Assessment of potential neuropathic changes in cattle after cautery disbudding

Highlights

• Weak evidence for an effect of disbudding on size distribution of nerve fibers in R. cornualis

• No evidence for an effect of disbudding on the proportion of TH-positive fibers in R. cornualis

• No evidence for an effect of disbudding on the percentage of ATF3-positive perikarya in trigeminal ganglia

• No evidence for an effect of disbudding on the average glial envelope of GFAP-positive perikarya in trigeminal ganglia

Abstract

Disbudding of calves is a standard husbandry procedure to reduce the risk of injuries to other cattle and to workers. Whereas acute pain resulting from disbudding has been studied extensively, little is known about chronic pain as a potential long-term consequence. The goal of the present study was to investigate possible morphological changes in the cornual nerve as a function of disbudding. Samples were collected from 17 randomly selected bulls and from 21 calves from a prospective clinical study. Among the calves, 13 were disbudded and 8 were sham-disbudded. Out of the disbudded calves, 4 showed signs of chronic pain. In all the animals, the infraorbital nerve was used as a methodological check. Morphological analysis included measuring minimal diameters of the axons present in both the cornual and infraorbital nerves. Sympathetic fibers were identified as based on the presence of Tyroxine hydroxylase (TH). TH-negative fibers were considered afferents. Trigeminal ganglia from the calves were immunostained for glial fibrillary acidic protein (GFAP) and Activating transcription factor 3 (ATF3). R. cornualis and N. infraorbitalis differed in terms of axon diameters and proportion of TH-positive fibers. Weak evidence (p > .091) of a difference in axon diameters between control and disbudded calves was found in R. cornualis, but the proportion of TH-positive fibers was alike in both groups. Average glial envelope and the percentages of ATF3-positive neurons revealed no difference between calves with and without signs of pain. Thus, available evidence is insufficient to support neuropathic changes as a result of disbudding in calves.

Introduction

Removing horn buds in young dairy and beef calves to reduce the risk of injuries to other cattle and farm workers is a common practice on cattle farms. Cattle without horns are easier to handle and require less space during transport and at the feed trough (Faulkner and Weary, 2000; Duffield et al., 2010; Stock et al., 2013). Moreover, it has been shown that injuries caused by horns may have a substantial impact on milk production and growth (Laden et al., 1985).

Depending on whether the horn bud is destroyed at an early stage or whether fully developed horns need to be amputated in mature cattle, different techniques are used. In young calves up to 8 weeks, when the horn buds are 5 to 10 mm long, animals are commonly disbudded with a heated disbudding iron (Stafford and Mellor, 2005), with a caustic paste in the first week of life, or by mechanical removal using a dehorning spoon (Stock et al., 2013). Regardless of the method, disbudding is painful for at least 2 h after the procedure (Petrie et al., 1996) and possibly up to 44 h (Faulkner and Weary, 2000). Acute pain or distress due to disbudding pain have been corroborated by changes in plasma cortisol (Petrie et al., 1996; Sutherland et al., 2002), heart rate (Grondahl-Nielsen et al., 1999; Stewart et al., 2008), eye temperature (Stewart et al., 2008), electroencephalogram (Gibson et al., 2007), calf behavior (McMeekan et al., 1999; Faulkner and Weary, 2000) and peripheral sensitization (Mirra et al., 2018). It has been shown that the cortisol response following hot iron disbudding without anesthesia or analgesia is lower than when either a dehorning spoon or a caustic paste are used (Stafford and Mellor, 2011).

The horn and the surrounding tissue are mainly innervated by the Ramus cornualis, a branch of the trigeminal nerve. Disbudding and dehorning are painful and, therefore, are regulated by animal welfare laws. To meet the Swiss legal requirements, local anesthesia is mandatory (cf. Art. 16 TSchG) and allows to alleviate acute pain (Laden et al., 1985; McMeekan et al., 1998; Stafford and Mellor, 2005). Cornual nerve block with local anesthetic is currently the accepted technique to anesthetize the horn bud prior to its removal (Fierheller et al., 2012), ideally in combination with a systemic analgesic to improve pain relief (McMeekan et al., 1998; McMeekan et al., 1999; Faulkner and Weary, 2000; Duffield et al., 2010).

It has long been recognized in human medicine that surgery often entails chronic pain (Kehlet et al., 2006; Macrae, 2008; Nikolajsen, 2012). Peripheral nerve injury is presumably the most frequent cause of postsurgical long-term pain. The distal end of a cut axon degenerates and is encased with inflammatory cells, a process which may lead to an abnormal activation of sensory neurons (Zimmermann, 2001) and pain hypersensitivity (Watkins and Maier, 2003). With respect to disbudding, research has concentrated on acute pain during and immediately after the procedure. However, little is known about chronic pain. Additionally, to date, there is no definitive method to detect chronic pain after disbudding in calves.

It is known from several studies in laboratory animals that functional changes of sensory neurons go along with a different expression of cellular markers (Brown et al., 1997; Matteis et al., 1998; Ohtori et al., 2000 Dec 28; Chiang et al., 2005). Based on this knowledge, it can be hypothesized that after disbudding, similar changes occur in the trigeminal nerves innervating the horn bud and its surrounding tissue due to peripheral nerve damage. Overall, neuropathic pain has been associated with hyperinnervation (Buonocore, 2013) and both sensory as well as tyrosine hydroxylase-reactive fibers have been shown to be involved in neurogenic pain (Lindqvist et al., 2000).

The goal of the present study was to investigate possible morphological changes in the cornual nerve as a result of cautery disbudding. The vast majority of the primary afferents from the horn base travel with the R. cornualis of the trigeminal nerve making this branch an ideal target for morphological analysis. The infraorbital nerve was selected as a methodological check as it may not be affected by the surgical treatment. To assess possible changes, different parameters were assessed and comparisons made between cornual and infraorbital nerves, between disbudded and sham-disbudded calves and between disbudded calves with and without signs of chronic pain. Parameters used included 1) the size distributions of minimal axon diameters in both the cornual and infraorbital nerves by means of fiber labelling with neuron-specific class III beta-tubulin (Tuj-1) as a pan-neuronal marker; 2) the proportion of sympathetic fibers in cornual and infraorbital nerves by means of fiber labelling with tyroxine hydroxylase (TH); 3) the average glial envelope, i.e. the glial fibrillary acidic protein (GFAP)-positive area per cell body in the trigeminal ganglia and 4) the percentage of cyclic adenosine monophosphate-dependent transcription factor 3 (ATF3)-positive neurons in the trigeminal ganglia. Therefore, the aim of this study was to evaluate potential histopathological effects of cautery disbudding on the R. cornualis taking into account evidence of chronic pain.