory (approximately 8.2% CP). Supplementation began at 167 d of gestation and continued through 1 wk before expected calving. Reported DMI was increased in hay-fed control cows compared with DDGS-supplemented cows while receiving similar NEm intake (Radunz et al., 2010). Unlike the present study, hay-fed and DDGS-supplemented cows did not differ in BCS near calving (Radunz et al., 2010). This further demonstrates that solely providing a supplementation source will not impact the maternal system, energy stores, or uteroplacental function. Further investigations are needed to determine how forage source, quality, and availability with supplementation allow for nutrient availability to the developing calf. This may not be simply explained by forage or dietary supplement characteristics alone but rather how that forage and/or supplement influences intake and feeding behaviors, which could impact overall metabolism and nutrient utilization.

In our study, DDGS supplementation provided greater MP to cows and, coupled with the increased intake, a greater overall plane of nutrition throughout the treatment period. These increased planes of nutrition conceivably influenced overall metabolism, including clearance of P4 and E2. This is supported by the elevated enzyme activity in the SUP group as well as their comparably lesser circulating E2 concentrations from d 214 onward and decreased circulating P4 concentrations from d 228 onward.

Increased hepatic enzyme activity in the SUP group suggests that supplementation influences the activity of these enzymes and that changes in uterine BF are, therefore, likely regulated by a more localized mechanism pertaining to the uterus. This could be better defined in future experiments by measuring local (uterine vein) hormone concentrations, effectively providing a more accurate measure of hormone production during pregnancy compared with those circulating in the peripheral blood.

In conclusion, supplementation of DDGS to cows fed a low-quality basal diet altered uterine hemodynamics, circulating E2 and P4 concentrations, and activity of hepatic steroid metabolizing enzymes. Increased uterine artery diameter and HR clearly facilitated a large increase in BF. The observation that calves born to DDGS-supplemented cows were heavier than those born to CON cows, despite no difference in any placental weights, may be related to the increased BF in the uterine arteries and potentially could be reflective of increased placental efficiency (as opposed to simply weight). Regardless of the need to further investigate the mechanisms behind these changes, DDGS supplementation modified some of the major contributors (i.e., maternal weight and endocrine profile) to a successful pregnancy in beef cows, which likely have downstream consequences for offspring growth in and out of the womb.




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