Measuring Response to Pain Mitigation for Ovariectomy in Bos taurus Yearling Beef Heifers

Modern society is concerned about the humane treatment of food animals. Pain mitigation is desirable and expected when surgical procedures are performed on cattle. More than a million Bos taurus and Bos indicus female cattle are surgically ovariectomized (spayed) annually in areas where extensive grazing management is practiced around the world, most commonly using the dropped ovary technique (DOT) which accesses the ovaries via a colpotomy (trans-vaginal) approach. This procedure prevents estrus cycling and pregnancy in cull females that will end up fattened for meat production rather than breeding stock. Producers can realize financial benefits, and heifers may experience better welfare when they are not pregnant on arrival at the feedlot. However, no analgesia for bovine ovariectomy has been described in the literature. The purpose of this research was to design an effective pain mitigation protocol for ovariectomy. Analgesic drugs that were easily administered, rapidly effective and did not cause recumbency were essential to minimize stress and enable administration while heifers were in the handling system for ovariectomy. Two experiments were performed, each using an injectable combination of the analgesic drugs butorphanol (0.01 mg/kg), xylazine (0.02 mg/kg) and ketamine (0.04 mg/kg) (BXK) that was administered by intramuscular injection while heifers waited in the chute race and the oral anti-inflammatory meloxicam (1 mg/kg) that was administered in the squeeze chute where ovariectomy was performed. To determine the efficacy of the protocol, behavioural (experiments 1 and 2) and physiological (experiment 2) data were collected. The objectives were to 1) determine the degree of stress and pain that ovariectomy causes in yearling beef heifers, 2) to determine whether BXK administered 5 min prior to ovariectomy could mitigate the procedural and immediate post-operative pain of ovariectomy, 3) to determine whether oral meloxicam administered at the time of ovariectomy could mitigate the post-surgical inflammatory pain of ovariectomy and 4) whether it was possible to administer the drug protocol in a ranch setting without hindering the spaying process. Experiment 1 was a randomized controlled clinical trial with a 2 × 2 factorial design that took place on a cattle ranch using 120 yearling beef heifers (302 ± 27 kg) scheduled for ovariectomy. Heifers were weighed, identified with ear tags and had activity data loggers affixed to a hind limb prior to spaying on d -1. Heifers were spayed on d 0 and randomly allocated to 4 treatment groups: control (no treatment), BXK, meloxicam, or BXK and meloxicam; then observed for the next 5 d. Behavioural data collected included visual analog scale (VAS) scoring and pain-related behaviour recording by observers during ovariectomy, and stride length, gait score, standing/lying behaviour and video recording for feeding duration measurement after ovariectomy. More BXK treated heifers (P = 0.002) walked after leaving the squeeze chute and lay down for a larger percentage of time (P < 0.01) at 4 h after ovariectomy compared to those not treated with BXK, and meloxicam treated heifers stood for a larger percentage of time at 7 h (P = 0.02), 8 h (P = 0.001) and 9 h (P = 0.02) after spaying compared to those not treated with meloxicam. On the day of ovariectomy, groups that had BXK (P = 0.03) and BXK and meloxicam (P = 0.002) had longer feeding event durations compared to controls. On the day after ovariectomy, heifers treated with meloxicam stood for a smaller percentage of the day (P = 0.01) and for shorter periods of time (P = 0.03) compared to heifers not treated with meloxicam. Behavioural measurements provided minimal evidence of pain from ovariectomy and its alleviation. Experiment 2 took place at a research facility using 45 yearling beef heifers (322 ± 27 kg) randomly allocated to 3 treatment groups: PALP (control group palpated but not spayed), SPAY (spayed without analgesia) and BXKM (spayed with analgesia). Continuous data was gathered over 7 d using accelerometers for activity and the GrowSafe System for feeding behaviour. Saliva and blood samples were harvested, rectal temperature was taken, and recording of flight speed, stride length and gait occurred on d -1, d 0 (time of palpation/ spaying), and 1, 2, 4, 24, 48, 96 and 168 h after palpation/spaying. Salivary cortisol concentrations were lower in BXKM heifers than SPAY heifers at 1 h (P = 0.01) and 2 h (P = 0.004) after ovariectomy. Serum haptoglobin concentrations were lower in BXKM heifers than SPAY heifers at 48 h (P = 0.01), 96 h (P < 0.001), and 168 h (P = 0.008) and lower than PALP heifers at 96 h (P < 0.001) after ovariectomy. Serum amyloid A concentrations were higher in BXKM heifers than PALP heifers (P = 0.04) at 1 h after ovariectomy and at 24 h after ovariectomy PALP heifers had lower concentrations than BXKM (P = 0.02) and SPAY (P = 0.05) heifers. Rectal temperatures were greater in BXKM heifers than PALP and SPAY heifers at 1 h (P < 0.001) and 2 h (P = 0.004) after ovariectomy. Results suggest that ovariectomy is acutely stressful and painful, BXK mitigated the procedural and immediate post-surgical pain of ovariectomy and meloxicam reduced the post-surgical inflammation and inflammatory pain for up to 7 days after ovariectomy

Prenatal Cocaine Disrupts Serotonin Signaling-Dependent Behaviors: Implications for Sex Differences, Early Stress and Prenatal SSRI Exposure

Prenatal cocaine (PC) exposure negatively impacts the developing nervous system, including numerous changes in serotonergic signaling. Cocaine, a competitive antagonist of the serotonin transporter, similar to selective serotonin reuptake inhibitors (SSRIs), also blocks dopamine and norepinephrine transporters, leaving the direct mechanism through which cocaine disrupts the developing serotonin system unclear. In order to understand the role of the serotonin transporter in cocaine’s effect on the serotonergic system, we compare reports concerning PC and prenatal antidepressant exposure and conclude that PC exposure affects many facets of serotonergic signaling (serotonin levels, receptors, transporters) and that these effects differ significantly from what is observed following prenatal SSRI exposure. Alterations in serotonergic signaling are dependent on timing of exposure, test regimens, and sex. Following PC exposure, behavioral disturbances are observed in attention, emotional behavior and stress response, aggression, social behavior, communication, and like changes in serotonergic signaling, these effects depend on sex, age and developmental exposure. Vulnerability to the effects of PC exposure can be mediated by several factors, including allelic variance in serotonergic signaling genes, being male (although fewer studies have investigated female offspring), and experiencing the adverse early environments that are commonly coincident with maternal drug use. Early environmental stress results in disruptions in serotonergic signaling analogous to those observed with PC exposure and these may interact to produce greater behavioral effects observed in children of drug-abusing mothers. We conclude that based on past evidence, future studies should put a greater emphasis on including females and monitoring environmental factors when studying the impact of PC exposure

Evaluation of atlas based mouse brain segmentation

Magentic Reasonance Imaging for mouse phenotype study is one of the important tools to understand human diseases. In this paper, we present a fully automatic pipeline for the process of morphometric mouse brain analysis. The method is based on atlas-based tissue and regional segmentation, which was originally developed for the human brain. To evaluate our method, we conduct a qualitative and quantitative validation study as well as compare of b-spline and fluid registration methods as components in the pipeline. The validation study includes visual inspection, shape and volumetric measurements and stability of the registration methods against various parameter settings in the processing pipeline. The result shows both fluid and b-spline registration methods work well in murine settings, but the fluid registration is more stable. Additionally, we evaluated our segmentation methods by comparing volume differences between Fmr1 FXS in FVB background vs C57BL/6J mouse strains

The sea urchin embryo as a model for mammalian developmental neurotoxicity: ontogenesis of the high-affinity choline transporter and its role in cholinergic trophic activity.

Embryonic development in the sea urchin requires trophic actions of the same neurotransmitters that participate in mammalian brain assembly. We evaluated the development of the high-affinity choline transporter, which controls acetylcholine synthesis. A variety of developmental neurotoxicants affect this transporter in mammalian brain. [3H]Hemicholinium-3 binding to the transporter was found in the cell membrane fraction at stages from the unfertilized egg to pluteus, with a binding affinity comparable with that seen in mammalian brain. Over the course of development, the concentration of transporter sites rose more than 3-fold, achieving concentrations comparable with those of cholinergically enriched mammalian brain regions. Dimethylaminoethanol (DMAE), a competitive inhibitor of choline transport, elicited dysmorphology beginning at the mid-blastula stage, with anomalies beginning progressively later as the concentration of DMAE was lowered. Pretreatment, cotreatment, or delayed treatment with acetylcholine or choline prevented the adverse effects of DMAE. Because acetylcholine was protective at a lower threshold, the DMAE-induced defects were most likely mediated by its effects on acetylcholine synthesis. Transient removal of the hyaline layer enabled a charged transport inhibitor, hemicholinium-3, to penetrate sufficiently to elicit similar anomalies, which were again prevented by acetylcholine or choline. These results indicate that the developing sea urchin possesses a high-affinity choline transporter analogous to that found in the mammalian brain, and, as in mammals, the functioning of this transporter plays a key role in the developmental, trophic activity of acetylcholine. The sea urchin model may thus be useful in high-throughput screening of suspected developmental neurotoxicants

GABA A Receptors Mediate Trophic Effects of GABA on Embryonic Brainstem Monoamine Neurons In Vitro

The inhibitory neurotransmitter GABA may act as a trophic signal for developing monoamine neurons in embryonic rat brain, because GABA neurons and their receptors appear in brainstem during generation of monoamine neurons. To test this hypothesis, we used dissociated cell cultures from embryonic day 14 rat brainstem, which contains developing serotonin (5-HT), noradrenaline (tyrosine hydroxylase; TH), and GABA neurons. Immunocytochemistry and reverse transcription-PCR (RT-PCR) revealed the presence of multiple alpha, beta, gamma, and delta subunits in these cultures. Competitive RT-PCR demonstrated high levels of beta3 subunit transcripts. Expression of functional GABAA receptors was demonstrated using 36Cl- flux assays. To investigate GABAergic regulation of neuronal survival and growth, cultures were treated for 1-3 d in vitro with 10 microM GABA and/or GABAA antagonist (bicuculline or the pesticide dieldrin). The effects of treatments were quantified by analysis of immunoreactive 5-HT, TH, and GABA neurons. GABAA receptor ligands differentially regulated neuronal survival and growth depending on neurotransmitter phenotype. GABA exerted positive effects on monoamine neurons, which were countered by bicuculline (and dieldrin, 5-HT neurons only). By itself, bicuculline produced inhibitory effects on both 5-HT and TH neurons, whereas dieldrin potently inhibited 5-HT neurons only. GABA neurons responded positively to both antagonists, but more strongly to bicuculline. Taken together, these results demonstrate that the activation/inhibition of GABAA receptors produces opposite effects on the development of embryonic monoamine and GABA neurons. This suggests that these neurotransmitter phenotypes may express GABAA receptors that differ in fundamental ways, and these differences determine the developmental responses of these cells to GABAergic stimuli

Early postnatal administration of 5,7-dihydroxytryptamine: Effects on substance P and thyrotropin-releasing hormone neurons and terminals in rat brain

Substance P, thyrotropin-releasing hormone (TRH) and serotonin are putative neurotransmitters which have been proposed to coexist in some brain neurons Our previous immunocytochemical and biochemical studies have demonstrated that 85–100% of all serotonin neurons are destroyed following neonatal 5,7-dihydroxtryptamine (5,7-DHT) treatment In this study, we have determined the effect of neonatal 5,7-DHT and desipramine (DMI) treatment on the biochemical content and immunocytochemical localization of substance P and TRH throughout the brain Interestingly, we have observed that virtually all substance P- and TRH-immunoreactive cells in the ventral pons-medulla are destroyed by the neurotoxin. However, peptide-containing neurons in other regions were not affected Additionally, we measured the peptide content and found that TRH is significantly decreased in the spinal cord (−50%) and pons-medulla (−20%), but not in other brain regions Substance P content was not significantly altered in any region, even after a greater than 90% reduction of serotonin These data indicate that the co-localized substance P and TRH forms a small proportion of the total peptide in brai

3-Dimensional Diffusion Tensor Imaging (DTI) Atlas of the Rat Brain

Anatomical atlases play an important role in the analysis of neuroimaging data in rodent neuroimaging studies. Having a high resolution, detailed atlas not only can expand understanding of rodent brain anatomy, but also enables automatic segmentation of new images, thus greatly increasing the efficiency of future analysis when applied to new data. These atlases can be used to analyze new scans of individual cases using a variety of automated segmentation methods. This project seeks to develop a set of detailed 3D anatomical atlases of the brain at postnatal day 5 (P5), 14 (P14), and adults (P72) in Sprague-Dawley rats. Our methods consisted of first creating a template image based on fixed scans of control rats, then manually segmenting various individual brain regions on the template. Using itk-SNAP software, subcortical and cortical regions, including both white matter and gray matter structures, were manually segmented in the axial, sagittal, and coronal planes. The P5, P14, and P72 atlases had 39, 45, and 29 regions segmented, respectively. These atlases have been made available to the broader research community

Evaluation of atlas based mouse brain segmentation

Magentic Reasonance Imaging for mouse phenotype study is one of the important tools to understand human diseases. In this paper, we present a fully automatic pipeline for the process of morphometric mouse brain analysis. The method is based on atlas-based tissue and regional segmentation, which was originally developed for the human brain. To evaluate our method, we conduct a qualitative and quantitative validation study as well as compare of b-spline and fluid registration methods as components in the pipeline. The validation study includes visual inspection, shape and volumetric measurements and stability of the registration methods against various parameter settings in the processing pipeline. The result shows both fluid and b-spline registration methods work well in murine settings, but the fluid registration is more stable. Additionally, we evaluated our segmentation methods by comparing volume differences between Fmr1 FXS in FVB background vs C57BL/6J mouse strains

An anxiety-like phenotype in mice selectively bred for aggression

Using selective bi-directional breeding procedures, two different lines of mice were developed. The NC900 line is highly reactive and attacks their social partners without provocation, whereas aggression in NC100 animals is uncommon in social environments. The enhanced reactivity of NC900 mice suggests that emotionality may have been selected with aggression. As certain forms of anxiety promote exaggerated defensive responses, we tested NC900 mice for the presence of an anxiety-like phenotype. In the open field, light-dark exploration, and zero maze tests, NC900 mice displayed anxiety-like responses. These animals were less responsive to the anxiolytic actions of diazepam in the zero maze than NC100 animals; diazepam also reduced the reactivity and attack behaviors of NC900 mice. The NC900 mice had reduced diazepam-sensitive GABAA receptor binding in brain regions associated with aggression and anxiety. Importantly, there was a selective reduction in levels of the GABAA receptor α2 subunit protein in NC900 frontal cortex and amygdala; no changes in α1 or γ2 subunit proteins were observed. These findings suggest that reductions in the α2 subunit protein in selected brain regions may underlie the anxiety and aggressive phenotype of NC900 mice. Since anxiety and aggression are comorbid in certain psychiatric conditions, such as borderline personality and posttraumatic stress disorder, investigations with NC900 mice may provide new insights into basic mechanisms that underlie these and related psychiatric conditions

An Invertebrate Model of the Developmental Neurotoxicity of Insecticides: Effects of Chlorpyrifos and Dieldrin in Sea Urchin Embryos and Larvae

Chlorpyrifos targets mammalian brain development through a combination of effects directed at cholinergic receptors and intracellular signaling cascades that are involved in cell differentiation. We used sea urchin embryos as an invertebrate model system to explore the cellular mechanisms underlying the actions of chlorpyrifos and to delineate the critical period of developmental vulnerability. Sea urchin embryos and larvae were exposed to chlorpyrifos at different stages of development ranging from early cell cleavages through the prism stage. Although early cleavages were unaffected even at high chlorpyrifos concentrations, micromolar concentrations added at the mid-blastula stage evoked a prominent change in cell phenotype and overall larval structure, with appearance of pigmented cells followed by their accumulation in an extralarval cap that was extruded from the animal pole. At higher concentrations (20-40 microM), these abnormal cells constituted over 90% of the total cell number. Studies with cholinergic receptor blocking agents and protein kinase C inhibitors indicated two distinct types of effects, one mediated through stimulation of nicotinic cholinergic receptors and the other targeting intracellular signaling. The effects of chlorpyrifos were not mimicked by chlorpyrifos oxon, the active metabolite that inhibits cholinesterase, nor by nonorganophosphate cholinesterase inhibitors. Dieldrin, an organochlorine that targets GABA(A )receptors, was similarly ineffective. The effects of chlorpyrifos and its underlying cholinergic and signaling-related mechanisms parallel prior findings in mammalian embryonic central nervous system. Invertebrate test systems may thus provide both a screening procedure for potential neuroteratogenesis by organophosphate-related compounds, as well as a system with which to uncover novel mechanisms underlying developmental vulnerability