Richard M. Hopper

Evaluation of Systemic Relaxin Blood Profiles in Horses as a Means of Assessing Placental Function in High-Risk Pregnancies and Responsiveness to Therapeutic Strategies

Placental insufficiency is regarded as the primary factor contributing to late‐term abortion and perinatal death of foals. Often when problems associated with late‐term pregnancy in the horse are manifest the condition is well‐advanced and therapeutic intervention may not be effective in rescuing the pregnancy. If a compromised pregnancy due to placental insufficiency could be identified early, the pregnancy might be sustained through medical intervention. Because the placenta is the sole source of circulating relaxin in the mare, we hypothesized that systemic relaxin may serve as a biomarker of placental function and fetal well‐being and a predictor of pregnancy outcome at delivery. To test this hypothesis we monitored plasma relaxin in mares (light breeds) with normal and problematic pregnancies from clinical cases presented to the veterinary hospital and in pregnant mares experimentally inoculated with Streptococcus equi zooepidemicus to induce uterine infection. Upon establishment of placentitis, mares were assigned to different therapeutic strategies and responsiveness was monitored. Blood was collected during the third trimester of pregnancy, and relaxin content was determined using a homologous equine relaxin radioimmunoassay. The results reported here show a positive relationship between low circulating relaxin and poor pregnancy outcome in mares with compromised placental function. While relaxin may have value as a diagnostic assay for identifying mares with high‐risk pregnancies associated with placental dysfunction, the variable results obtained from mares undergoing drug treatment for experimentally induced placentitis make it difficult to determine the reliability of relaxin for evaluating therapeutic efficacy.

Ex vivo bioluminescence imaging of late gestation ewes following intrauterine inoculation with lux-modified Escherichia coli.

Our objectives were to develop an ovine model for Escherichia coli-induced preterm delivery, and monitor E. coli (lux modified for photonic detection) invasion of the fetal environment--ewes (124+/-18d of gestation) received intrauterine inoculations using E. coli-lux as follows: control (n = 5), 1.2 x 10(6) CFU/ml (n = 5), 5.6 x 10(6) CFU/ml (n = 5) E. coli-lux. Preterm delivery occurred between 48 and 120 h post-inoculation in 60%, 60% of ewes infected with 1.2, and 5.6 x 10(6) CFU/ml E. coli-lux, respectively, with presence of emitting bacteria confirmed by real-time imaging of lamb tissues. In summary, preterm delivery and/or fetal distress were observed in a majority of inoculated ewes. Finally, the use of photonic bacteria with imaging was a feasible means to monitor bacterial presence ex vivo.

Horse species symposium: a novel approach to monitoring pathogen progression during uterine and placental infection in the mare using bioluminescence imaging technology and lux-modified bacteria.

Uterine and placental infections are the leading cause of abortion, stillbirth, and preterm delivery in the mare. Whereas uterine and placental infections in women have been studied extensively, a comprehensive examination of the pathogenic processes leading to this unsatisfactory pregnancy outcome in the mare has yet to be completed. Most information in the literature relating to late-term pregnancy loss in mares is based on retrospective studies of clinical cases submitted for necropsy. Here we report the development and application of a novel approach, whereby transgenically modified bacteria transformed with lux genes of Xenorhabdus luminescens or Photorhabdus luminescens origin and biophotonic imaging are utilized to better understand pathogen-induced preterm birth in late-term pregnant mares. This technology uses highly sensitive bioluminescence imaging camera systems to localize and monitor pathogen progression during tissue invasion by measuring the bioluminescent signatures emitted by the lux-modified pathogens. This method has an important advantage in that it allows for the potential tracking of pathogens in vivo in real time and over time, which was hitherto impossible. Although the application of this technology in domestic animals is in its infancy, investigators were successful in identifying the fetal lungs, sinuses, nares, urinary, and gastrointestinal systems as primary tissues for pathogen invasion after experimental infection of pregnant mares with lux-modified Escherichia coli. It is important that pathogens were not detected in other vital organs, such as the liver, brain, and cardiac system. Such precision in localizing sites of pathogen invasion provides potential application for this novel approach in the development of more targeted therapeutic interventions for pathogen-related diseases in the equine and other domestic species.

Maternal nutrient restriction alters uterine artery hemodynamics and placentome vascular density in Bos indicus and Bos taurus

The objective was to examine uterine artery blood flow (UBF) as well as macroscopic and microscopic placentome vascular density in nutrient-restricted Angus and Brahman heifers. Angus (n = 6) and Brahman (n = 6) heifers were bred to a single sire and pregnancy confirmed at 30-d postbreeding. Heifers were randomly assigned to 1 of 2 dietary treatments consisting of 100% (control-fed; CON; n = 6) or 60% (total nutrient-restricted; RES; n = 6) based from net energy requirements for gestating heifers. Nutritional treatments were imposed from days 50 to 180 of gestation. On day 175 of gestation, UBF was collected ipsilateral and contralateral to the conceptus via Doppler ultrasonography. Heifers underwent Cesarean sections for collection of 2 adjacent placentomes on day 180 of gestation. The primary cotyledonary artery of 1 placentome was perfused with Alexa Fluor 647 Con A conjugate to examine macroscopic cotyledonary vascular density via an in vivo imaging system. The second placentome was fixed for microscopic immunofluorescence labeling of capillaries and separated into maternal (caruncle) and fetal (cotyledon) components for determination of angiogenic factor mRNA expression. Main effects of nutritional treatment and breed are reported in the absence of a significant nutritional treatment by breed interaction. Ipsilateral UBF was decreased (P < 0.05) by 48% in RES vs. CON, whereas breed did not influence ipsilateral UBF. Contralateral UBF was not different between nutritional treatments; however, contralateral UBF was decreased (P < 0.05) by 63% in Brahman vs. Angus cattle. Macroscopic cotyledonary vascular density was increased (P < 0.05) by 36% in RES vs. CON and 82% in Brahman vs. Angus heifers. Percent capillary area and capillary perimeter were increased (P < 0.05) in RES vs. CON and increased (P < 0.05) in Brahman vs. Angus heifers. Dietary treatments did not alter angiogenic factor expression; however, transcript abundance of caruncle and cotyledon ANGP1, FLT1, and KDR was increased (P < 0.05) in Brahman vs. Angus heifers. In summary, these data indicate compensatory responses in macroscopic and microscopic placentome blood vessel density during maternal nutrient restriction-induced reductions in UBF. Moreover, a greater macroscopic density of cotyledonary blood vessels was observed in Brahman vs. Angus heifers.

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

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.