Impacts of Flunixin Meglumine injection on pain responses of either rubber ring castrated and tail docked lambs or surgically castrated and docked lambs

INTRODUCTION

Pain is defined as an aversive sensory or emotional event that causes an animal to experience stress (Molony and Kent, 1997). Stress is a biochemical response that can occur from a physical or psychological response on the individual (Eberhard and Veisser, 2007). There are multiple methods of castration and docking that livestock producers utilize, and each one can cause different levels and types of stress. The rubber ring (RR) method is commonly used in the sheep industry for castration and docking, whereas the cattle industry performs either surgical or the RR method for castration (Daly, 2015). Warnock et al. (2012) and Molony and Kent (1997) compared different methods of either castration or docking in cattle and sheep. Warnock et al. (2012) reported that castration method had no effect on performance traits, whereas Molony and Kent (1997) reported castration method may affect behavioral responses in sheep. Thornton and Waterman-Pearson (1999) observed that analgesics may decrease behavioral stress and cortisol concentrations in lambs. One potential analgesic to use during castration and docking is Flunixin Meglumine (FM). Flunixin Meglumine is a non-narcotic, nonsteroidal analgesic agent with anti-inflammatory and antipyretic activity (Banamine Intervet/Merck Animal Health, Madison, NJ). However, we are not aware of research that evaluates the comparison of castration and docking methods in lambs, with or without the use of analgesics. Therefore, our hypothesis was that administering FM to lambs that were either RR castrated and docked or surgical castrated and emasculator docked would decrease behavioral stress, serum cortisol, and haptoglobin concentrations, but increase average daily gain.

MATERIALS AND METHODS

All procedures were approved by the Animal Care and Use Committee of North Dakota State University.

Behavioral and Performance Responses

One hundred and eighty-one Rambouillet ram lambs (12.5 ± 5.5 d of age) were allocated in a completely randomized design with a 2 × 2 factorial arrangement of treatments to determine the effects on behavioral and physiological stress responses. Lambs were attained from the NDSU Hettinger Research Extension Center (HREC) flock and were housed in the lambing barn at the HREC. Lambs were allotted to treatment by stratifying over time to ensure technicians could collect data accurately. Lambs were manually restrained by trained personnel for weight and blood collection. Lambs were randomly allotted to one of four treatments at the time of docking and castration (n = 42 lambs per treatment): 1) rubber ring castrated and tail docked (RR) receiving an injection of saline (S) 15 to 30 min prior to banding (2.2 mg/kg of BW SQ; RR-S) or 2) Flunixin Meglumine 15 to 30 min prior to banding (2.2 mg/kg of BW SQ; RR-FM), and 3) lambs surgically castrated (SC) and emasculator docked receiving an injection of saline 15 to 30 min prior to procedure (2.2 mg/kg of BW SQ; SC-S) or 4) Flunixin Meglumine 15 to 30 min prior to procedure (2.2 mg/kg of BW SQ; SC-FM). Rubber band castration and tail docking was placement of a rubber band at the distal end of caudal fold and at the base of the scrotum. Surgical castration used surgical scissors to remove the bottom third of the scrotum and manually removing the testicles. Emasculator docking crushed the tail at the distal end of the caudal fold, followed by cutting of the tail. Lambs were vaccinated at the time of castration and docking and at weaning (CD-T; Bar Vac CD/T; Boehringer Ingelheim, Ridgefield, CT).

Lambs were selected from ewe lambing pens so that all lambs used were of similar age (between 7 and 18 d of age). Lambs were weighed, randomly assigned to treatment, and given the respective dosage of the treatment followed by their treatment procedure. Lambs were injected using an insulin syringe (29 gauge, VWR International). To determine behavioral responses, the scale described by Molony et al. (1993, 1995) was utilized. Postural indices were used for measurements, including various forms of lying and standing categorized further by the positions of the limbs and head. These postures were divided into 1) normal lying, ventral (sternal) with the head down (V1), with the head up (V2), both occurring frequently in control lambs; 2) abnormal lying, ventral with partial extension of one leg (V3) or full extension of one or more legs (V4), and lateral lying with a shoulder down and the head up (L1) or down (L2); 3) standing postures including normal standing /walking (S1), abnormal standing/walking with just detectable ataxia, swaying or abnormal stance (S2), grossly abnormal standing/walking with stilted gait walking on knees or walking backward (S3), and immobile or statue These measurements were taken immediately post-castration and tail docking. Observations were also taken at 30, 60, 90, 120, 150, and 180 min post-treatment. Lambs were weighed every 2 wk until weaning (approximately 74 d of age), and average daily gain was calculated at the end of the trial as the difference in final and initial weight divided by 42 to 56 d.

Physiological Responses

Blood was drawn −30, 30, 90, and 140 min after castration and docking. A trained handler extended the neck of the lamb and another trained personnel drew 3 mL of blood via jugular venipuncture using vacutainers (SST, VWR International) with a 21-gauge, 2.54-cm needle for the evaluation of serum cortisol. Additionally, a subset of 25 lambs from each treatment group were randomly selected for the determination of haptoglobin concentration. An additional 3 mL of blood was collected at the same time previously described using jugular vacutainers (EDTA, VWR International). Blood was cooled at 4°C for 2 h and centrifuged (3,640 × g, 15 °C, 20 min), and serum was harvested and stored (−20 °C). Cortisol serum was analyzed at North Dakota State University using the Immulite 1000 (Siemens Healthcare Diagnostics). Haptoglobin samples was analyzed at University of Guelph using the Roche Cobas 6000 c501 biochemistry analyzer (J. G. Skinner laboratory).

Statistical Analysis

For analysis purposes, lamb behavioral responses were placed on a zero- to four-pain scale to distinguish no pain, little, moderate, moderately high, and high amounts of pain. Group 0 was classified as V2 and S1. Group 1 is V1 due to the lamb having the head being down. Group 2 includes V3 and S2 because it indicates moderate pain. Group 3 includes L1 and S3 as it is the next level of pain and more stressful than the previous level. Group 4 indicates severe levels of pain and stress the lamb is enduring including S4, V4, and L2. Repeated measures were present for behavioral responses, cortisol, and haptoglobin levels. Treatment, time, and treatment × time interaction were included as fixed effects. Age was included as a fix covariate. Behavioral traits, physiological traits, and average daily gain were analyzed in PROC GENMOD (SAS Inst. Inc., Cary, NC) due to the lack of normality in the residuals when analyzed in PROC MIXED.

RESULTS AND DISCUSSION

Behavioral and Performance Responses

Pain Behavioral Scale.

A treatment × time interaction (P < 0.001) occurred for lamb pain behavioral scale, where scores at 30-, 60-, and 180-min after castration and docking exhibited differences among treatments (Figure 1). The RR treated lambs (both with and without FM) experienced the highest pain level rating at 30 min post-castration with a 40% increase in lambs standing in abnormal positions At 30 and 60 min post-treatment, there was a castration and docking method difference; the first 60 min post-castration and docking the RR group experienced higher levels of stress. At 90 min all treatment groups beside SC-S group experienced over 47% chance at pain level zero (i.e., no pain). Pain behavioral scores continued to decrease over time with 55% to 70% chance all groups’ average scores being 1 or less (little pain exhibited) at 120 min after procedures were applied. Only the SC-S group exhibited 36% of pain level 2 when compared with less than 25% with other treatments at 180 min post-treatment (P < 0.05). Lambs treated with FM had no significant difference (P > 0.05) with behavioral stress. Our results disagree with those of Marini et al. (2017) and Small et al. (2014), as lambs in their trials that were administered analgesics exhibited a reduced painful posture. However, our results were similar to those of Meleches et al. (2007) who reported that the surgical and burdizzo methods resulted in more expression of pain in lambs when compared with the RR and handled groups. For our trial at different time points, there was a difference between method of castration and docking; however, our results conclude that FM did not have an effect on painful behavior.

Figure 1.

The effect of castration and docking technique and administration of Flunixin Meglumine on lamb behavior (Molony et al., 1993, 1995). Treatments include RR-FM (rubber ring-Flunixin Meglumine), RR-S (rubber ring-saline), SC-FM (surgical castration-Flunixin Meglumine), and SC-S (surgical castration-saline). Means with different superscripts (A–C) differ (P ≤ 0.05) within each time point. Scale for behavior ranges from 0 to 4, 0—no pain and 4—highest level of pain.

Performance.

Average daily gain did not exhibit a treatment × time interaction (P = 0.22) or treatment main effect (P = 0.50). Initial and final weights were similar across treatments (P ≥ 0.09), resulting in an average daily gain of 0.21 kg/d (SEM = 0.02; Table 1). Similar results have been reported for the effects of castration technique for performance traits in cattle (Bretschneider, 2005; Warnock et al., 2012) and sheep (Melches et al., 2007).

Table 1.

The effect of castration and docking technique and administration of Flunixin Meglumine on growth performance

Item	Treatment1				SEM	P-value2
	RR-FM	RR-S	SC-FM	SC-S
Initial weight, kg	7.40	7.50	7.40	7.10	0.18	0.34
Final weight, kg	15.80	16.30	16.30	17.00	0.35	0.09
Final ADG3, kg/d	0.21	0.23	0.19	0.22	0.02	0.72

¹Treatments include RR-FM (rubber ring-Flunixin Meglumine), RR-S (rubber ring-saline), SC-FM (surgical castration-Flunixin Meglumine), and SC-S (surgical castration-saline).

²  P-value across treatments (n = 42).

³ADG = average daily gain.

Physiological Response

Cortisol Concentration.

Serum cortisol concentration exhibited a treatment × time interaction (P = 0.002; Figure 2). Baseline cortisol concentration was taken at −30 min prior to castration and docking. Serum cortisol concentration peaked at 30 min post-castration and docking, with SC-S exhibiting the highest concentration and RR-S the lowest. At 90 min post-castration, there was a difference between use of FM in the SC group (P = 0.004) with the SC-FM group having lower cortisol concentration when compared to their saline cohorts. As time progressed to 140 min post-treatment, the SC-S remained significantly higher (P < 0.001) than all other treatments, indicating the FM decreased cortisol concentration compared with saline, similar to SC-S in the present trial. Results found for the SC-treated lambs were similar to Molony et al. (1995) in cattle, who reported SC castration was more stressful than RR castration. The surgically castrated with FM administer lambs decrease cortisol levels by 48% compared with SC-S lambs at 140 min.

Figure 2.

The effect of castration and docking technique and administration of Flunixin Meglumine on serum cortisol concentration. Treatments include RR-FM (rubber ring-Flunixin Meglumine), RR-S (rubber ring-saline), SC-FM (surgical castration-Flunixin Meglumine), and SC-S (surgical castration-saline). Means with different superscripts (A–B) differ (P ≤ 0.05) within each time point.

Haptoglobin Concentration.

Haptoglobin did not exhibit a treatment × time interaction (P = 0.99) or time main effect (P = 0.85); however, treatment (P = 0.001) and age (P = 0.004) did influence serum haptoglobin concentration. The RR-S lambs had an increase in haptoglobin concentration (P = 0.001; Table 2) compared with other treatments, with SC-treated lambs receiving FM being intermediate. Our results are similar to results reported by Marini et al. (2017) who observed that flunixin fed lambs had lower levels in haptoglobin concentration than the control. Haptoglobin was numerically but not significant different (P = 0.07) when comparing the RR-saline treatment group; however, further research needs to be conducted to evaluate levels of haptoglobin because research from Marini et al. (2017) reported levels significant linear increase after 12 h.

Table 2.

The effect of castration and docking technique and administration of Flunixin Meglumine on serum haptoglobin concentration

Treatment1	N	Haptoglobin (g/L)	SEM	P-value2
RR-FM	25	1.25	0.21	0.20
RR-S	27	2.89	0.39	<0.01
SC-FM	27	1.48	0.23	0.01
SC-S	25	1.30	0.24	0.16

¹Treatments include RR-FM (rubber ring- Flunixin Meglumine), RR-S (rubber ring-saline), SC-FM (surgical castration-Flunixin Meglumine), and SC-S (surgical castration-saline).

IMPLICATIONS

Castration and docking practices have been utilized by producers for many years in domestic livestock production. Our data suggest that RR castration and docking caused an increase in acute behavioral stress compared with surgical techniques in lambs, but surgical castration and docking may have caused more physiological stress than RR castration and docking. The administration of FM appeared to decrease both behavioral and physiological stress of the castration and docking experience for ram lambs. It was also observed that average daily gain from birth to weaning was not affected by castration and docking technique or through the administration of FM, indicating that use of the analgesic does not indicate production performance outside of health and welfare. Future research should evaluate additional long-term health implications in lambs due to castration and docking techniques and the use of different analgesics.

Conflict of interest statement. None declared.