Assessment of response variables in bovine models of pain and stress, with and without meloxicam

Doctor of PhilosophyDepartment of Diagnostic Medicine and PathobiologyRonette GehringThe absence of pain management for common husbandry procedures, such as dehorning and castration of cattle, is considered to be an important animal welfare consideration, but there are currently no drugs approved by the FDA for the purpose of providing pain relief in cattle. The FDA’s Center for Veterinary Medicine (CVM) recognizes the need for the availability of pain-relieving drugs and has encouraged research into the development of behavioral and physiologic measures which can reliably demonstrate the effectiveness in that species. The USDA has also recognized this need by providing grants for research into investigating pain models for cattle that can be used for the development of pain mitigation methods. The studies reported in this dissertation were funded by the USDA and the American Association of Bovine Practitioners. They add to the body of knowledge from which a pain model in cattle may eventually be validated for use in the drug approval process and also contribute to knowledge base for a candidate non-steroidal anti-inflammatory drug for convenient use in cattle. The study reported in Chapter 2 was conducted to support research of a candidate pain-assessment variable, substance P. This study provides future researchers with recommended sample handling procedures for obtaining reliable and repeatable results, which is important if substance P is to be validated as pain biomarker in cattle. The study in Chapter 3 investigated the use of several variables for use in a pain model. The results provided researchers, veterinarians and policy-makers with evidence to support the common practice of castrating and dehorning calves at the same time rather than as individual procedures separated by a healing interim. The study in Chapter 4 investigated the pharmacokinetics of oral meloxicam when administered to juvenile ruminant and pre-ruminant calves. This study added to the growing knowledge base of the pharmacokinetics of oral meloxicam in cattle and also provided practitioners with practical information concerning the administration of the drug in milk replacer. Chapter 5 investigated the use of oral meloxicam in a production setting and indicated that meloxicam administration prior to surgical castration may reduce the incidence of respiratory disease in the post-surgical period

Assessment of behavioral changes associated with oral meloxicam administration at time of dehorning in calves using a remote triangulation device and accelerometers

Background: Dehorning is common in the cattle industry, and there is a need for research evaluating pain mitigation techniques. The objective of this study was to determine the effects of oral meloxicam, a non-steroidal anti-inflammatory, on cattle behavior post-dehorning by monitoring the percent of time spent standing, walking, and lying in specific locations within the pen using accelerometers and a remote triangulation device. Twelve calves approximately ten weeks of age were randomized into 2 treatment groups (meloxicam or control) in a complete block design by body weight. Six calves were orally administered 0.5 mg/kg meloxicam at the time of dehorning and six calves served as negative controls. All calves were dehorned using thermocautery and behavior of each calf was continuously monitored for 7 days after dehorning using accelerometers and a remote triangulation device. Accelerometers monitored lying behavior and the remote triangulation device was used to monitor each calf’s movement within the pen. Results: Analysis of behavioral data revealed significant interactions between treatment (meloxicam vs. control) and the number of days post dehorning. Calves that received meloxicam spent more time at the grain bunk on trial days 2 and 6 post-dehorning; spent more time lying down on days 1, 2, 3, and 4; and less time at the hay feeder on days 0 and 1 compared to the control group. Meloxicam calves tended to walk more at the beginning and end of the trial compared to the control group. By day 5, the meloxicam and control group exhibited similar behaviors. Conclusions: The noted behavioral changes provide evidence of differences associated with meloxicam administration. More studies need to be performed to evaluate the relationship of behavior monitoring and post-operative pain. To our knowledge this is the first published report demonstrating behavioral changes following dehorning using a remote triangulation device in conjunction with accelerometers

Pharmacokinetics and effect of intravenous meloxicam in weaned Holstein calves following scoop dehorning without local anesthesia

Background: Dehorning is a common practice involving calves on dairy operations in the United States. However, less than 20% of producers report using analgesics or anesthetics during dehorning. Administration of a systemic analgesic drug at the time of dehorning may be attractive to dairy producers since cornual nerve blocks require 10 – 15 min to take effect and only provide pain relief for a few hours. The primary objectives of this trial were to (1) describe the compartmental pharmacokinetics of meloxicam in calves after IV administration at 0.5 mg/kg and (2) to determine the effect of meloxicam (n = 6) or placebo (n = 6) treatment on serum cortisol response, plasma substance P (SP) concentrations, heart rate (HR), activity and weight gain in calves after scoop dehorning and thermocautery without local anesthesia. Results: Plasma meloxicam concentrations were detectable for 50 h post-administration and fit a 2-compartment model with a rapid distribution phase (mean T[subscript ½α] = 0.22 ± 0.087 h) and a slower elimination phase (mean T[subscript ½β] = 21.86 ± 3.03 h). Dehorning caused a significant increase in serum cortisol concentrations and HR (P < 0.05). HR was significantly lower in the meloxicam-treated calves compared with placebo-treated calves at 8 h (P = 0.039) and 10 h (P = 0.044) after dehorning. Mean plasma SP concentrations were lower in meloxicam treated calves (71.36 ± 20.84 pg/mL) compared with control calves (114.70 ± 20.84 pg/mL) (P = 0.038). Furthermore, the change in plasma SP from baseline was inversely proportional to corresponding plasma meloxicam concentrations (P = 0.008). The effect of dehorning on lying behavior was less significant in meloxicam-treated calves (p = 0.40) compared to the placebo-treated calves (P < 0.01). Calves receiving meloxicam prior to dehorning gained on average 1.05 ± 0.13 kg bodyweight/day over 10 days post-dehorning compared with 0.40 ± 0.25 kg bodyweight/day in the placebo-treated calves (p = 0.042). Conclusions: To our knowledge, this is the first published report examining the effects of meloxicam without local anesthesia on SP, activity and performance of calves post-dehorning. These findings suggest that administration of meloxicam alone immediately prior to dehorning does not mitigate signs of acute distress but may have long term physiological, behavior and performance effects

Unmitigated Surgical Castration in Calves of Different Ages: Cortisol Concentrations, Heart Rate Variability, and Infrared Thermography Findings

The objective was to characterize physiological responses to unmitigated surgical castration in calves of varying ages. Thirty male Holstein calves of three ages [<6 w (6W); 3 m (3M); 6 m (6M); n = 10] underwent a simulated castration treatment (SHAM) followed 24 h later by castration (CAST). For both treatments, heart rate variability, eye temperature, and cortisol were measured over time from treatment to specified end points to capture the acute response period. Interactions between treatment and age (p = 0.035) and time and age (p < 0.001) were noted for cortisol. The 6W calves had lower cortisol compared to 6M calves at SHAM and CAST. Cortisol of 6W calves decreased from peak to pre-treatment levels faster than 6M calves. An interaction between time and age was reported in squared differences of inter-beat-intervals (RMSSD; p = 0.02) and high-frequency power (HFP; p = 0.05), whereby both responses decreased in 6W calves during the sampling period which was not seen in 3M and 6M calves. Average eye temperature (AET) differed by age (p = 0.0018) whereby 6W calves had lower AET than 6M calves (p = 0.0013) regardless of treatment and time. The findings suggest that responses to unmitigated surgical castration seem to be mediated by the autonomic nervous system in an age-related manner

Finding sRNA generative locales from high-throughput sequencing data with NiBLS

Background: Next-generation sequencing technologies allow researchers to obtain millions of sequence reads in a single experiment. One important use of the technology is the sequencing of small non-coding regulatory RNAs and the identification of the genomic locales from which they originate. Currently, there is a paucity of methods for finding small RNA generative locales. Results: We describe and implement an algorithm that can determine small RNA generative locales from high-throughput sequencing data. The algorithm creates a network, or graph, of the small RNAs by creating links between them depending on their proximity on the target genome. For each of the sub-networks in the resulting graph the clustering coefficient, a measure of the interconnectedness of the subnetwork, is used to identify the generative locales. We test the algorithm over a wide range of parameters using RFAM sequences as positive controls and demonstrate that the algorithm has good sensitivity and specificity in a range of Arabidopsis and mouse small RNA sequence sets and that the locales it generates are robust to differences in the choice of parameters. Conclusions: NiBLS is a fast, reliable and sensitive method for determining small RNA locales in high-throughput sequence data that is generally applicable to all classes of small RNA

RNA interference in the nucleus: roles for small RNAs in transcription, epigenetics and beyond

A growing number of functions are emerging for RNA interference (RNAi) in the nucleus, in addition to well-characterized roles in post-transcriptional gene silencing in the cytoplasm. Epigenetic modifications directed by small RNAs have been shown to cause transcriptional repression in plants, fungi and animals. Additionally, increasing evidence indicates that RNAi regulates transcription through interaction with transcriptional machinery. Nuclear small RNAs include small interfering RNAs (siRNAs) and PIWI-interacting RNAs (piRNAs) and are implicated in nuclear processes such as transposon regulation, heterochromatin formation, developmental gene regulation and genome stability

Small RNA sorting: matchmaking for Argonautes

Small RNAs directly or indirectly impact nearly every biological process in eukaryotic cells. To perform their myriad roles, not only must precise small RNA species be generated, but they must also be loaded into specific effector complexes called RNA-induced silencing complexes (RISCs). Argonaute proteins form the core of RISCs and different members of this large family have specific expression patterns, protein binding partners and biochemical capabilities. In this Review, we explore the mechanisms that pair specific small RNA strands with their partner proteins, with an eye towards the substantial progress that has been recently made in understanding the sorting of the major small RNA classes - microRNAs (miRNAs) and small interfering RNAs (siRNAs) - in plants and animals