S. T. Willard

Effect of neonatal rat bisphenol a exposure on performance in the Morris water maze.

Bisphenol A (BPA), an environmental estrogen, is a component of many food and beverage containers and can leach into the container contents over time. Due to its estrogenic properties, exposure to BPA during development could alter the appropriate maturation of pathways essential for normal cognitive function at later ages. To investigate this, the effects of repeated postnatal exposure of male and female rats to BPA on spatial learning and memory were investigated using a Morris water maze. Breeders and offspring were maintained on a standard phytoestrogen-free diet. Oral administration of 72 microg/kg 17 beta-estradiol (E(2)), 100 microg/kg BPA (low BPA), 250 microg/kg BPA (high BPA), or the safflower oil vehicle was performed daily from postnatal d 1 (PND1) through PND14. There were no treatment-related effects on swimming ability or motivation (PND33) or on acquisition of maze solution (PND34-37). However, acquisition of maze performance was significantly better in control males than in control females. Treatment with E(2) and low BPA disrupted this normal gender-dependent pattern of acquisition, while treatment with high BPA did not. In a probe trial (PND40), females treated with high BPA spent significantly less time in the escape quadrant. These data indicate that E(2) and low dosages of BPA can alter the normal gender-dependent pattern of acquisition, while higher dosages of BPA alter the retention of spatial information without significantly affecting acquisition.

The effects of GnRH administration postinsemination on serum concentrations of progesterone and pregnancy rates in dairy cattle exposed to mild summer heat stress

The objective of this study was to evaluate whether administration of GnRH postinsemination would improve reproductive performance in heat-stressed dairy cattle. Estrous cycles of Holstein cows were synchronized using the OvSynch protocol and cows were artificially inseminated. Cows were then administered the following treatments: control (no GnRH; n=37), GnRH (100 microg) on Day 5 (GnRH-D5; n=34), or GnRH (100 microg) on Day 11 (GnRH-D11; n=34) postinsemination. Cows were provided access to both fans and sprinklers, and environmental data was collected hourly. Rectal temperatures and blood samples were obtained from cows on Days -9, -2, 0 (AI) and on alternate days from Day 5 to Day 19 postinsemination. Blood serum was collected for the analysis of progesterone (P(4)) by RIA. In a subset of cows (n=6/treatment) ultrasonography was performed on alternate days from Day 5 to Day 19 postinsemination to assess numbers of corpora lutea (CL) and CL cross-sectional areas. Pregnancy status of cows was confirmed at Day 30 postinsemination. Environmental data indicated that cows experienced mild heat stress during the trials (mean daily THI=73-77). Serum P(4) was greater (P<0.05) after Day 9 for GnRH-D5 cows and after Day 15 for GnRH-D11 cows through Day 19 postinsemination. The number of CLs present for GnRH-D5 cows was greater (P<0.05) on Day 17 than in either the control or GnRH-D11 treatment groups. On Day 17 postinsemination, both the GnRH-D5 and GnRH-D11 cows were observed to have greater (P<0.05) total CL tissue area than control cows. The interval from insemination to when serum P(4) returned to <1 ng/ml (i.e. luteolysis and return to estrus) did not differ (P>0.10) among treatment groups. Control cows (19%) tended to exhibit lower pregnancy rates (P<0.08) compared to the GnRH-D5 and GnRH-D11 treatment groups combined (35%). In summary, the treatment of heat-stressed dairy cows with GnRH postinsemination (Day 5 or 11) results in the appearance of more CL tissue, increased serum concentrations of P(4) and a tendency toward greater pregnancy rates.

Evaluation of the early conception factor (ECF™) test for the detection of nonpregnancy in dairy cattle

The ability to detect conception and/or conception failure in cattle would be beneficial to producers in formulating reproductive management plans. A new diagnostic test, the early conception factor (ECF) test, has been developed forthis application yetthe accuracy of this test has not been adequately determined. The objectives of this study were to evaluate the effectiveness of the ECF test for detecting the nonpregnant cow, and to compare the reliability of serum versus milk ECF tests relative to actual pregnancy rates. In Trial 1, Holstein heifers were synchronized, the animals were bred (timed-AI), and serum ECF tests were performed 72 h later. Heifers exhibiting a negative ECF test after AI were re-synchronized, bred again, and re-tested for ECF for up to three services. Relative to actual pregnancy rates, a negative ECF test was correct (i.e., true negative) 38.5% of the time over the three services. In Trial II, Holstein heifers were bred (AI) after observed estrus and serum ECF tests conducted between Days 1 and 3 and Days 7 and 9 after AI. In this trial, only 44.4% and 55.6% of the confirmed nonpregnant heifers were identified correctly by serum ECF analysis at Days 1 to 3 and Days 7 to 9 post-AI respectively. In Trial III, 40 lactating cows were synchronized, the animals were bred (AI), and serum and milk ECF tests were performed on Days 3, 9, 15, 21 and 30 after AI. Pregnancy diagnosis (ultrasound on Day 30 and palpation on Day 51) confirmed that 50% of the cows were pregnant to AI, while serum and milk ECF analysis indicated a 100% and 37.5% predicted pregnancy rate, respectively, at 30 d post-AI. Moreover, results of the serum and milk ECF tests disagreed with one another 36.9% of the time overall, while agreement between ECF and actual pregnancy rates were 50.6% and 45.6% for milk and serum respectively. Additionally in Trial III, a negative ECF result only identified 5% and 28.8% of nonpregnant cows overall for serum and milk tests respectively (i.e., true negatives), with a high incidence of false positive ECF results noted (47.5% and 31.3% for serum and milk, respectively). Collectively, these data indicate that the current ECF test cannot accurately identify the nonpregnant cow with the precision needed by the dairy producer.

Temperament influences endotoxin-induced changes in rectal temperature, sickness behavior, and plasma epinephrine concentrations in bulls.

This study was designed to determine the influence of temperament on endotoxin-induced changes in body temperature, sickness behavior, and stress hormone concentrations in cattle. Brahman bulls were selected based on temperament score measured 28 d prior to weaning. In dwelling recording devices were used to monitor rectal temperature, and jugular catheters were used to collect blood samples to determine cortisol and epinephrine concentrations before and after LPS administration (0.5 μg/kg body weight). Temperamental bulls had the lowest peak rectal temperature and sickness behavior scores relative to the Calm and Intermediate bulls. Prior to the administration of LPS, Temperamental bulls had greater cortisol and epinephrine concentrations than Calm or Intermediate bulls. Cortisol concentrations increased following LPS administration but were not affected by temperament. Epinephrine concentrations peaked 1 h after LPS administration in Calm bulls. Temperamental bulls did not exhibit an epinephrine response to LPS challenge. These data demonstrate that the temperament of calves can modulate the physiological, behavioral, and endocrine responses of pre-pubertal Brahman bulls to endotoxin challenge. Specifically, temperament differentially affected the rectal temperature, sickness behavior and epinephrine, but not cortisol, responses to LPS challenge.

Application of quantum dot nanoparticles for potential non-invasive bio-imaging of mammalian spermatozoa.

BackgroundVarious obstacles are encountered by mammalian spermatozoa during their journey through the female genital tract, and only few or none will reach the site of fertilization. Currently, there are limited technical approaches for non-invasive investigation of spermatozoa migration after insemination. As the knowledge surrounding sperm behavior throughout the female genital tract still remains elusive, the recent development of self-illuminating quantum dot nanoparticles may present a potential means for real-time in vitro and in vivo monitoring of spermatozoa.ResultsHere, we show the ability of boar spermatozoa to harmlessly interact and incorporate bioluminescent resonance energy transfer-conjugated quantum dot (BRET-QD) nanoparticles. The confocal microscope revealed in situ fluorescence of BRET-QD in the entire spermatozoon, while the ultra-structural analysis using the transmission electron microscope indicated BRET-QD localization on the sperm plasma membrane and intracellular compartment. In controlled-in vitro assays, bioluminescent imaging demonstrated that spermatozoa incubated with BRET-QD and luciferase substrate (coelenterazine) emit light (photons/sec) above the background, which confirmed the in situ fluorescence imaging. Most importantly, sperm motility, viability, and fertilizing potential were not affected by the BRET-QD incorporation when used at an appropriated ratio.ConclusionsOur results demonstrate that pig spermatozoa can incorporate BRET-QD nanoparticles without affecting their motility and capacity to interact with the oocyte when used at an appropriated balance. We anticipate that our study will enable in-depth exploration of the male components of in vivo migration, fertilization, and embryonic development at the molecular level using this novel approach.

Assessment of pregnancy in the late-gestation mare using digital infrared thermography

The objective of this study was to investigate use of digital infrared thermal imaging (DITI) to determine whether surface temperature gradient differences exist between pregnant and nonpregnant mares as a noncontact method to determine pregnancy status. On the day measurements were collected, each pregnant mare (n=10; beginning at 292.4+/-1.4 d of gestation) was paired with a nonpregnant mare (n=17). Ambient temperature, DITI measurements (left and right flank, wither temperatures [i.e., animal surface control] and background temperature), and rectal temperatures were obtained every 7 d for 5 wk before parturition and for 3 wk after parturition. There were no differences (P>0.10) in temperature of the left and right side within groups; therefore, data were pooled. Pregnant mares had a higher (P<0.01) flank temperature than that of nonpregnant mares (36.0+/-0.2 degrees C vs. 34.2+/-0.2 degrees C, respectively). Moreover, the difference (2.4 degrees C) in flank temperatures between the pregnant and nonpregnant mares was greater when the ambient temperature was <19 degrees C. Flank and wither temperatures were positively correlated (R=0.72; P<0.01) and were positively correlated with ambient temperature (R=0.48 and 0.64, respectively; P<0.01). However, wither temperatures (skin control site) did not differ (P>0.10) between pregnant and nonpregnant mares. In conclusion, late-gestation mares had higher flank temperatures than those of nonpregnant mares, regardless of environmental conditions, however discriminating abilities were greater when ambient temperature was lower. We inferred that DITI may have value in confirming mid- to late-gestation pregnancies in some species by noncontact means, as observed in the mare.

Use of glycerol as an optical clearing agent for enhancing photonic transference and detection of Salmonella typhimurium through porcine skin

The objective of this study was to evaluate glycerol (GLY) and GLY + dimethyl sulfoxide (DMSO) to increase photonic detection of transformed Salmonella typhimurium (S. typh-lux) through porcine skin. Skin was placed on 96-well plates containing S. typh-lux, imaged (5 min) using a CCD camera, and then completely immersed in PBS, GLY, DMSO, GLY+DMSO in a dose- and time-dependent manner and re-imaged (5 min). The percent of photonic emissions detected (treated or untreated skin relative to no skin controls) was used for analysis. Treatment for 4 h with 50% GLY-PBS and 50:30:20% GLY:DMSO:PBS increased photonic detection compared to untreated skin, 100% PBS, or 30:70% DMSO:PBS. Treatment with 50% GLY in the presence of 20 and 40% DMSO (v/v with PBS) increased photonic detection compared to 50% GLY alone and in the presence of 10% DMSO: 50% GLY (v/v with PBS). Data indicate that GLY and GLY+DMSO are effective optical clearing agents on porcine skin (2-3 mm thick) when treated for 4 h to increase detection of emitted photons. Clearing agents such as GLY have the potential to minimize effects of porcine skin tissue as one of the photon transmittance barriers (i.e., skin, fat, muscle, and visceral tissues) in vivo.

THERMOREGULATORY RESPONSES ASSOCIATED WITH LYING AND STANDING IN HEAT-STRESSED DAIRY COWS

The purpose of this study is to characterize the thermoregulatory responses of unrestrained heat-stressed dairy cows within a freestall environment using fan and spray configurations for cooling cows while lying or standing. An experimental treatment sprayed individual cows lying in freestalls from 11:00 to 15:00 (stall cooling period) during hot-humid weather (average THI of 82.4) over a five-day trial period, using ultrasound transceivers to detect their presence. Core body temperatures were continuously monitored with vaginal temperature loggers. To assess behavioral responses, cows were visually monitored during the stall cooling period. Respiration rates and dorsal surface temperatures were recorded when the cow lay down or stood up in a stall. Core body temperature of lying cows rises at a rate of 0.6°C h-1 when exposed to fan cooling alone. Adding spray cooling to fan cooling slows the rate of rise to 0.3°C h-1. With or without freestall spray cooling, cows stand and seek cooling when their core body temperatures reach 38.9°C. Core body temperature is a more reliable indicator than either dorsal skin temperatures or respiration rates for predicting when cows stand and seek cooling. Core body temperatures of cows fall at a rate of 0.7°C h-1 while standing under feed line spray and fan cooling, while core body temperatures of cows standing under fans without spray remain unchanged. To cool heat-stressed cows, water spray is required in addition to fans while the cows are standing. Fans alone are inadequate.

Photonic Monitoring in Real Time of Vascular Endothelial Growth Factor Receptor 2 Gene Expression under Relaxin‐Induced Conditions in a Novel Murine Wound Model

Abstract: Relaxin is known to promote vascular endothelial growth factor (VEGF) expression in reproductive tissue, and successful wound healing depends on good vascularization of wound sites, a process that relaxin may facilitate. Thus, the objective of this study was to evaluate the efficacy of relaxin on the development of vascular tissue at wound sites in a novel VEGF receptor 2‐luc (VEGFR2‐luc) transgenic mouse wound model by monitoring the rate of VEGFR2‐luc‐mediated gene expression using bioluminescence and real‐time imaging. To this end, 12 FVB/N VEGFR2‐luc transgenic male mice were assigned to treatments (six per group): saline alone or relaxin (1 g/6 h/14 days) administered intraperitoneally (i.p.). On day 0, a set of full‐thickness wounds (6‐mm punch) were generated under anesthesia on the dorsal aspect of each mouse. Photonic emissions were recorded (5‐min collection of photons) from wound sites 10 min after the administration of luciferin (150 mg/kg i.p.) on day 0 and on days 1, 2, 4, 7, 9, 11, and 14 postwounding to quantify luciferase activity using an IVIS 100 biophotonic imaging system. Animals were sacrificed (three per group) on day 7 or 14, and wound tissue specimens were recovered for molecular and histologic analyses. Although photonic emission from wound sites increased (P <.001) over time with peak values obtained by day 7, no significant (P >.05) effect of relaxin treatment on VEGFR2‐luc gene expression was noted at wound sites. Whereas measuring relaxins effect on angiogenesis indirectly via the VEGFR2 model was not successful, photonic imaging provides an exciting new tool using alternative models (i.e., VEGF‐luc mouse) to study relaxin‐induced gene expression in normal (i.e., wound healing) or tumorigenic tissues in real time.

Continuous Measurements of Vaginal Temperature of Female Cattle Using A Data Logger Encased in a Plastic Anchor

Vaginal temperatures of 20 pregnant dairy cows were continuously recorded using data loggers with built in temperature sensors for 3 weeks. Plastic anchors were designed and produced to hold data loggers inside the vagina of cows. The anchors were manufactured from plastisol - a flexible translucent plastic that could be molded to a desired configuration. The material is contact compatible with dairy products but is not known if it qualifies for a long-term implant. The plastic cures by heating to 160°C, which takes about one hour in an oven at a temperature of 215°C. The volume of material necessary to produce one anchor is about 90 cc. The system and procedure was reliable, accurate, low cost, easy to implant and does not fall out, and had no ill effects to the health of the cows or disturb their behavior/activities. Rectal temperatures were recorded using a high performance digital thermometer. The vaginal temperatures were compared to the rectal temperatures and the results matched to within 0.06 ± 0.015°C.