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. 2021 Feb 15;99(3):skab050. doi: 10.1093/jas/skab050

Effects of administering exogenous bovine somatotropin to beef heifers during the first trimester on conceptus development as well as steroid- and eicosanoid-metabolizing enzymes

Carla D Sanford 1, Megan P T Owen 2, Nicola Oosthuizen 3, Pedro L P Fontes 4, Kimberly A Vonnahme 5, Megan Nelson 5, Arshi Reyaz 5, Caleb O Lemley 6, Nicolas DiLorenzo 7, Graham Cliff Lamb 3,✉
PMCID: PMC7999624  PMID: 33587143

Abstract

The aim of this study was to evaluate the effect of bovine somatotropin (bST) on fetal and placental development during the first third of gestation in beef heifers. Angus heifers (n = 97) were randomly assigned to either receive a 500-mg injection of bST (BST) biweekly on days 0, 15, 29, 43, and 57 of gestation or not receive bST (CTL) throughout the experiment. Body weight (BW) was assessed on days −9, −3, 0, 15, 22, 29, 43, 50, 57, 64, and 77, while blood samples were collected on days 0, 22, 50, and 64. Pregnancy status was determined via transrectal ultrasonography on days 29 and 64. A subset of pregnant heifers (BST, n = 7; CTL, n = 5) were harvested on day 84, and complete gravid reproductive tracts and liver tissue were collected for analysis. Cytochrome P450 1A (CYP1A), 2C (CYP2C), 3A (CYP3A), and uridine 5′-diphospho-glucuronosyltransferase (UGT) activities were determined. Mean change in BW and average daily gain of heifers between fixed-time artificial insemination (day 0) and day 77 did not differ between treatments (P ≥ 0.05). Mean concentrations of insulin-like growth factor 1 (IGF-1) were greater (P < 0.001) in BST (347 ± 27.7 ng/mL) compared with CTL (135 ± 32.8 ng/mL) heifers. Mean placental weight, fetal membrane weight, uterine weight, and ovarian and corpus luteum (CL) weights, as well as fetal morphometric data, did not differ (P ≥ 0.05) between treatments. However, BST heifers had greater (P = 0.03) quantities of combined fetal fluid compared with CTL (521.6 ± 22.9 vs. 429.6 ± 27.14 g, respectively). Tendencies were observed for BST heifers to have reproductive tracts with fewer placentomes (P = 0.08) and fetuses with greater umbilical diameters (P = 0.09) compared with CTL. The activity of CYP1A did not differ (P ≥ 0.05) within the maternal and fetal liver, caruncle, cotyledon, or CL tissue samples between treatments. Furthermore, CYP3A activity was only observed in maternal liver samples and was not different between treatments (P ≥ 0.05). Interestingly, CYP2C activity was greater (P = 0.01) in the liver of BST vs. CTL heifers, and UGT activity was greater (P = 0.02) in the CL from BST heifers compared with CTL. In conclusion, the administration of bST during the first third of gestation increased plasma concentrations of IGF-1, which resulted in an increase in fetal fluid, decrease in placentome number, and greater umbilical diameter, but failed to alter fetal development.

Keywords: conceptus development, insulin-like growth factor 1, placental development, recombinant bovine somatotropin

Introduction

Offspring survival and performance are major determinants of profitability in livestock production systems. Fetal programming plays a large role in both survival and future performance, as nutrient availability to the fetus during gestation has lasting impacts on offspring development (Barker and Osmond, 1986; Barker et al., 1993; Ford and Long, 2012). Fetal growth is supported by the placenta, which secretes hormones including estrogens, progesterone (P4), placental lactogen (PL), and placental growth hormone (GH), and growth factors that alter intrauterine development (Gootwine, 2004). The GH/insulin-like growth factor (IGF) system is primarily responsible for regulating growth in all vertebrates. The binding of GH to its receptors, primarily located in the liver, causes an increase in synthesis and secretion of IGF-1 (Le Roith et al., 2001), which, along with in IGF-2, promote the growth of the offspring both before and after birth (Sferruzzi-Perri et al., 2011).

Uterine blood flow (BF) is linked to fetal growth, development, and survival of offspring (Reynolds and Redmer, 1995; Lang et al., 2003; Reynold et al., 2006). Research has indicated that chronic restrictions in uterine BF cause intrauterine growth restriction due to the reduced availability of oxygen and nutrient supply to the conceptus (Lang et al., 2003). Supplementation of steroid hormones, particularly estradiol-17β, has been observed to increase BF to various uterine tissues (Rosenfeld et al., 1973; Ford and Chenault, 1981). Progesterone is the primary steroid hormone produced during pregnancy and is predominantly metabolized in the liver; however, it has been noted to be metabolized in extrahepatic tissues as well (Iwano et al., 2001; Darwish et al., 2010; Girolami et al., 2016). Eicosanoids regulate vasoconstriction and vasodilation; therefore, changes in metabolism could regulate placental blood perfusion. Local extrahepatic metabolism of steroids and eicosanoids could potentiate placental nutrient transport and uteroplacental BF influencing fetal growth. Recently, uteroplacental secretion of steroids was associated with alterations in placental hormone-metabolizing activity in an ovine model of nutrient restriction (Lemley et al., 2018).

This study was designed to evaluate the administration of bovine somatotropin (bST) and its effects on fetal and placental development as well as steroid- and eicosanoid-metabolizing enzyme activity when administered during the first third of gestation in beef heifers. We hypothesized that bST administration would increase circulating concentrations of IGF-1 and consequently increase fetal and placental development occurring during early gestation in beef heifers. In addition, we hypothesized that bST would decrease the activity of enzymes associated with steroid and eicosanoid metabolism resulting in greater local concentrations of steroids and eicosanoids to maintain pregnancy.

Materials and Methods

All procedures for the study conducted at the North Florida Research and Education Center in Marianna, FL, were reviewed and approved by the University of Florida Institutional Animal Care and Use Committee (number: 201609600).

Experimental design

Angus-based crossbred beef heifers (n = 97) that ranged from 15 to 18 mo of age were enrolled in a completely randomized design at the North Florida Research and Education Center in Marianna, FL. Heifers were maintained according to standard operating procedures of the facility where they were housed in 7.7 ha bahiagrass (Paspalum notatum) pastures, supplemented with a finishing diet, and had ad libitum access to water. The finishing diet was fed ad libitum and consisted of (dry matter basis) 10% fiber pellets, 41.2% corn gluten, 40.8% soy hull pellets, 5% vitamin supplement, and 3% whole soybeans. Additionally, heifers were offered Tifton 85 bermudagrass (Cynodon dactylon) hay ad libitum. All heifers were exposed to the 7-d CO-Synch + controlled internal P4-releasing insert (CIDR; 1.38 g P4; Zoetis Animal Health) estrus synchronization control protocol followed by fixed-time artificial insemination (TAI; day 0), as described by Lamb et al. (2006), to a single sire. Heifers were then randomly assigned to receive one of the two treatments: 1) heifers received a 500-mg injection of bST (Posilac, sometribove zinc, Elanco Animal Health, Greenfield, IN) subcutaneously in the neck at TAI, followed by 500-mg injections of bST every other week until day 57 (BST; n = 47) or 2) heifers received no bST and served as untreated controls (CTL; n = 50). Heifer body weight (BW) was recorded on days −9, −3, 0, 15, 22, 29, 43, 50, 57, 64, and 77. Initial BW at the time of study enrollment was 430 ± 21.3 kg. Pregnancy status was determined via transrectal ultrasonography with a portable ultrasound equipped with a 5.0-MHz linear multi-frequency transducer (Ibex, E.I. Medical Imaging, Loveland, CO) on days 29 and 64 after TAI.

Blood sampling analysis

To determine the concentrations of plasma IGF-1, blood samples were collected on days 0, 22, 50, and 64 relative to TAI via jugular venipuncture using ethylene diamine tetra acetic acid-treated Vacutainers (BD Diagnostics, Franklin Lakes, NJ). Blood samples were placed on ice until centrifugation (1,500 × g at 4 °C for 15 min). Plasma was collected post centrifugation, aliquoted, and then stored at −20 °C until analysis. Concentrations of plasma IGF-1 were determined with an immunoassay system (Immulite 1000 Version 5.22; Siemens Healthcare Diagnostics, Malvern, PA) as previously reported for bovine samples (Leiva et al., 2017; Lippolis et al., 2017).

Harvesting and tissue collection

On day 84, all heifers were transported to a commercial abattoir for harvest. At the time of harvest (day 85), a subset of the heifers (BST, n = 7; CTL, n = 5) were selected and used for the assessment of fetal and placental characteristics. Complete gravid reproductive tracts and dam livers were collected immediately following harvest. Subsequently, the fetus was removed from the placenta at the umbilicus, and fetal crown-to-nose length (CNL), crown-to-rump length (CRL), heart girth (HG), BW, brain weight, and liver weight data were collected. Extraembryonic measurements included: umbilical cord diameter, the combined volume of the chorioallantoic and amniotic fluids, fetal membrane weights after removal of all placentomes, placentome weights, and total placentome number. Fetal fluid weight, fetal membrane weight, and fetal weight were measured utilizing a scale, while umbilical cord diameter was obtained utilizing a digital caliper. One placentome near the umbilicus was selected per tract, trimmed to a 1-cm cube, and immersion fixed in 10% neutral-buffered formalin for histological analysis. Tissues were stored in a 70% ethanol (EtOH) solution prior to being embedded in paraffin. In addition, gravid uterine weight, empty uterine weight, ovarian weight, and corpus luteum (CL) weight were recorded. Approximately 1 g of maternal liver, fetal liver, caruncle, cotyledon, and CL was collected and placed in pre-labeled 1.5 mL cryovials (Wheaton, DWK Life Sciences, Millville, NJ) and snap-frozen in liquid nitrogen. All tissue samples were collected within 2 h after exsanguination. Caruncle and cotyledon tissues were collected adjacent to the umbilicus, and the CL was collected ipsilateral to the fetus. Following tissue collection and measurement, the placentome number was determined. On day 89, carcass quality grade (as determined by USDA Standards), carcass yield, and carcass weight data were obtained from harvested heifers.

Tissue processing

Approximately, 200 mg of wet tissue was placed into a polypropylene tube with 1,000 µL of potassium phosphate (KPO4) buffer (400 mM, pH = 7.4). Samples were then mechanically homogenized using a Tissue Tearor (Biospec Products, Bartlesville, OK) according to the manufacturer’s protocol. Tissue homogenate samples were placed into microcentrifuge tubes and then centrifuged at 10,000 × g for 10 min at 4 °C. The supernatants were stored at −80 °C until enzyme assays were conducted. Protein concentrations of the supernatants were determined by a Coomassie Plus (Bradford) protein assay according to the manufacturer’s protocol (Thermo Scientific, Rockford, IL). Following protein analysis, samples were diluted to a similar concentration of 4,000 µg/mL.

Cytochrome P450 enzymes

Assay kits for Cytochrome P450 1A (CYP1A), 3A (CYP3A), and 2C (CYP2C) and nicotinamide adenine dinucleotide phosphates (NADPH) regeneration solution were purchased from Promega Corporation (Madison, WI), and assays were conducted according to Hart et al. (2014) with minor adaptations. Briefly, reconstitution buffer was added to luciferin detection reagent. Luciferin 6’-chloroethyl ether (luceiferin-CEE; CYP1A), luciferin isopropyl acetal (luciferin-IPA; CYP3A), and 6’-deoxyluciferin (luciferin-H; CYP2C) homogenates (28 µg of protein per well) and enzyme-specific luciferin substrate were added to 96-well plates in duplicate. Plates were then preincubated for 10 min (CYP1A), 30 min (CYP3A), or 90 min (CYP2C) at 37 °C. Following the incubation, NADPH regeneration solution was added to each well, and plates were incubated for 30 min (CYP1A and CYP2C) at 37 °C or 10 min (CYP3A) at room temperature. After the incubation, luciferin detection reagent was added to each well, and plates were protected from light and incubated for an additional 20 min at room temperature. Plates were then placed into a Promega Multi-Plus plate reader, and luminescence was measured.

Uridine 5′-diphospho-glucuronosyltransferase

The uridine 5’-diphospho-glucuronosyltransferase (UGT) assay kit was purchased from Promega Corporation, and the assay was performed according to Hart et al. (2014) with minor adaptations. Briefly, uridine diphospho-glucoronic acid was added to half of the plates to act as reaction wells, and distilled water was added to the other half as control wells. The UGT reaction mixture, containing UGT multienzyme substrate, was then combined with tissue homogenates (28 µg of tissue protein per well), and the plates were preincubated for 90 min at 37 °C. After incubation, detection reagent was added to each of the wells, followed by an incubation period of 20 min at room temperature while protected from light. The plates were then analyzed using a Promega Multi-Plus plate reader with luminescence detection mode.

Luteal tissue P4 assay

Concentrations of P4 in luteal tissue were determined using radioimmunoassay procedures based on the manufacturer’s recommendations (MP Biomedicals, Costa Mesa, CA, 92626). Approximately, 50 mg of luteal tissue was placed into a 15-mL conical tube with 5 mL of phosphate-buffered saline (PBS). Samples were then mechanically homogenized using a Tissue Tearor (Biospec Products, Bartlesville, OK). Tissue homogenate samples were centrifuged at 1,500 × g for 15 min at 4 °C, and then the supernatant was placed into a 5-mL polypropylene tube. The supernatant was vortexed and diluted (1:20) in PBS and was used to analyze luteal concentrations of P4. The inter- and intra-assay coefficients of variation were 4.6% and 5.8%, respectively. Concentrations of P4 are reported as microgram P4 per gram of CL as well as total P4 content of CL, which was calculated by multiplying the P4 concentration (microgram of P4 per gram of CL) by the CL weight at harvest.

Immunofluorescence staining

Placentome samples were trimmed to a width of 0.5 cm and embedded in paraffin to be utilized for placentome sectioning (3 µm thickness). Placentome sections were deparaffinized, rehydrated in descending concentrations of EtOH (100%, 95%, 70%, and 50% EtOH), and rinsed in distilled water. Heat-induced antigen retrieval was performed utilizing 10 mM sodium citrate buffer (pH = 6.0), and the samples were exposed to 121 °C for 20 min under pressure to expose target proteins (2100 Antigen Retriever; Aptum Biologics; Southampton, UK). Tissues were blocked in 10% normal goat serum (NGS; Vector Labs; Burlingame, CA, USA) for 60 min at 25 °C. Placentome sections were incubated with 20 µg/mL biotinylated lectin Dolichos biflorus agglutinin (Vector Labs; Burlingame, CA, USA) at similar conditions. Texas Red Avidin D (20 µg/mL; Vector Labs; Burlingame, CA, USA) was added to tissues and then held at 25 °C for 60 min in a dark room under similar conditions. This process was completed to allow for fluorescent staining of binucleate cells (BNC; Figure 1). Subsequently, 20 µg/mL Fluorescein-labeled Griffonia (Bandeiraea) simplicifolia lectin I (BS1-FITC; Vector Labs; Burlingame, CA, USA) was added to tissues, which were held at a similar condition as before. This process was completed to allow for fluorescent staining of the cotyledon area. All reagents were diluted in a mixture of 1x Tris-buffered saline and 1% NGS and were prepared at least 15 min prior to the addition to the slides.

Figure 1.

Figure 1.

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Image of fluorescent staining of BNC in the cotyledon area of bovine cells at day 85 of gestation post addition of 20 µg/mL Fluorescein-labeled Griffonia (Bandeiraea) simplicifolia lectin I (BS1-FITC; Vector Labs; Burlingame, CA, USA). Image captured with ZEN 2 pro software using a Zeiss Inverted Axio Observer Z1 fluorescence microscope equipped with a Plan-Apochromat 20× objective and an Axiocam 506 monochrome camera (Carl Zeiss; Thornwood, NY, USA).

Imaging and image analysis

Five representative images of each slide were acquired with ZEN 2 pro software using a Zeiss Inverted Axio Observer Z1 fluorescence microscope equipped with a Plan-Apochromat 20× objective and an Axiocam 506 monochrome camera (Carl Zeiss; Thornwood, NY, USA). Image analyses were performed using Image-Pro Premier 9.1 software (Media Cybernetics; Rockville, MD, USA) to obtain BNC number, size, and cotyledon area from each slide. Each image per slide was analyzed and the average was calculated. The average cotyledon and BNC number per heifer were used to calculate the percentage of BNC per cotyledon area.

Statistical analysis

Data were analyzed as a completely randomized design and heifer was considered the experimental unit. The SAS (version 9.4; SAS/STAT, SAS Inst. Inc., Cary, NC) statistical package was used for all statistical analyses. All continuous data were analyzed by the PROC MIXED procedure. Heifer BW and plasma concentrations of IGF-1 were analyzed as repeated measures. The models included the fixed effects of treatment, day, and the treatment × day interaction. The remaining continuous response variables were analyzed with the fixed effect of treatment. Pearson correlation coefficients were determined using the PROC CORR procedure of SAS to analyze the relationships between CL weight, microgram of P4/g of CL, total CL P4, and enzyme activity with respect to each tissue. Two tissues were removed from the BNC analysis as one tissue was biologically inconsistent, and the other tissue collected was not placental tissue. The PROC CORR procedure was used to evaluate the relationship between BNC numbers and conceptus size. Least squares means and standard errors are reported. Statistical significance was declared at P ≤ 0.05, and tendencies discussed when 0.05 < P ≤ 0.10.

Results and Discussion

Somatotropin is a peptide hormone produced by the anterior pituitary that regulates growth through a direct response or mediates a response through the induction of IGF-1, which acts in a variety of tissues, including bone, adipose, and muscle. Bovine somatotropin has been commonly used by the dairy industry to increase milk yield, where a greater yield of milk per lactation period is proposed to be a result of the antagonizing actions of insulin and a shift in nutrient partitioning (Bauman et al., 1999). When low doses of bST were administered at AI in dairy cows, there was improved fertility, reduced embryonic loss, and enhanced conceptus development (Ribeiro et al., 2014). In both beef cows and heifers, the potential use of bST prior to AI, as well as concurrently with AI, for the improvement of pregnancy risk has been studied (Mercadante et al., 2016; Oosthuizen et al., 2018). However, no studies have investigated the effects of bST on fetal and placental development in gestating beef heifers. Thus, this study was conducted to evaluate the effects of bST administration during the first trimester of gestation on fetal and placental development in beef heifers. Mean change in BW (71.9 ± 7.40 kg) and average daily gain (ADG; 0.95 ± 0.11 kg; Table 1) of the heifers from TAI to day 77 of gestation did not differ between treatments (P ≥ 0.05). In the present study, concentrations of IGF-1 were greater (treatment × day; P < 0.001) in BST heifers when compared with CTL (Figure 2), which is in agreement with past reports in both beef (Cooke et al., 2013; Mercadante et al., 2016, Oosthuizen et al., 2018) and dairy females (Bilby et al., 2004). In addition, no differences were noted between treatments in regard to carcass quality grade, yield, or weight (P = 0.88, 0.35, and 0.99, respectively; Table 1). The results of this study differ from previous reports where bST altered the performance of finishing cattle. When Simmental beef heifers received either 320 or 640 mg of bST every other week until the time of slaughter, there was a tendency for an increase in ADG and a reduction in intramuscular fat (Schwarz et al., 1993). In addition, Angus-based heifers that received bST every other week for 196 d had similar ADG but had reduced backfat thickness (Cooke et al., 2013).

Table 1.

Growth performance and carcass parameters for gestating beef heifers treated with recombinant bST or not1

Variable CTL
(n = 5)		 BST
(n = 7)		 SEM2 P-value
Mean change in BW, kg 73.66 71.89 8.380 0.88
ADG, kg/d 0.97 0.95 0.098 0.88
Carcass quality grade3 2 2 0.25 0.88
Carcass yield grade 3 3 0.25 0.35
Carcass weight, kg 147.71 147.73 3.03 0.99
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1Angus-based heifers were randomly assigned to either BST or CTL after being exposed to the 7-d CO-Synch + CIDR estrus synchronization protocol.

2Pooled SE of treatment means. BST, n = 7; CTL, n = 5.

3Heifers were harvested and carcass quality was measured by USDA Standards.

Figure 2.

Figure 2.

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Plasma concentrations of IGF-1 in beef heifers during early gestation. Heifers in the BST treatment group received 500 mg of bST biweekly from TAI to day 57, whereas the CTL heifers received no bST treatment. *P < 0.001.

Mean placentome weight, fetal membrane weight, gravid and empty uterine weight, as well as ovarian and CL weights did not differ (P > 0.10) between treatments (Table 2). In addition, microgram P4/g of CL and total luteal concentrations of P4 (40.7 ± 12.3 µg/g and 192.9 ± 64.4 µg, respectively) did not differ (P ≥ 0.05) between treatments. These results are contrary to those previously reported in bovine granulosa cells (Spicer et al., 1993), which indicated that P4 production by granulosa cells (which transition to luteal cells at luteinization and are responsible for P4 production) increased after exposure to increased IGF-1. However, those results were observed in short-term in vitro cultures that had 0 to 200 ng/mL of IGF-1 present. Thus, this represents an acute dose and not a continuously elevated or chronic concentration of IGF-1 as carried out in the current study.

Table 2.

Effects of experimental treatment on extraembryonic, maternal, and fetal development measurements in gestating beef heifers treated with recombinant bST or not1

Item CTL
(n = 5)		 BST
(n = 7)		 SEM3 P-value2
Fetal membrane weight, kg 0.27 0.24 0.134 0.90
No. of placentomes 73 54 6.8 0.08
Placentome weight, g 73.61 61.30 6.754 0.24
Combined fetal fluid, mL 430 522 25 0.03
Gravid uterine weight, kg 1.31 1.49 0.078 0.16
Empty uterine weight, kg 0.64 0.53 0.057 0.19
Ovarian weight, g 15.86 14.34 1.691 0.54
CL weight, g 4.94 4.62 0.372 0.56
Brain weight, g 2.72 2.34 0.269 0.35
CNL, cm 3.84 4.08 0.243 0.50
CRL, cm 13.18 13.28 0.237 0.76
Fetal BW, g 60.08 59.40 2.464 0.85
HG, cm 9.20 9.22 0.147 0.90
Fetal liver weight, g 3.27 3.01 0.243 0.48
Umbilical cord diameter, mm 6.66 8.90 0.831 0.09
BNC number 98.20 112.57 5.90 0.13
BNC size, μm 441.14 524.46 40.95 0.19
BNC per cotyledon area, % 9.43 11.45 1.14 0.25
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1Angus-based heifers were randomly assigned to either BST (n = 7) or CTL (n = 5) after being exposed to the 7-d CO-Synch + CIDR estrus synchronization protocol.

2Statistical significance was declared at P ≤ 0.05, while a statistical tendency was declared at 0.05 < P ≤ 0.10.

3Pooled SE of treatment means.

The activity of CYP1A was not different between treatments within maternal liver (P = 0.19), fetal liver (P = 0.53), caruncle (P = 0.32), cotyledon (P = 0.63), or CL (P = 0.76; Figure 3) tissue. The activity of CYP2C was greater (P = 0.01) in the maternal liver of BST vs. CTL heifers; however, the activity was not different between treatments in caruncle (P = 0.91) or CL (P = 0.97; Figure 3) tissue. Increased CYP2C may indicate greater P4 metabolism in the dam’s liver, but no differences were observed in CL P4 parameters. Concentrations of serum P4 did not differ in ovariectomized, non-lactating dairy cattle supplemented with P4 in the form of a CIDR in addition to bST supplementation on days 0, 9, and 18 of the estrous cycle (Aboin et al., 2013). In pregnant ewes, concentrations of serum P4 at day 19 were lesser in those supplemented with bST beginning on day 0 of the estrous cycle (Fukui et al., 2000). The activity of UGT tended to be greater (P = 0.09) in CL samples from BST compared with CTL heifers; however, the activity was not different between treatments in maternal (P = 0.49) or fetal liver (P = 0.95) samples and was not detected in the caruncles or cotyledons (Figure 3).

Figure 3.

Figure 3.

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Results for enzymatic activity of steroid- and eicosanoid-metabolizing enzymes in beef heifers on day 84 of gestation. The BST treatment group (n = 7) received 500 mg of bST biweekly from TAI to day 57, whereas the CTL heifers (n = 5) received no bST treatment. Open bars represent CTL and closed bars represent BST. (A) Enzyme activity of CYP1A in maternal and fetal tissues. (B) Enzyme activity of CYP2C in maternal and fetal tissues. (C) Enzyme activity of UGT in maternal and fetal tissues. Means with an asterisk (*) represent a difference (P ≤ 0.05) between treatments. RLU, relative light units for A, B, and C and ND, not detectable in tissue.

Fetal CRL, CNL, BW, HG, and liver weight did not differ between treatments (P ≥ 0.05). These results are in contrast to those in dairy cattle where females that received bST had longer conceptuses on day 19 (Bilby et al., 2004) and larger fetuses on day 40 of gestation (Ribeiro et al., 2014). The activity of CYP2C was not detected in the fetal liver or cotyledons. The activity of CYP3A was only observed in the maternal liver and did not differ between treatments (P = 0.82). Treatments had no differences in BNC number (P = 0.13), BNC size (P = 0.19), or percentage of BNC per cotyledon area (P = 0.25). However, quantities of combined fetal fluid collected from those heifers that received bST were greater (P = 0.03) compared with CTL heifers. Furthermore, there was a tendency for BST heifer reproductive tracts to have fewer placentomes (P = 0.08) and greater umbilical diameters (P = 0.09) than CTL. It should also be noted that Costine et al. (2005) reported no difference in day 25 chorioallantoic weight after GH administration at the time of breeding in sheep but did observe cotyledonary differences at day 80 of gestation. Perhaps an observation difference in placentome number would have been noted by mid-pregnancy in the present study if the collection would have occurred later in gestation with the presence of continued growth in size and number of placentomes. There is the potential that bST treatment during early pregnancy could enhance BNC numbers and perhaps placentome growth to aid in nutrient delivery to the calf as they function in forming the interface needed for implantation and subsequent cotyledonary growth in the placentome (Wooding, 1992). The observed tendency for an increase in umbilical cord diameter in bST-treated heifers indicates that nutrient delivery may have increased if gestation had continued. An increased number of BNC are expected to increase PL secretion and, therefore, fetal weight (Schoknecht et al., 1991; Hossner et al., 1997).

The present study indicates that there is no change in fetal weight during early pregnancy when bST is supplemented to gestating first-calf beef heifers. This contradicts findings when used with an adolescent ewe model and supplementation with bST during the placental growth phase, which reported altered nutrient partitioning that favored uteroplacental and fetal growth when assessed on day 81 of gestation (Wallace et al., 2004). These differences could be due to species differences or the dosage of bST administered. An increase in fetal fluid volume with no difference in fetal weight after GH treatment has also been observed in over-nourished sheep (Wallace et al., 2004). Reduced amniotic fluid volume has been reported in pregnancies with small-for-gestational-age human fetuses (Roberts et al., 2018). It is plausible that a compensatory function of individual placentomes allowed for similar transplacental function in comparison to the lesser number of placentomes observed in the CTL group. Therefore, although concentrations of IGF-1 were increased in heifers that received biweekly administration of bST from TAI to day 57 of gestation, the overall fetal development parameters did not differ between treatments.

In the present study, the administration of 500 mg of bST every other week through early gestation was successful at increasing circulating concentrations of IGF-1 in gestating beef heifers. However, no effects were detected on BW, ADG, carcass quality grade, yield, or weight between BST and the CTL heifers. Likewise, the treatment failed to alter measured fetal parameters or ovarian and uteroplacental weights. There was a tendency for extraembryonic samples from BST tracts to contain greater quantities of fetal fluid and to have greater umbilical diameters, yet to have fewer placentomes. The activity of CYP1A was not different between treatments within the maternal liver, fetal liver, caruncle, cotyledon, or CL samples, while the activity of CYP2C was greater in the maternal liver of BST heifers. Furthermore, the activity of UGT tended to be greater in CL samples from BST. Further research is required to further understand the potential placental function benefits of an increase in GH by IGF-1 activity.

Acknowledgments

Together, we wish to thank the Florida Cattle Enhancement Board as well as the USDA Agriculture and Food Research funded Biophotonic Initiative # 58-6402-3-018 for providing research funding; Zoetis Animal Health (Parsippany, NJ) for donating the following products: PGF2α (Lutalyse), GnRH (Factrel), and CIDR inserts (EAZI-BREED CIDR); and FPL Food, LLC (Augusta, GA), for allowing sample collection in the commercial abattoir. We also wish to thank R. Heiple for coordinating the harvest for sampling; J. Kirsch, A. Siomka, M. Vasquez, A. Tanner, K. McCarty, M. Steichen, C. McGee, C. Hart, and L. Cangiano for their assistance with data collection and analysis; and P. Folsom, M. Foran, D. Jones, D. Thomas, and C. Wood for their assistance with data collection at the University of Florida North Florida Research and Education Center Beef Unit.

Glossary

Abbreviations

ADG

average daily gain

BCS

body condition score

BF

blood flow

BNC

binucleate cells

bST

bovine somatotropin

BW

body weight

CIDR

controlled internal progesterone-releasing insert

CL

corpus luteum

CNL

crown-to-nose length

CRL

crown-to-rump length

GH

growth hormone

HG

heart girth

IGF

insulin-like growth factor

NADPH

nicotinamide adenine dinucleotide phosphates

P4

progesterone

PBS

phosphate-buffered saline

PL

placental lactogen

TAI

fixed-time artificial insemination

UGT

uridine 5′-diphospho-glucuronosyltransferase

Conflict of interest statement

The authors have no conflict of interest to declare.

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