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. 2018 Sep 8;96(12):5100–5111. doi: 10.1093/jas/sky363

Effect of chronic melatonin supplementation during mid to late gestation on maternal uterine artery blood flow and subsequent development of male offspring in beef cattle

Keelee J McCarty 1, Megan P T Owen 1,✉, Caitlin G Hart 1, Robyn C Thompson 1, Derris D Burnett 1, E Heath King 2, Richard M Hopper 2, Caleb O Lemley 1,✉
PMCID: PMC6276587  PMID: 30203092

Abstract

The objective of the current study was to examine the effects of supplemental melatonin implants on uterine artery blood flow from mid to late gestation in beef cattle and subsequent development of their male offspring. Commercial beef heifers (n = 32) and cows (n = 25) were bred via artificial insemination and assigned to 1 of 2 groups supplemented with melatonin implants (MEL) or without (CON) at day 180, 210, and 240 of gestation. Uterine artery blood flow was determined using color Doppler ultrasonography. A subset of 12 crossbred heifers (n = 6 MEL; n = 6 CON) underwent Cesarean sections on day 243 ± 2 of gestation to allow for placentome collection. Maternal and fetal serum were collected to analyze melatonin concentrations. The remaining cattle were allowed to calve and at weaning (195 ± 2 d of age), bull calves (n = 15) were castrated and testicular tissue harvested for seminiferous tubule analysis. Heifer uterine artery blood flow was increased (P = 0.009) at day 240 of gestation in MEL compared with CON heifers. Cow uterine artery blood flow was increased (P = 0.003) in MEL compared with CON cows irrespective of gestational day. Maternal and fetal concentrations of melatonin were increased (P < 0.05) in MEL compared with CON heifers. The percent of placentome capillary area per mm2 was decreased (P = 0.019) in MEL compared with CON heifers, while cotyledonary ANGPT1 mRNA tended to increase (P = 0.095) in MEL compared with CON heifers. At weaning, body weight of male offspring and their scrotal circumference were increased (P < 0.05) in calves born to MEL compared with CON dams, while seminiferous tubule diameter and area were not different (P > 0.40) between treatments. In summary, melatonin supplementation increased uterine artery blood flow in mid to late gestating cattle, but this was not accompanied by an increase in fetal weight. Alterations in postnatal development of bulls, including increased body weight and scrotal circumference, warrants future investigations related to attainment of puberty and subsequent fertility of offspring born to melatonin supplemented dams.

Keywords: beef cattle, bull development, fetal programming, melatonin, placenta

INTRODUCTION

Uteroplacental blood flow is imperative for normal fetal development in livestock species. Oxidative stress during pregnancy may disrupt fetal development leading to intrauterine growth restriction and decreased birth weights (Robinson, 2017; Sultana et al., 2017). To combat these disorders, researchers are evaluating the mediators of uterine hemodynamics during pregnancy. Uteroplacental blood flow plays a role in successful growth and development of the fetus (Reynolds and Redmer, 1995; Vonnahme and Lemley, 2012). Efficiency of placental transport is directly related to uteroplacental blood flow and transplacental exchange relies on increased placental growth during early gestation followed by increased uteroplacental vasculature during late gestation (Reynolds and Redmer, 1995). The impact of exogenous therapeutics on uterine hemodynamics and how they interact with endogenous factors have become a more recent area of study (Yunta et al., 2015; Brockus et al., 2016a; Kennedy et al., 2017). Specifically, those that could improve placental efficiency to increase gas exchange and nutrient transport. In an ovine model of intrauterine growth restriction, melatonin supplementation increased umbilical artery blood flow compared to nonsupplemented control ewes (Lemley et al., 2012). Brockus et al. (2016a) demonstrated that dietary melatonin supplementation to Holstein heifers during mid to late gestation increased uterine artery blood flow. Additionally, calves born from melatonin-treated heifers had an increase in calf growth compared to calves born from control heifers (Brockus et al., 2016b). While programming has been researched throughout prenatal and perinatal life, there is limited research on the postnatal development of male offspring in cattle. In respect to the testis, exogenous melatonin upregulates gene expression of spermatogenesis-related genes (Yang et al., 2014). However, a paucity of information exists on prenatal programming of testicular development, which may persist into the postnatal and mature stages of bull development. We hypothesized that chronic melatonin supplementation, via melatonin ear implants, would increase uterine artery blood flow and placental angiogenic factor expression compared with nonsupplemented control beef cattle. In addition, we hypothesized that chronic maternal melatonin supplementation during late gestation would increase male progeny size and increase seminiferous tubule development in the testis during postnatal development.

MATERIALS AND METHODS

Animal Care and Treatments

Animal care and use were according to protocols approved by the Mississippi State University Institutional Animal Care and Use Committee (#16-036). All animals were bred using a timed artificial insemination (TAI) protocol. Heifers and cows were inseminated at the H. H. Leveck Animal Research Center at Mississippi State University. Heifers (n = 87) were bred on December 10, 2015 and cows (n = 65) on December 21, 2015. At approximately 120-d post-AI, both groups underwent transrectal ultrasonography to determine pregnancy and those who conceived to AI were enrolled in the study; 32 heifers and 25 cows. Breeds consisted of 8 Hereford, 28 Angus, and 21 predominantly Angus breeding crossbreds.

A randomized complete block design was utilized to block animals based on breed and body weight. On day 180 of gestation, heifers and cows were assigned to 1 of 2 treatments: melatonin implants (MEL; n = 29) or no melatonin implant control (CON; n = 28) starting on day 180 of gestation and ending on day 270. Melatonin treatment consisted of 2 subdermal ear implants containing 24 mg of melatonin each (Melatonin Implants, Conroe, TX) to slow-release melatonin into peripheral circulation over a 30-d period. Melatonin implants were administered every 30 d, occurring on day 180, 210, and 240 of gestation. The last implant, at day 240 of gestation, was expected to provide an increased concentration of melatonin until day 270 of gestation. Dams did not receive any further treatment after final administration at day 240. Nontreatment CON animals involved sticking the ear with a sterilized needle. Following parturition, dams and calves received no additional treatments. Heifers and cows were housed in similar pastures with ad libitum access to water. Pastures were made up of summer perennials, composed of Bermudagrass, Dallisgrass, and Bahaigrass, in addition to tall fescue. After parturition, cow/calf pairs were moved to a common pasture of similar makeup until weaning. As forage became limited, the paddocks received ad libitum access to hay from the same pasture composition. An outline of animal number and management of heifers, cows, and subsequent offspring are depicted in Fig. 1, which takes into account calves removed from the study as a result of twins or predation.

Figure 1.

Figure 1.

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Animal numbers of dams and heifers throughout the experimental period. Dams (heifers and cows) underwent timed artificial insemination (TAI) and were randomly assigned to melatonin implants or no melatonin (Control). Cesarean sections were performed on 12 heifers, while the remaining heifers and cows were allowed to calve. Male offspring born to heifers and cows were analyzed for morphometric measurements at birth and following castration at 195 ± 2 d of age.

Color Doppler Ultrasonography

Hemodynamic measurements of the uterine arteries ipsilateral and contralateral to the conceptus were determined on day 180, 210, and 240 of gestation following the techniques described by Brockus et al. (2016a). Color Doppler ultrasonography (MicroMaxx; SonoSite, Inc., Bothell, WA) was performed using a transrectal probe (Linear Endorectal L52x probe, Sonosite, Inc.). Examinations were approximately 30 min per animal between 0500 and 1200 h. Cardiac cycle waveforms from 2 independent ultrasound scans were used to calculate systolic velocity (s; cm/s), diastolic velocity (d; cm/s), s:d ratio, pulsatility index (PI), and resistance index (RI) using preset functions on the Doppler ultrasound. Mean velocity (MnV) was calculated using the equation: (s − d)/PI. Blood flow was calculated using the equation: (MnV * vessel area * 60 s). Total uterine artery blood flow was calculated as the summation of both the right and left uterine arteries. Ipsilateral and contralateral uterine artery blood flow is presented as the vessels on the same and opposite side of the fetus, respectively.

Cesarean Sections

A subset of 12 crossbred heifers (n = 6 MEL; n = 6 CON) underwent Cesarean section on day 243 ± 2 of gestation to allow for placentome collection and fetal size measurements. A blood sample was collected from all heifers via venipuncture of the coccygeal vein immediately preceding surgery. Briefly, samples were centrifuged at 10,000 × g for 15 min at 4 °C. The supernatant was aliquoted and stored at −80 °C long term.

Surgeries were performed at the H. H. Leveck Animal Research Center (Mississippi State, MS); therefore, animals were not transported prior to surgery. Cesarean sections were performed with the dam standing following a paravertebral or inverted-L block with 2% lidocaine. After the skin surrounding the incision site was prepared for aseptic surgery, an incision was made 10 to 15 cm ventral to the transverse processes of the lumbar vertebrae midway between the last rib and tuber coxae and extended sufficiently to allow extraction of the fetus. A left oblique celiotomy approach was utilized for standing cows. The abdominal wall incision extended cranioventrally at a 45° angle. This surgical approach permitted easier access to the gravid uterus than more traditional vertical incisions in the paralumbar fossa. One of the fetal limbs was identified and used as a handle to deliver the uterus to the abdominal incision. After the uterus was incised, the umbilical cord was located and clamped off in 2 locations approximately 10 cm from the fetus and 10 cm prior to the major branch points of the cotyledons and cut between the 2 clamps. The fetus was weighed immediately following successful removal and testis weight was recorded in male offspring (MEL, n = 3; CON, n = 3). Fetal blood samples were collected at exsanguination to evaluate peripheral concentrations of melatonin. In addition, a placentome was immediately excised from the uterine wall following successful removal of the fetal calf. A 1 cm by 1 cm placentome section was placed in optimal cutting temperature (OCT) tissue embedding media (Fisher Scientific, Pittsburgh, PA), and frozen by submersion in supercooled isobutene and stored at −80 °C. The remaining placentome was separated into caruncle (maternal) and cotyledon (fetal) portions and snap frozen in liquid nitrogen and stored at −80 °C for later processing of angiogenic factor mRNA expression.

Melatonin, Antioxidant, and Nitrate Analysis

Blood samples were analyzed from fetuses of crossbred heifers (n = 12), as well as the associated dam. Melatonin concentrations were determined with an ELISA kit (TECAN, Morrisville, NC) following manufacturer’s instructions. Samples were compared to a melatonin standard curve (0 to 1000 pg/mL), with a sensitivity of 1.0 pg/mL. The intra-assay CV for the melatonin assay was 11.8%. Total antioxidant capacity (TAC) was determined with a colorimetric assay kit (Cayman Chemical Co., Ann Arbor, MI) following the techniques described by Brockus et al. (2016a). Antioxidant capacity, reported as mM Trolox equivalents, was analyzed against a Trolox standard curve (0 to 0.33 mM), with a sensitivity of 0.01 mM. The intra-assay CV for the total antioxidant assay was 14.6%. Total nitrates of the caruncle (n = 6 per treatment) and cotyledon (n = 6 per treatment) were determined using QuantiChrom Nitric Oxide Assay Kit (BioAssay Systems, Inc., Haward, CA) following the methods of Lemley et al. (2013). Briefly, samples were deproteinized and quantified following reduction of total nitrates to nitrites using the Griess method and analyzed against a linear nitrites standard curve (0 to 100 µM). The intra-assay CV was 5.4%.

Placentome Immunohistochemistry

Placentomes embedded in OCT molds were sectioned into four 10-μm cryosections using a CRYOSTAR NX50 (Thermo Scientific, Waltham, MA), and positioned on positively charged microscope slides. For immunofluorescence imaging of blood vessels, slides were blocked for 30 min with 10% goat serum in PBS with the addition of 0.2% Tween-20. Next, slides were treated with the primary antibody, Anti-Von Willebrand Factor (ab6994; Abcam, Cambridge, MA) followed by secondary antibody, Goat Anti-Rabbit IgG H&L (Alexa Fluor 594; ab150080; Abcam). Slides were incubated with 10 μg/mL Fluorescein-labeled Griffonia Simplicifolia Lectin I (FL-1101; Vector Laboratories, Burlingame, CA) to stain the trophoblast layer, similar to other reports in the ovine placentome (Eifert et al., 2015; Carr et al., 2016). This technique allowed for differentiation of caruncle versus cotyledon capillary densities of the ovine placentome; however, photomicrographs of the bovine placentome showed abundant FITC staining around the fetal villous tree and caruncular epithelial cell layer. Therefore, differential examination of caruncle versus cotyledon capillary densities was not performed in the bovine placentome. Lastly, tissue sections were treated with Fluoroshield mounting medium with DAPI (ab104139; Abcam) for nuclear staining. Images were captured using an EVOS microscope (AMAFD1000; Life Technologies, Carlsbad, CA) with 10× magnification. At least 10 representative photomicrographs were captured per animal. Images captured with the EVOS Texas Red light cube (Alexa Fluor 594) were analyzed using ImageJ (https://imagej.nih.gov/ij/download.html). Total capillary number per tissue area (vessel number per mm2), percent capillary area (%/mm2), average capillary size (μm2), and capillary perimeter per tissue area (mm/mm2 or mm−1) were recorded.

Placentome RT–PCR

Expression of genes of interest in the placenta were performed on caruncle (n = 12) and cotyledon (n = 12) tissues collected on day 243 ± 2 of gestation. Approximately 1 g of placental tissue was homogenized with 1 mL of PBS. Nucleic acids were isolated by the placental homogenate using 700 µL of QIAzol Lysis Reagent (QIAGEN, Hilden, Germany) followed by purification using a miRNeasy Mini Kit (QIAGEN, Hilden, Germany). Extracted total RNA was quantified using a NanoDrop One spectrophotometer (Thermo Scientific, Waltham, MA). Samples exhibiting 260 nm/280 nm ratios between 1.9 and 2.1 were deemed acceptable for downstream procedures and stored at −80 °C. For each sample, triplicate cDNA synthesis reactions were conducted using 100 ng of total RNA using High-Capacity cDNA Reverse Transcription Kit (Thermo Fischer Scientific, Auburn, AL). Real-time PCR was performed using Custom TaqMan Gene Expression Assays (Thermo Fischer Scientific, Auburn, AL) according to the manufacturer protocol. The genes of interest for placental membranes were angiopoietin 1 (ANGPT1), vascular endothelial growth factor A (VEGFA), kinase insert domain containing receptor (KDR), and fms-like tyrosine kinase 1 (FLT1). Beta-actin (ACTB) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) served as housekeeping genes. Duplexing of genes were executed per manufacturer’s instruction; briefly, 1 µL of labeled TaqMan gene was substituted for 1 µL of nuclease-free water per reaction mixture. Genes labeled with the VIC dye were paired for multiplexing with a gene labeled with the FAM dye. Approximately 1 µL of FAM-labeled TaqMan Gene and 1 µL of VIC-labeled TaqMan gene were present in each reaction. Assays for the genes of interest (Table 1) were validated for efficiency and specificity prior to qPCR. Primer efficiencies were determined by plotting the threshold cycle versus the log of the input concentrations for 6, serial dilutions of pooled caruncle or cotyledon tissue cDNA in duplicate. Efficiency was calculated by raising 10 to the power of −1 divided by the slope of the line (E = 10[−1/slope]). Primer sets and assays with efficiencies between 1.8 and 2.2 (90% to 110%) were considered acceptable for real-time PCR analysis. Complementary DNA was amplified in triplicate using TaqMan fast advanced master mix (Thermo Scientific, Waltham, MA). A master mix containing TaqMan fast advanced master mix and gene assay was pipetted along with 100 ng of cDNA into a 96-well MicroAmp Fast Optical Reaction Plate (Applied Biosystems, Foster City, CA). The plate was then covered with Masterclear real-time PCR Film (Eppendorf, Hamburg, Germany), briefly vortexed and centrifuged, then placed in QuantiStudio 3 (Applied Biosystems, Foster City, CA) for real-time qPCR analysis. Thermal cycling parameters used consisted of: a hold stage at 50 °C for 2 min; polymerase activation hold at 95 °C for 20 s; PCR stage with step 1 at 95 °C for 1 s followed by step 2 at 60 °C for 20 s for 40 cycles. Replicate CT values were averaged and used for relative quantification using the 2−ΔΔCT method.

Table 1.

Assays, accession, amplicon length, and efficiency (10[−1/slope]) of primers used for TaqMan real-time PCR quantification of placental gene expression

Gene Assay ID Accession Amplicon Efficiency
Caruncle Cotyledon
ACTB Bt03279174 NP_776404.2 141 2.05 2.04
GAPDH Bt03210913 NP_001029106.1 66 2.04 2.09
ANGPT1 Bt03249559 NP_001070265.1 147 2.10 2.05
VEGFA Bt03213282 NP_776641.1 59 2.20 2.14
KDR Bt03258885 NP_001103470.1 68 2.09 2.06
FLT1 Bt04302190 NP_001178061.2 101 2.04 2.08
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Postnatal Measurements of Male Offspring

At birth, singleton male calves from MEL dams (n = 9) and CON dams (n = 14) were given tag and tattoo identification, naval iodine dip, and evaluated for overall health. Morphometric measurements recorded included body weight, heart girth, abdominal girth, hip height, and curved-crown rump length (CCR). Ponderal index was calculated from CCR and body weight measurements as (body weight [kg]/CCR [m]3). A subset of male offspring (MEL, n = 6; CON, n = 9) were castrated at 195 ± 2.3 d of age. Calves were restrained in a squeeze chute and scrotal circumference was measured prior to castration. An epidural administration of lidocaine (0.17 mg/kg) was performed. The distal one-third of the scrotum was transected, and each testicle removed by slow, continuous traction. Exposed scrotal area was sprayed with commercially available wound spray. Calves received an oral dose of 1 mg/kg of Meloxicam before leaving the chute. Testicular morphometric measurements such as weight, average length, and total width were taken immediately following castration. A cross section of testicular parenchyma was placed in a microvette histology cassette (Thermo Fisher Scientific, Auburn, AL). Cassettes were placed in 10% formalin for short-term storage. Tissue sections were prepared for histological processing by the Mississippi State University College of Veterinary Medicine Department of Pathobiology and Population Medicine. Briefly, paraffin sections were sliced into 5 µm sections then stained with hematoxylin eosin to determine seminiferous tubule diameter and area. Approximately 100 circular seminiferous tubules were measured and averaged per animal.

Statistical Analysis

Uterine blood flow was analyzed using repeated measures of ANOVA (SAS software version 9.4, SAS Institute, Cary, NC). The model statement included: day of gestation, treatment, day by treatment interaction, breed, and calf sex. Autoregressive was the type of covariance structure selected based on the fit of statistical parameters in the model. Uterine artery blood flow of cows and heifers were analyzed separately. Placental and fetal measurements at Cesarean sections were analyzed using ANOVA (SAS software version 9.4, SAS Institute, Cary, NC), where the model statement included: treatment, fetal sex, and their respective interactions. Male calf morphometric measurements were analyzed using ANOVA (SAS software version 9.4, SAS Institute, Cary, NC). The model statement included: treatment, parity, and breed. Gestation length of each calf served as a covariate. Testicular morphometric and seminiferous tubule measurements were analyzed using ANOVA (SAS software version 9.4, SAS Institute, Cary, NC). The model statement included: treatment and breed. Least square means and standard error of the means are reported. Statistical significance was declared at P ≤ 0.05 while a tendency was declared at 0.05 < P ≤ 0.10.

RESULTS

Maternal Body Weight

Heifer and cow body weight increased (P < 0.0001) as gestational day increased (data not shown). Heifer body weight was not different (P = 0.951) between treatments and averaged 512.0 ± 3.9 kg. Cow body weight tended to be increased (P = 0.086) in MEL cows (580.0 ± 16.3 kg) compared with CON cows (572.0 ± 16.5 kg).

Uterine Artery Hemodynamics

Heifer ipsilateral uterine artery blood flow tended to have a treatment × day interaction (P = 0.06) where MEL had increased (P = 0.023) blood flow at day 240 compared with CON (Fig. 2A). Diameter of the ipsilateral uterine artery tended to have a treatment × day interaction (P = 0.06) where MEL (0.99 ± 0.05 cm) heifers had larger (P = 0.003) diameter compared with CON (0.84 ± 0.04 cm) heifers at day 240. No effect of treatment was observed for MnV (P = 0.329), s/d ratio (P = 0.516), RI (P = 0.587), or PI (P = 0.621) of the ipsilateral uterine artery of heifers (data not shown). Cow ipsilateral uterine artery blood flow was increased (P = 0.01) in MEL compared with CON (Fig. 2B). A main effect of gestational day (P < 0.0001) was observed for ipsilateral uterine artery blood flow which increased (P < 0.0001) as gestational day increased in cows (Fig. 2B). Ipsilateral MnV was increased (P = 0.02) in MEL (215 ± 13 cm/s) versus CON (170 ± 13 cm/s) cows. Diameter of the ipsilateral uterine artery tended to exhibit a treatment × day interaction (P = 0.07) where MEL (1.01 ± 0.05 cm) cows tended to have larger (P = 0.078) diameter compared with CON (0.88 ± 0.05 cm) cows at day 210, however, did not differ (P = 0.991) at day 240. No effect of treatment was observed for s/d ratio (P = 0.678), RI (P = 0.557), or PI (P = 0.669) of the ipsilateral uterine artery of cows (data not shown).

Figure 2.

Figure 2.

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Ipsilateral uterine artery blood flow (BF) in heifers (A) and cows (B), contralateral uterine artery BF in heifers (C) and cows (D), and total uterine artery BF in heifers (E) and cows (F) treated with (MEL) or without melatonin (CON) from day 180 to 270 of gestation. An asterisk (*) signifies a significant difference at P ≤ 0.05 while a dagger (†) signifies a tendency at 0.05 < P ≤ 0.10.

Heifer contralateral uterine artery blood flow exhibited a treatment × day interaction (P = 0.032) where MEL was increased (P = 0.012) at day 240 compared with CON (Fig. 2C). Diameter of the contralateral uterine artery tended (P = 0.09) to be increased in MEL (0.50 ± 0.03 cm) versus CON (0.45 ± 0.03 cm) heifers. No effect of treatment was observed for MnV (P = 0.288), s/d ratio (P = 0.222), RI (P = 0.218), or PI (P = 0.229) of the contralateral uterine artery of heifers (data not shown). Cow contralateral uterine artery blood flow (Fig. 2D) was not different (P > 0.10) between treatments or gestational day. Moreover, no effect of treatment was observed for MnV (P = 0.752), diameter (P = 0.873), s/d ratio (P = 0.966), RI (P = 0.624), or PI (P = 0.663) of the contralateral uterine artery of cows (data not shown).

Total uterine artery blood flow exhibited a treatment × day interaction (P = 0.025) where MEL heifers had increased (P = 0.009) blood flow at day 240 compared with CON heifers (Fig. 2E). Heart rate tended to have a treatment × day interaction (P = 0.096) where heart rate did not differ (P = 0.603) at day 210; however, MEL (87 ± 3 bpm) heifers had increased (P = 0.028) heart rate compared with CON (80 ± 3 bpm) heifers at day 240. Total uterine artery blood flow was increased (P = 0.003) in MEL cows compared with CON cows (Fig. 2F). Total uterine artery blood flow increased (P < 0.0001) as gestational day increased in cows (Fig. 2F). There was no effect of treatment (P = 0.161) on cow heart rate; however, heart rate tended to increase (P = 0.059) from day 210 (78 ± 1 bmp) compared with day 240 (81 ± 1 bmp) of gestation in cows.

Cesarean Section Analysis

Fetal body weight was not different (P = 0.561) between treatments and averaged 23.5 ± 0.8 kg. Moreover, sex of the fetus did not influence (P = 0.845) fetal body weight at time of Cesarean section. Fetal testis weight was not different (P = 0.396) between MEL (6.1 ± 1.1 g) and CON (4.7 ± 0.9 g). Maternal concentrations of melatonin were increased (P = 0.021) in MEL (27.3 ± 4.2 pg/mL) compared with CON (15.8 ± 4.2 pg/mL) heifers. Fetal concentrations of melatonin were increased (P = 0.028) in MEL (49.8 ± 9.6 pg/mL) compared with CON (25.2 ± 9.6 pg/mL) heifers. Total antioxidant capacity was not different (P > 0.20) in fetal or maternal serum between treatments (data not shown).

Placentome Analysis

Representative photomicrographs of capillary staining are illustrated in Fig. 3. Total number of placentome capillaries per tissue area was not different between treatments (Table 2). Percent area of capillaries was decreased (P = 0.019) in MEL compared with CON heifers. Average capillary size and total capillary perimeter were not different (P > 0.30) between treatments. Concentrations of nitrates in cotyledon and caruncle tissues were not different (P > 0.50) in MEL compared with CON heifers (data not shown). Caruncular ANGPT1, VEGFA, KDR, and FLT1 mRNA were not different (P > 0.10) between treatments (Table 2). Cotyledonary ANGPT1 mRNA tended to increase (P = 0.100) in MEL heifers compared with CON (Table 2). Cotyledonary VEGFA, KDR, and FLT1 mRNA were not different (P > 0.30) between treatments (Table 2). Weight of sampled placentomes were not different (P = 0.73) between treatments and averaged 66.6 ± 11.8 g (data not shown).

Figure 3.

Figure 3.

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Representative immunofluorescence images of heifer placentomes at 240 d of gestation. Cryosectioned placentomes were stained for capillaries with Anti-Von Willebrand Factor (red, Alexa Fluor 594), caruncular and chorionic epithelium (green, FITC), and nuclei (blue, DAPI). Panel (A) represents a negative control treated without Anti-Von Willebrand Factor. Panel (B) represents a fully stained image with all 3 fluorescent channels overlaid and Panel (C) represents the Texas Red channel only from Panel (B). The white scale bar represents 400 μm.

Table 2.

Placentome blood vessel analysis and relative transcript abundance of angiogenic factors of cotyledon and caruncles from C-section heifers treated with melatonin (MEL; n = 6) or without melatonin (CON; n = 6) from day 180 to 243 ± 2 of gestation

Item CON MEL SE P-value
Trt
Placentome
 Capillary number, number per mm2 998 1009 96 0.934
 Percent capillary area, %/mm2 8.8 7.5 0.3 0.019
 Average capillary size, μm2 113 98 10 0.302
 Capillary perimeter, mm−1 43.2 39.2 2.6 0.301
Caruncle
 ANGPT1 1.08 1.26 0.39 0.649
 VEGFA 4.58 0.90 2.25 0.141
 KDR 4.15 1.27 2.58 0.297
 FLT1 0.88 1.12 0.30 0.459
Cotyledon
 ANGPT1 0.87 1.41 0.29 0.100
 VEGFA 1.10 1.14 0.17 0.810
 KDR 1.07 1.16 0.10 0.451
 FLT1 1.09 0.90 0.19 0.346
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Postnatal Measurements of Male Offspring

Morphometric measurements of male offspring at birth are depicted in Table 3. Birth weight was not different (P = 0.772) in calves from MEL compared CON dams. Heart girth, abdominal girth, and hip height were not different (P > 0.20) between treatments. Curved-crown rump length was increased (P = 0.031) in calves from MEL compared with CON dams, while PI was decreased (P = 0.029) in calves from MEL compared with CON dams.

Table 3.

Morphometric measurements of male calves at birth from heifers and cows treated with melatonin (MEL; n = 9) or without (CON; n = 14)

Item CON MEL SE P-value
Trt
Body weight, kg 31.1 31.8 1.8 0.772
Heart girth, cm 72.7 73.2 1.7 0.815
Abdominal girth, cm 79.2 81.2 1.9 0.405
Curved-crown rump length, cm 79.8 86.3 2.3 0.031
Hip height, cm 72.7 75.1 1.8 0.286
Ponderal index, BW (kg)/CCR (m)3 59,207 51,680 2,582 0.029
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A representative image of seminiferous tubules from bull calves are illustrated in Fig. 4. Testicular morphometric and seminiferous tubule measurements at 195 ± 2 d of age are depicted in Table 4. At castration, body weight was increased (P = 0.006) in bull calves from MEL compared with CON dams. Scrotal circumference was increased (P = 0.032) in calves from MEL compared with CON dams. Total testis weight, average length, and total width were not different (P > 0.10) between treatments. Seminiferous tubule diameter and area were not different (P > 0.40) between treatments.

Figure 4.

Figure 4.

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A representative image of seminiferous tubules in bull calves at 195 ± 2 d of age. The white scale bar is (A) 200 µm (B) 60 µm.

Table 4.

Testicular morphometric measurements of castrated calves at 195 ± 2 d of age from heifers and cows treated with melatonin (MEL; n = 6) or without (CON; n = 9)

Item CON MEL SE P-value
Trt
Body weight, kg 195.5 221.5 7.3 0.006
Scrotal circumference, cm 18.7 19.6 0.4 0.032
Testis
 Total weight, g 129.6 148.0 11.0 0.128
 Average length, mm 68.4 71.0 3.2 0.424
 Total width, mm 66.3 70.5 2.5 0.113
Seminiferous tubule
 Diameter, µm 157.4 148.2 11.1 0.428
 Area, µm2 20,228 19,098 2,285 0.633
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DISCUSSION

An increase in uterine blood flow is vital in late gestation to maintain oxygen supply and nutrient delivery to the growing fetus (Ford, 1995; Redmer et al., 2004; Reynolds et al., 2006). Moreover, specific increases in placental blood flow could combat the negative consequences of pregnancy disorders such as preeclampsia or intrauterine growth restriction. In the present study, total uterine artery blood flow was increased in MEL implanted compared with CON heifers and cows. Similar results were observed by Brockus et al. (2016a), after dietary melatonin supplementation to Holstein heifers during mid to late gestation where total uterine artery blood flow was increased in melatonin-treated heifers compared with nontreated control. In contrast, in an ovine model of intrauterine growth restriction, melatonin supplementation did not change uterine artery blood flow but instead increased umbilical artery blood flow compared to nonsupplemented control ewes (Lemley et al., 2012). Collectively, therapeutic supplementation of melatonin during gestation tends to increase uteroplacental hemodynamics in sheep and cattle. Alterations of uteroplacental blood flow during melatonin supplementation may be mediated by 1) improved endothelial function via decreased vascular oxidative stress or 2) activation of cardiovascular melatonin receptors 1 or 2, which have been shown to alter vascular reactivity depending on vessel origin (Shibata et al., 1989; Weekley, 1993). Melatonin increases blood flow by directly modulating local vascular tone and numerous studies have demonstrated both vasoconstrictor and vasorelaxant properties (Hashimoto et al., 1989; Weekley, 1993; Reiter et al., 1995; Viswanathan et al., 1995; Geary et al., 1998). Additionally, increased umbilical blood flow by melatonin treatment may be due to increased sensitivity of placental vessels to bradykinin-induced vasorelaxation (Shukla et al., 2014). A portion of these melatonin-induced uteroplacental blood flow responses in sheep have been reversed by chronically infusing the melatonin receptor antagonist, luzindole, during mid gestation (Lemley et al., 2013). Moreover, the bovine placenta expresses melatonin receptor 2, which is increased in cotyledonary versus caruncle tissue throughout pregnancy (Lemley and Vonnahme, 2017). Therefore, the increase in bovine uterine artery blood flow could be independent of melatonin antioxidant pathways and directly related to placental vascular melatonin receptor pathways.

In sheep, numerous models of compromised pregnancies have decreased vascularity due to underfeeding or overfeeding adolescents, underfeeding adults, or multiple pregnancies (Reynolds et al., 2010). Moreover, placental capillary development has been associated with placental angiogenic factor expression (Reynolds et al., 2005). In the current study, placentome capillary number and capillary perimeter were not different between treatments. However, the percent capillary area was decreased in MEL compared with CON heifers, while cotyledonary ANGPT1 mRNA tended to be increased in MEL compared with CON-treated heifers. These opposing responses may be related to chronic increases in uterine artery blood flow in MEL heifers, leading to offsetting alterations in the percent capillary area of the placentome. Vonnahme et al. (2007) observed an increase in transcript abundance of VEGF and FLT1 in both caruncle and cotyledon tissues when beef cows were nutrient restricted from day 30 to 125 of gestation; however, no difference in vascularity of the placentome was observed. Similar studies by McLean et al. (2017) observed decreased VEGF expression in the intercaruncle of restricted-fed Angus heifers compared with control-fed heifers during early gestation. However, nutrient restriction in ewes caused an increase in cotyledon and caruncle morphological change to occur earlier in gestation, thus, altering placental formation (Vonnahme et al., 2006), as well as increased angiogenic factors in caruncle tissue (Vonnahme et al., 2007).

Melatonin concentrations in some livestock species participate as a biological clock to mediate onset of puberty, seasonal breeding, and reproductive cycles (Yellon and Longo, 1987). In the current study, following implant supplementation, melatonin concentration increased 98% in fetal serum and 73% in maternal serum of MEL heifers compared with CON heifers. In previous dietary models of melatonin supplementation, a 6-fold increase in serum concentrations of melatonin in sheep supplemented with 5 mg of melatonin per day (Lemley et al., 2012) versus a 4-fold increase in serum concentrations of melatonin in dairy heifers supplemented with 20 mg of melatonin per day (Brockus et al., 2016a) were observed. Surgically implanting melatonin-loaded Alzet mini osmotic infusion pumps into the pregnant uterus of sheep increased both maternal and fetal concentrations of melatonin by 25% and 125%, respectively (Lemley et al., 2013). Although not measured in the current study, the dosage of melatonin needed to alter behavior (i.e., sedation, hypnotic activity, and/or anti-anxiety effects) is much greater compared with doses required for altering endocrine profiles (Golombek et al., 1996).

In the current study, birth weights of male calves were similar irrespective of maternal treatment, while other measurements of male calf size at birth were altered by maternal melatonin supplementation. For example, CCR was increased and PI decreased in bull calves from MEL dams compared to calves from CON dams at birth. Overall, bull calves exposed to chronic supplementation of melatonin in utero were longer and leaner compared to calves from control-treated dams, with no difference in body weight between treatments. However, at weaning and castration, bull calves born to MEL dams were heavier compared to bull calves born to CON dams. This supports previous findings of Brockus et al. (2016b) where dairy calves born from MEL-treated heifers had similar birth weights and an increase in calf growth compared to calves born from CON heifers. In contrast to previous work with dairy heifers that were immediately removed from the dam and fed milk replacer (Brockus et al., 2016b), beef calves from the current study were allowed to remain with the dam until weaning. Milk yield of the dam could contribute to the increase in body weight of the calves, which should be investigated in future studies.

Melatonin receptors have been found in the testis of various species such as the mouse and rat (Liu et al., 2009; Zhang et al., 2012). Melatonin has been proposed as a recovering agent for testicular damage, but limited studies have evaluated the direct action of melatonin on testicular histology in cattle. For example, in sheep, melatonin reduces oxidative injury in seminiferous tubules that occurs during transplantation (Gholami et al., 2015). Prior experiments in mice examined similar histological properties and determined melatonin reduced harmful effects on seminiferous tubule structure due to the freezing and re-thawing process (Gholami et al., 2015). Sertoli cells play a significant role in spermatogenesis (Gholami et al., 2015) within the seminiferous tubules. Melatonin receptors MT1 and MT2 have been found in bovine Sertoli cells and exogenous melatonin upregulates gene expression of spermatogenesis-related genes (Yang et al., 2014). In the present study, maternal melatonin supplementation from mid to late gestation did not alter body weight of the fetus or fetal testis weight. However, at the time of castration, postnatal body weight and scrotal circumference were increased in calves born to MEL dams compared with CON. Interestingly, the bulls from MEL dams may be reaching sexual maturity sooner and future studies will be designed to further establish this relationship. During postnatal development Holstein bulls experience a 4-fold increase in tubule diameter from birth to 18 mo of age (Fossland, 1954). The seminiferous tubules of beef bulls finish developing postnatally by 30 wk of age (Evans et al., 1996). In the current study, at approximately 27 wk of age, seminiferous tubule diameter and area were unaltered by maternal melatonin supplementation.

In conclusion, melatonin supplementation increased uterine artery blood flow in mid to late gestating cattle but this was not accompanied by an increase in fetal weight. Although angiogenic factor expression in caruncle and cotyledon tissues did not significantly differ between treatments, the percent area of placentome capillaries was decreased during melatonin supplementation. In contrast to our previous studies, these pathways were not associated with changes in serum antioxidant capacity during melatonin supplementation. Therefore, a more direct effect of melatonin on vascular melatonin receptors may contribute to these in vivo responses. Finally, in utero melatonin supplementation did not alter testicular development, specifically, the seminiferous tubules did not differ. Compared to previous studies in our laboratory involving the impacts of melatonin supplementation, additional studies are warranted to determine if milk yield of the dams contributed to the change in body weight and composition observed in subsequent offspring growth. Moreover, a larger number of offspring will need to be studied during development to fully understand these programming pathways.

ACKNOWLEDGMENTS

This publication is a contribution of the Mississippi Agricultural and Forestry Experiment Station. This material is based upon work that is supported by the National Institute of Food and Agriculture, U.S. Department of Agriculture, Hatch project under accession number 1011100. Additional funding was provided by the U.S. Department of Agriculture, Agricultural Research Service, Biophotonic Initiative number 58-6402-3-018.

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