Stress and pain response after oligofructose induced-lameness in dairy heifers

Animal welfare studies have focused on the distress that animals might experience and have highlighted pain as one of the most important aspects to be addressed [37]. Lameness in dairy cows is a serious welfare issue. Over the last few decades, the importance of lameness in dairy cattle has been recognized, and it is becoming one of the most important economic problems afflicting the dairy industry [23]. Lameness is a response to pain caused by trauma, metabolic disorders and infections [15]. Lameness in cattle is a debilitating condition that is often associated with tissue damage, pain and discomfort. This conduction produces a major impact on the productivity of dairy cattle, reducing milk production, decreasing fertility, and increasing the likelihood of other diseases such as mastitis [14]. The notion that chronic pain is a phenomenon distinct from acute pain is widely accepted as common sense among veterinarians and the public [27]. In contrast, questions regarding whether acute pain causes chronic pain have not been completely resolved, although it is known that some stimuli or continuous nociceptive processes provide the impetus for chronic pain to develop [38].

Blood biomarkers correspond to well defined and specific biochemical substances that have proven useful in the diagnosis and treatment of specific conditions [19]. Pain studies in cattle often use the activity of the hypothalamus-pituitary-adrenal (HPA) axis and the autonomous nervous system (ANS) as indicators of stress or pain. Cortisol has been extensively employed as a stress biomarker in lame cattle [18, 32]. Nevertheless, it is well known that cortisol plasma concentration is affected by handling procedures or by the sampling process [21]. Norepinephrine has also been used as a marker of stress or acute pain [16, 31], and increases in the serum concentration in lame cattle have previously been reported [18]. Haptoglobin is an acute phase protein synthetized by hepatocytes in response to macrophage mediated secretion of cytokines that increases in serum after inflammatory processes, trauma and stress [7, 32]. Substance P has arisen as one of the most specific biomarkers of pain because it regulates the excitability of spinal cord neurons after injury and has been used to assess pain after castration in beef calves [5]. Beta-endorphin is an endogenous opioid peptide synthetized by the hypophysis that is considered to be an important component of the anti-nociceptive system, generating analgesia during painful conditions. Although a very important mediator of pain, its concentrations have only been evaluated in pregnant cows [24] and during slaughter [36]. This study was conducted to evaluate clinical signs and plasma concentration of several pain and stress biomarkers after oligofructose-induced lameness in dairy heifers.

The Ethics and Bioethics Committee on Animal Use of the Universidad Austral de Chile approved the present study (approval no. 42-2012).

The main objective of this study was to evaluate clinical signs and plasma concentrations of several pain and stress biomarkers after oligofructose-induced lameness in dairy heifers. We hypothesized that oligofructose overload and subsequent development of ruminal acidosis would induce lameness in treated heifers. It is well known that metabolic disorders are associated with the onset of inflammatory like laminitis [34]. Increases in heart rate were similar to those previously reported after induction of lameness using the oligofructose model [33]. Although Thoefner et al. [33] considered the increase in HR to be related to dehydration and metabolic acidosis, we did not observe signs of dehydration in our animal. We believe that the increases in HR reported here are related to pain, and further associated with increased activity of the sympathetic nervous system (SNS), which was also reflected here by increases in plasma norepinephrine concentration. Several authors have concluded that variations in heart rate and respiratory rate may be valuable indicators of pain [2]. Nonetheless, their values are highly variable and may not be affected by other pathological process of physiological conditions [9]. The reduction of RF is a characteristic sign of acute ruminal acidosis that has been reported in all models of oligofructose overload [6, 33]. This decrease in RF further confirms that our oligofructose overload model was successful in inducing ruminal acidosis. All our animals develop lameness as early as 6 h after oligofructose administration. These results differ from those reported previously [33], in which 4 out of 6 animals developed clinically identified lameness. Another important factor that could explain these results is the fact that our lameness scoring analysis was performed from video recordings. According to Whay et al. [39], few farmers could positively identify lame cows in their herds [39]. Moreover, scoring analysis disadvantages include being hard to evaluate, low repeatability and the need for expertise to correctly interpret the results [8].

Oligofructose overload induced acute lameness-associated pain. Acute pain induces activation of the hypothalamus-pituitary-adrenal (HPA) axis; therefore, the acute cortisol response can be used to quantify the stress response associated with pain induced by common veterinary practices [21]. Here, cortisol baseline values were higher than those previously reported for sound cows (4.7-7.5 µg/L) [32]. Moreover, we describe increases in plasma cortisol concentration of treated heifers starting at 6 h after oligofructose overload that remained elevated for up to 48 h. This increase indicates that the oligofructose model induces pain and inflammation. Our findings are concordant with those previously reported by Ting et al. [35] and Pang et al. [25], who described increases in plasma cortisol 24 and 48 h after castration, respectively. Increases as early as 30 min after castration have also been reported [30].

Baseline values for haptoglobin were lower than reference values [32]. The higher haptoglobin plasma concentrations observed here in the treated group are related to the acute insult induced by oligofructose overload. Increases in plasma haptoglobin concentrations have been associated with conditions such as severe sole ulcers, white line abscesses and interdigital dermatitis, which cause a more generalized acute phase reaction [13, 32]. Moreover, our results are in agreement with those of Horadagoda et al. [11], who concluded that acute phase proteins are useful for differentiating acute from chronic conditions because of their higher sensibility and specificity. This view is supported by Tadich et al. [32], who concluded that increases in haptoglobin concentrations were related to lower nociceptive thresholds as lameness score increased, suggesting that secondary lesions may represent persistent inflammatory processes.

Norepinephrine values were similar to those previously reported for ewes and cattle with chronic lameness [17, 18]. Plasma catecholamines, which have been used in cattle as a stress biomarker, possess a short half-life [10]. Nevertheless, most studies that describe catecholamines concentrations are associated with short-term painful stimuli such as castration [31] and dehorning [20]. To the best of our knowledge, this is the first study to examine plasma norepinephrine concentrations after oligofructose overload and lameness-associated pain. Here, we describe a significant increase in plasma concentration of norepinephrine 6 h after oligofructose overload. These findings could be associated with stimulation of the SNS in response to pain. Mellor et al. [20] previously reported that plasma norepinephrine concentrations peaked at 30 min after dehorning of calves. Moreover, we believe that oligofructose overload induced constant nociceptive stimulation characterized by a marked increase in HR that corresponded with a significant increase of norepinephrine. It is well known that during nociceptive stimulation, norepinephrine modulates and generates pain inhibition [26].

To the best of our knowledge, this is the first report that evaluates changes in plasma concentrations of substance P and beta-endorphin after oligofructose overload and lameness induction. We report significant increases in plasma substance P concentration starting 6 h after oligofructose overload, with the maximum values occurring at 12 h. These results are in agreement with those reported by Coetzee et al. [5], who described significant increases in the plasma concentration of substance P in beef calves 45 min after castration. Moreover, significant increases in substance P have been reported after dehorning in calves that did not receive analgesia, with concentrations reaching maximum values 120 h after dehorning [1]. Nonetheless, our results are discordant with the findings reported by Repenning et al. [28], who did not find significant differences in the plasma substance P concentration of calves subjected to band castration and their respective controls. Although our results are similar to those of previous studies, several authors have mentioned that sampling time and handling procedures are critical to obtaining reliable substance P concentrations [28, 22].

The beta-endorphin concentration for controls were similar to those previously reported for pregnant cows [24]. Our results showed significant differences between control and treated heifers, with values for the treated group peaking 24 h after induction of lameness by oligofructose overload. Endogenous opioids are relevant to understanding the transition between acute and chronic pain, although the role that beta-endorphins may play as part of the endogenous opioid antinociceptive system is still the subject of debate [3]. According to Chen et al. [4], lower analgesia levels associated with dysfunction of the endogenous opioid system are observed in chronic neuropathic pain patients and related to the loss of inhibition induced by µ-opioid receptors in the spinal cord. These opioid receptors are located in the primary afferent fibers and the dorsal horn of the spinal cord, and their primary function is to inhibit the release of excitatory neurotransmitters, specifically substance P [12, 40]. These findings suggest that the increases in beta-endorphins observed in our study could be associated with response mechanisms designed to regulate the secretion of Substance P.

We conclude that lameness induced using the oligofructose overload model in heifers was associated with variations in the evaluated clinical parameters. Moreover, the use of specific pain and stress biomarkers was useful in the detection of lameness-associated acute pain characterized by increases in the plasma concentration of cortisol, haptoglobin, norepinephrine, substance P and beta-endorphins. Although the use of these biomarkers could be useful in identifying the mechanisms that mediate the transition between acute and chronic pain in lame cattle, further studies are necessary to confirm our findings.