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Journal of Animal Science - Article

 

 

This article in

  1. Vol. 87 No. 10, p. 3187-3195
     
    Received: Feb 19, 2009
    Accepted: June 23, 2009
    Published: December 5, 2014


    2 Corresponding author(s): bernadette.earley@teagasc.ie
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doi:10.2527/jas.2009-1905

Effects of banding or burdizzo castration of bulls on neutrophil phagocytosis and respiratory burst, CD62-L expression, and serum interleukin-8 concentration1

  1. W. Y. Pang*†,
  2. B. Earley* 2 ,
  3. T. Sweeney,
  4. S. Pirani*,
  5. V. Gath and
  6. M. A. Crowe
  1. Teagasc, Animal Bioscience Research Centre, Dunsany, Co. Meath, Ireland; and
    School of Agriculture, Food Science & Veterinary Medicine, University College Dublin, Belfield, Dublin 4, Ireland

Abstract

The objective was to investigate measures of neutrophil function in response to banding or burdizzo castration of bulls. Thirty-two Holstein-Friesian bulls (14 mo old, 505 ± 7.8 kg of BW) were assigned to 1 of 4 treatment groups: 1) sham-handled control (CON); 2) banding castration alone (BAND); 3) burdizzo castration alone (BURD); or 4) cortisol infusion (CORT) as a further control group. For each group on d –14, 8 animals (2 animals/treatment) were tied up in tie stalls (day of treatment = d 0). At −2, 2, 6, 12, 24, 48, 72, and 144 h relative to treatment time, blood samples were collected for analyses of neutrophil phagocytosis and respiratory burst, neutrophil CD62-L expression, and serum IL-8 concentration. Leukocyte counts, phagocytosis activity, and CD62-L expression were similar (P > 0.05) among the 4 treatment groups. The BURD castrates had greater burst activity compared with BAND castrates (P = 0.048) and CON (P = 0.01) at 72 h posttreatment. The BURD castrates had a greater percentage of granulocyte positive leukocytes (Gr%; P < 0.01) at 2 h posttreatment compared with CON and CORT bulls. The BURD castrates had greater (P < 0.05) Gr% compared with BAND, CON, and CORT animals at 24, 48, and 72 h posttreatment. The BURD and BAND castrates had greater Gr% (P < 0.05) compared with CORT bulls at 144 h posttreatment. In general, BAND, BURD, and CORT did not affect serum IL-8 concentration. Banding castration, BURD, and CORT did not induce leukocytosis, whereas BURD induced a modest neutrophilia. Neutrophil functioning in terms of phagocytosis and respiratory burst and serum IL-8 concentration were not compromised by BAND, BURD, and CORT. These findings indicate nonsurgical castration is unlikely to induce a severe acute systemic inflammatory response in terms of neutrophil function.


INTRODUCTION

The application of rubber rings or tightened latex bands (referred to as banding; Fell et al., 1986; Chase et al., 1995) and the use of a burdizzo instrument to crush the testicular cords (Robertson et al., 1994) are 2 methods routinely used to castrate cattle. Castration by these techniques induces physiological stress indicated by acutely increased plasma cortisol concentrations (Fisher et al., 1996; Pang et al., 2006). Observational studies have described relationships between increasing cortisol concentrations and altered trafficking, phagocytic ability, and l-selectin (CD62-L) expression of blood neutrophils around parturition-related stress (Burton and Kehrli, 1995; Lee and Kehrli, 1998; Waisman et al., 1998; Barton et al., 2000). A profound leukocytosis, marked by neutrophilia, has often been observed in association with elevated glucocorticoids in the circulation (Nakagawa et al., 1998), particularly in cattle (Burton et al., 1995; Preisler et al., 2000). Mechanisms by which tissue injury occurs have been elucidated; these include a premature activation of neutrophils while still in the migration stage, overactivity during tissue remodeling, and the extracellular release of antimicrobial products (Smith, 1994). A combination of reactive oxygen species generated during the neutrophil respiratory burst and the proteolytic enzymes released during degranulation provide the maximum potential for destruction (Weiss, 1989). There are currently no data available in the literature on the effects of banding or burdizzo castration on neutrophil functioning (phagocytosis, respiratory burst, or CD62-L and IL-8).

The hypothesis of this study was that banding or burdizzo castration, which increases cortisol concentrations, would affect phagocytosis and respiratory burst, CD62-L expression in neutrophils, and serum IL-8 concentration. Cortisol infusion was used as a control in the study to mimic the castration-induced secretion of cortisol (Ting et al., 2004).

MATERIALS AND METHODS

All procedures were conducted under experimental license from the Irish Department of Health in accordance with the Cruelty to Animals Act 1876 and the European Communities (Amendment of Cruelty to Animals Act, 1876; SI No. 566/2002) Regulations, 1994.

Animals and Treatments

Thirty-two Holstein-Friesian bulls (14 mo old, 505 ± 7.8 kg of BW) were blocked by BW into 4 time-based blocks and assigned to 1 of 4 treatments (n = 8 bulls/treatment): 1) sham-handled control (CON); 2) banding castration alone (BAND); 3) burdizzo castration alone (BURD); or 4) cortisol infusion (CORT). On d −14, 8 bulls (2 bulls/treatment) were tied into tie stalls per week (day of treatment = d 0). The experiment was completed in 4 wk. Bulls had ad libitum access to water and grass silage.

Experimental Procedures

Each week of the study, 8 bulls were catheterized (jugular vein) on d –1 after the procedure of Ting et al. (2004). The BAND animals were castrated (time = 0 min) with latex bands applied and tensioned to the neck of the scrotum using the Callicrate Smart Bander (No-Bull Enterprises, St. Francis, KS) following instrument guidelines. Burdizzo castration (time = 0 min) was performed in the BURD bulls following the procedure of Fisher et al. (1996). As part of the castration procedure, gentle manual restraint of the bulls was used to facilitate the operator. Bulls that received cortisol were administered with intravenous doses of aqueous 0.2% (wt/vol) hydrocortisone sodium succinate (Solu-Cortef, Pharmacia and Upjohn Ltd., Knowhill, Milton Keynes, UK) solution prepared in sterile 0.9% (wt/vol) saline. Each CORT bull received 16 mg of hydrocortisone at 0 h (time of castration in BAND and BURD groups), 8 mg at 0.25 and 0.5 h, and 4 mg at 0.75 and 1 h, followed by 2 mg at 1.5, 2, 3, 3.5, 5, 5.5, 6, 7, and 8 h. After each hydrocortisone injection, 5 mL of sterile saline was administered to flush the catheters. Animals in BAND, BURD, and CON groups were given an equivalent volume of sterile 0.9% saline via the catheter at the same time CORT animals received hydrocortisone. Animals in the CON group were sham handled for a period equivalent to the time required to perform the castration procedure in the castration groups.

At −2, 2, 6, 12, 24, 48, 72, and 144 h relative to treatment, heparin anti-coagulated blood samples (approximately 6 mL) were collected for neutrophil phagocytosis and respiratory burst assays and for cortisol assay; acid-citrate-dextrose blood samples were collected for granulocyte (Gr-1) and CD62-L flow cytometric assay; serum samples were collected for IL-8 assay. Two further heparinized blood samples were collected at 0.5 and 1 h relative to the time of castration for cortisol measurement. Plasma and serum samples were collected after centrifugation at 1,600 × g at 8°C for 15 min and stored at −20°C until analyzed. The other samples were processed immediately after collection.

Assay Procedures

Plasma cortisol concentrations were determined using a commercially available RIA kit (Corti-cote, ICN Pharmaceuticals, Orangeburg, NY) adapted and validated for bovine plasma (Fisher et al., 1996). The intraassay CV (n = 4) for pooled quality control serum samples containing 1.5 and 16.4 ng/mL of cortisol were 19.0 and 8.3%, respectively; the interassay CV (n = 3) for the same samples were 17.2 and 7.6%. Standards accompanying the kit were used in determining the intra- and interassay CV.

Immunostaining and fluorescence-activated flow cytometry were used to analyze neutrophil surface CD62-L and percentage of neutrophils. Immunostaining was performed in 96-well microtiter plates (VWR Scientific, Chicago, IL) using a whole blood assay modified by Weber et al. (2001). Briefly, a 1-mL aliquot of whole blood was pipetted into a 5-mL test tube (Sarstedt, Nümbrecht, Germany). Tubes were centrifuged at 250 × g for 5 min at 4°C. After aspiration of supernatants, 3 mL of BD Facs lysing solution (BD Biosciences, Oxford, UK) was added into each tube to lyse red blood cells for 10 min at room temperature. The remaining leukocytes were suspended in 1.5 mL of sheath fluid (Coulter Isoton II Diluent, Beckman Coulter UK Ltd., High Wycombe, UK) and counted using a Z1 Coulter Particle Coulter (Beckman Coulter, LABPLAN Ltd., Galway, Ireland); the counts were recorded as white blood cells (WBC). One hundred-microliter aliquots of cell suspension (1 × 106 cells) were added to a series of 3 wells (per sample), one that received 100 μL of PBS (pH 7.2) containing 0.01% BSA (PBS-BSA), one that received 100 μL of anti-G1 monoclonal antibody (clone MM20A, IgG1, VMRD, Pullman, WA), and the third that received 100 μL of anti-CD62-L monoclonal antibody (clone BAQ92A, IgG1, VMRD). Both monoclonal antibodies were diluted to 14 μL/mL in PBS-BSA before addition to the plate wells. Plates were sealed and incubated for 30 min at 4°C. After the incubation, 100 μL of cold PBS-BSA was added to each well and plates centrifuged at 250 × g for 5 min at 4°C. Using the same centrifugation step, all wells were washed twice using 200 μL of cold PBS-BSA per well, with gentle aspiration of supernatants between washes. After washes, 100 μL of PE-conjugated goat-anti-mouse IgG1 antibody (1:400 diluted in PBS-BSA; Caltag Laboratories, Burlingame, CA) was added and plates sealed and incubated for 15 min at 4°C. After addition of 100 μL of cold PBS-BSA to each well, plates were centrifuged as before and supernatants aspirated. After 2 washes and centrifugations, the cell pellet was resuspended in 200 μL of sheath fluid and then further diluted in 800 μL of sheath fluid for immediate flow cytometric acquisition of neutrophil Gr-1 and CD62-L expression data on a Partec CyFlow Flow Cytometer (Partec Gmbh, Munster, Germany).

The percentage of neutrophils was determined by calculating the proportion of the granulocyte-positive (Gr-1) stained cells in the gate set for neutrophils (Burton and Kehrli, 1995) and the percentage of gated cells (the neutrophil gate) in the whole leukocyte population. Surface CD62-L expression was recorded as percentage of CD62-L staining positive cells and the mean fluorescence intensity in the gated neutrophils in which at least 90% of the cells were Gr-1 positive. The PBS-BSA-treated leukocytes were used to set a threshold for positive staining for the Gr-1 and CD62-L analysis. Data were collected from 30,000 cells in each assay, and analysis was done using FloMax software (Partec GmbH).

Phagocytosis and respiratory burst were analyzed using the Phagotest kit and Bursttest (Phagoburst) kit (Orpegen Pharma, Heidelberg, Germany) with whole, heparinized blood following the manufacturer’s instructions with the modifications by Kampen et al. (2004), on a Partec CyFlow Flow Cytometer. Samples were analyzed in duplicate. Data were collected from 30,000 cells per sample, and analysis was done using FloMax software. The results of the phagocytosis assay are presented as percentage of phagocytosing cells and the mean fluorescence intensity in the gate set for neutrophils. The results of the Bursttest assay are presented as percentage of phagobursting cells and the mean fluorescence intensity in the neutrophil gate.

Serum IL-8 concentrations were measured using a human IL-8 DuoSet ELISA (R&D Systems, Abingdon, UK) following the manufacturer’s instructions. Each plate had standards and samples that were run in duplicate. The optical density values were obtained using a TECAN SUNRISE absorbance reader (TECAN Austria GmbH, Grödig, Austria). The reader was set to 450 nm, with a reference wavelength of 570 nm. The standard curves (4 parameter logistic curve-fit) were generated and IL-8 concentrations automatically calculated by using WIACALC software (Pharmacia-LKB, Uppsala, Sweden). To validate the assay for bovine serum samples, a spike and recovery test was carried out according to an experimental protocol (R&D Systems Europe). The recovery of spiked IL-8 standard was 92.1% (should be in the range of 80 to 120%), and the assay had linearity for bovine serum samples. Two bovine serum samples were included in each ELISA assay as a quality control. The intraassay CV (n = 2) for samples containing 30.3 and 111.5 pg/mL of IL-8 were 6.3 and 9.8%, respectively; the interassay CV (n = 7) for the same samples were 8.4 and 7.5%, respectively.

Statistical Analyses

All statistical analyses were performed using SAS (SAS Institute Inc., Cary, NC). The log10 of WBC number and all other data were analyzed using repeated measure analysis of SAS (PROC MIXED). The effects of treatment, time, and treatment × time interactions were listed in the model statement. There was no treatment × replicate interaction; thus, it was not included in the statistical model. A matched-pair t-test was used to detect treatment differences. Pretreatment values (−2 h) were included as covariates for serum IL-8 concentrations because a significant pretreatment difference was found among treatments. A probability of P < 0.05 was chosen as the level of significance for the statistical tests. Data are represented as least squares means (Lsmeans ± SEM), and differences between LSmeans for the effect of treatment by bleed were tested using the PDIFF option in SAS.

RESULTS

Plasma Cortisol

There was an effect of time (P < 0.0001), treatment (P < 0.0001), and treatment × time interaction (P < 0.0001) for plasma cortisol concentrations. The AUC data were calculated, but no differences were found among treatments. Cortisol concentrations were not different from baseline throughout the experimental period in CON bulls (Table 1). After BAND, BURD, and CORT, cortisol concentration increased (P < 0.05) at 0.5 and 1.0 h after treatment. The CORT bulls had greater cortisol concentrations than castrated animals at 0.5 h posttreatment; BURD bulls had a secondary peak 24 to 48 h after castration.

Phagocytosis Assay

There was an effect of time (P = 0.0003) but no effect of treatment (P = 0.80) or a treatment × time interaction (P = 0.84) for percentage of phagocytosing cells. The values were similar (P ≥ 0.91) among treatment groups at 2 h before and after treatment at all time points evaluated up to 144 h (Table 2).

There was an effect of time (P < 0.0001) but no effect of treatment (P = 0.72) or a treatment × time interaction (P = 0.36) for the mean fluorescent intensity. The mean fluorescence intensity of phagocytosing neutrophils was similar (P > 0.41) among all treatment groups at all time points examined (Table 3).

Respiratory Burst Assay

There was an effect of time (P < 0.0001) but no effect of treatment (P = 0.57) or a treatment × time interaction (P = 0.09) for respiratory burst activity. The percentage of phagobursting cells was similar (P ≥ 0.20) among treatment groups at all time points after treatment except at 72 h. At 72 h, BURD castrates had greater percentage of phagobursting cells compared with BAND castrates (P = 0.048) and CON (P = 0.01; Table 4).

There was a treatment × time interaction (P = 0.03) but no effect of treatment (P = 0.09) or time (P = 0.10) for the mean fluorescent intensity. The mean fluorescence intensity of neutrophils that performed respiratory burst was similar (P > 0.12) among all treatments at all time points (Table 5).

Gr-1 Assay

There was an effect of time (P = 0.009), treatment (P = 0.006), and a treatment × time interaction (P = 0.03) for the percentage of Gr-1 positive cells. The percentage of Gr-1 positive cells was increased (P ≤ 0.04) in BURD compared with CON at 2, 24, and 72 h postcastration, and was greater (P ≤ 0.04) than that for CORT bulls at 2, 24, 48, 72, and 144 h posttreatment. The BURD castrates tended (P = 0.058) to have a greater percentage of Gr-1-positive cells than CON animals at 144 h posttreatment (Table 6). Otherwise, the percentage of Gr-1-positive cells was similar among treatment groups.

There was an effect of time (P < 0.0001), and a treatment × time interaction (P = 0.04), but no effect of treatment (P = 0.09), for mean fluorescence intensity of Gr-1 positive neutrophils. After treatment, mean fluorescence intensity was less in BAND castrates compared with BURD castrates (P = 0.009), and CON bulls (P = 0.02), at 2 h posttreatment (Table 6). Mean fluorescence intensity was also less at 48 h posttreatment in BAND castrates compared with BURD castrates (P = 0.02), CON bulls (P = 0.004), and CORT bulls (P = 0.004). At 144 h posttreatment, mean fluorescence intensity was less in BURD castrates than in CON (P = 0.04), and CORT (P = 0.02) bulls (Table 7).

CD62-L Assay

There was an effect of time (P = 0.002), but no effect of treatment (P = 0.63) or a treatment × time interaction (P = 0.21) for the percentage of CD62-L-positive cells. The percentage of CD62-L positive cells was similar (P ≥ 0.35) among treatment groups at 2 h before and after treatment at all time points evaluated up to 144 h (Table 8).

There was an effect of time (P = 0.02), but no effect of treatment (P = 0.85) and no treatment × time interaction (P = 0.34), for mean fluorescence intensity of CD62-L-positive neutrophils. The mean fluorescence intensity of CD62-L-positive neutrophils was similar (P ≥ 0.29) among all treatment groups at these time points (Table 9).

Serum IL-8 Concentration

There was an effect of time (P = 0.0001) and a treatment × time interaction (P = 0.01), but no effect of treatment (P = 0.41), for serum IL-8 concentrations (Table 10). There was no difference among BURD castrates, CON, and CORT bulls, when the pretreatment concentrations were included as covariates. Serum IL-8 concentrations were less in BURD castrates than in CON (P = 0.026) and CORT (P = 0.035) bulls at 2 h posttreatment, whereas serum IL-8 concentrations were similar between BAND and BURD castrates. Serum IL-8 concentrations were less (P < 0.05) in BAND and BURD castrates, and in CON, than in CORT bulls at 48 h posttreatment.

WBC

There was an effect of time (P = 0.005) and a treatment × time interaction (P = 0.05), but no effect (P > 0.05) of treatment, for WBC number. The WBC number in bulls assigned to BURD, BAND, CORT, and CON was similar (P ≥ 0.24) at all times except at 12 h, at which BURD bulls had greater (P = 0.04) WBC than BAND bulls (data not shown).

DISCUSSION

Studies on parturition (Burton and Kehrli, 1995; Weber et al., 2001) indicated that cortisol-associated responses influence neutrophil function. Because BAND and BURD are known to increase circulating concentrations of cortisol (Fisher et al., 1996; Pang et al., 2006), our objective in the present experiment was to determine whether castration would affect neutrophil function. A CORT model was used to examine the direct effects of cortisol on neutrophil function. The cortisol response profile of BURD calves was successfully mimicked through hydrocortisone infusion, but no effect was found of castration or cortisol mimic on leukocyte numbers after treatment from d 1 to 35. However, infusion of pharmacological dosages of cortisol resulted in a transient suppression of IFN-γ production, as well as increased plasma glucose, insulin, and GH concentrations (Ting et al., 2004). In the present study, greater dosages of hydrocortisone were injected in an attempt to induce a greater plasma cortisol concentration so as to investigate the effect of cortisol on neutrophil function. A greater peak concentration was achieved by hydrocortisone injection than that induced by BAND or BURD, thus indicating that, at physiological concentrations induced in these experiments, cortisol per se does not directly affect neutrophil function. It is likely that the lack of a severe inflammatory response in CORT bulls compared with castrated bulls may have contributed to a return to basal cortisol concentrations at 6 h in the CORT group.

There was no difference in WBC among castrates or CORT and CON bulls. This is in agreement with Ting et al. (2003b, 2004), but contrary to the reports of Chase et al. (1995), Ting et al. (2003a), and Pang et al. (2006). Burdizzo castration induced a greater percentage of granulocytes in the leukocyte population compared with control bulls. Similarly, in previous studies, a greater neutrophil percentage was observed on d 2 after BURD (Murata, 1997). However, Ting et al. (2003b) and Pang et al. (2006) found no difference in granulocyte percentage (GR%) after BURD. It is unclear why no neutrophilia was found in BAND castrates because there was no difference in the cortisol response profile between BAND and BURD animals. Hydrocortisone infusion had no effect on the percentage of granulocytes (GR%) in the leukocyte population compared with controls. It may be that increased cortisol concentration is not the only reason for neutrophilia after castration. In contrast, severe glucocorticoid challenge by dexamethasone administration has been reported to cause neutrophilia (Weber et al., 2001). There was also a decrease in mean Gr-1 expression per cell indicated by the mean Gr-1 fluorescence intensity in BAND and BURD castrates. The reason for such a decrease is not clear, and this may warrant further exploration into the functions of Gr-1 expression in neutrophils.

There was no difference in the percentage of cells expressing CD62-L or the mean CD62-L expression level per cell (indicated by the mean CD62-L fluorescence intensity) among the 4 treatment groups. It seems that this is the first time CD62-L has been studied in the bovine castration model. The results presented indicate BAND or BURD would not affect the capacity of neutrophils to adhere to blood vessel endothelia, and hydrocortisone infusion on its own would not affect neutrophil trafficking. However, physiological and pharmacological stressors such as parturition and dexamethasone injection result in gradual and chronic downregulation of CD62-L on the surface of blood neutrophils in cattle (Weber et al., 2001). A decreased surface expression of CD62-L in neutrophils was also observed after transportation (Yagi et al., 2004).

There was no difference in neutrophil phagocytosis among the 4 treatment groups. Similarly, no difference in the percentage of phagocytosing neutrophils was observed between ACTH-administered and control sheep (Paltrinieri et al., 2002). In the present study, there was no difference in neutrophil respiratory burst among the 4 treatment groups. These findings indicate that those 2 functions are not likely to be affected by castration (BAND or BURD) or hydrocortisone infusion. There are no previous reports in the literature regarding phagocytosis and respiratory burst after castration. In contrast, there are a few reports showing decreased phagocytosis in horses after transportation (Raidal et al., 1997) and after ACTH injection in dairy cows (Paape et al., 1981). Increased phagocytosis was reported in exercise-induced stress in humans (Ortega, 2003), after minor surgical stress in children (Romeo et al., 2002), and by foot-shock stress in rats (Shurin et al., 1994). Whereas increased oxidative burst was observed in stress caused by minor surgery in children (Romeo et al., 2002), decreased superoxide production was found during stress in calves (Henricks et al., 1987). Furthermore, unchanged or enhanced oxidative burst activity has been reported after transportation of horses (Raidal et al., 1997). Perhaps these findings altogether indicate that there is no nonspecific stress response that applies to all stressors, in general agreement with Mason (1975), who demonstrated that different stressors elicit very different types of biological response.

In general, BAND, BURD, and CORT did not affect serum IL-8 concentration. This finding may indicate that a mild degree of localized injury or stress after BAND, BURD, and CORT had no effect on serum IL-8 concentration. Interleukin-8 is produced by many cell types in response to inflammatory stimuli and plays a pivotal role in neutrophil recruitment (Sozzani et al., 2005). In the present study, there was no difference among BURD castrates, CON, and CORT bulls, when the pretreatment concentrations were taken as covariates in the statistical analysis, because the pretreatment values tended to be different comparing BURD animals with CON and CORT bulls. Similarly, plasma IL-8 concentration remained unchanged after surgical stress (laparoscopic and open gastric bypass; Nguyen et al., 2002). However, an increase in the concentration of IL-8 was observed at tension (mesial) sites at 1 and 24 h and 6 and 10 d after tooth movement (Tuncer et al., 2005). There would likely be an IL-8 concentration gradient between blood and inflamed local tissue, so neutrophils could be chemo-attracted to inflammatory sites. In contrast, surgical cardiac stress elicits an increase in IL-8 serum concentration (Alcaraz et al., 2005). In conclusion, BAND, BURD, and CORT did not induce leukocytosis, whereas BURD induced a modest neutrophilia; neutrophil functioning in terms of phagocytosis and respiratory burst and serum IL-8 concentration were not compromised by BAND, BURD, or CORT. These findings indicate nonsurgical castration is unlikely to induce a severe acute systemic inflammatory response in terms of neutrophil functioning. The results of the present study indicate that immunosuppression may not be directly due to malfunctioning of neutrophils. Further studies are needed to elucidate the underlying mechanism of immune suppression postcastration.

1 This study was supported by a Teagasc Walsh Fellowship to W. Y. Pang. The authors thank D. Prendiville, J. Larkin, M. Murray, and B. MacDonnell (Teagasc, Animal Bioscience Research Centre) and N. Hynes (Veterinary Sciences Centre at University College Dublin) for excellent technical help and assistance during the study. The authors also acknowledge the help and the excellent support of graduate student V. Bzik from National University of Ireland Maynooth, Co. Kildare, Ireland. Many thanks are due to the farm staff at Teagasc, Grange Research Centre, for care and management of the animals.


View Full Table | Close Full ViewTable 1.

Mean plasma cortisol concentration (least squares means ± SEM; ng/mL) of bulls banding castrated (BAND), burdizzo castrated (BURD), left untreated (CON), or injected with cortisol (CORT)

 
–2 6.9 ± 2.63 8.0 ± 1.18 11.1 ± 7.04 7.5 ± 1.35
0.5 6.7 ± 0.75b 24.7 ± 3.88a 32.2 ± 2.08a 61.2 ± 7.67c
1 5.0 ± 1.27c 27.2 ± 4.14ab 21.5 ± 2.05a 31.0 ± 3.93b
2 11.2 ± 3.00 22.7 ± 5.25 10.0 ± 3.36 20.1 ± 7.49
6 5.1 ± 2.05 2.6 ± 1.39 12.1 ± 6.47 6.4 ± 2.06
12 5.4 ± 1.40a 8.0 ± 3.26ab 8.5 ± 3.34ab 16.0 ± 4.77b
24 4.1 ± 1.88a 3.5 ± 1.33a 13.5 ± 4.42b 8.0 ± 3.45ab
48 7.0 ± 2.23b 11.1 ± 2.73ab 14.7 ± 3.08a 8.6 ± 2.12ab
72 8.4 ± 3.27 8.7 ± 2.92 10.1 ± 2.22 8.4 ± 1.61
144 7.7 ± 2.82 12.0 ± 2.75 7.8 ± 1.42 11.2 ± 2.21
a–cLeast squares means within a row that do not have common superscripts differ (P < 0.05); the P-value for the treatment × time interaction was P < 0.0001.


View Full Table | Close Full ViewTable 2.

The percentage of granulocyte cells that underwent phagocytosis (least squares means ± SEM) of bulls castrated by banding (BAND), burdizzo (BURD), or left untreated (CON), or injected with cortisol (CORT)1

 
–2 53.3 ± 4.9 50.3 ± 5.0 53.1 ± 5.1 55.4 ± 5.0 0.91
2 52.1 ± 3.9 57.2 ± 4.0 55.2 ± 3.9 56.4 ± 4.0 0.89
6 49.1 ± 5.0 48.1 ± 5.1 52.6 ± 5.0 52.4 ± 5.1 0.81
12 52.8 ± 3.1 56.0 ± 3.1 52.8 ± 3.3 54.5 ± 3.1 0.92
24 52.0 ± 3.3 51.7 ± 3.2 56.6 ± 3.1 59.3 ± 3.1 0.27
48 56.4 ± 2.9 55.4 ± 3.0 59.2 ± 2.9 58.4 ± 3.0 0.71
72 48.7 ± 3.1 51.9 ± 3.0 51.8 ± 3.0 51.6 ± 3.1 0.89
144 53.5 ± 4.3 56.0 ± 4.3 57.5 ± 4.2 51.6 ± 4.3 0.88
1The P-value for the treatment × time interaction was P = 0.84.


View Full Table | Close Full ViewTable 3.

The mean fluorescence intensity (PhagoMean) of granulocyte cells that underwent phagocytosis (least squares means ± SEM) of bulls castrated by banding (BAND), burdizzo (BURD), or left untreated (CON), or injected with cortisol (CORT)1

 
–2 8.6 ± 0.6 9.2 ± 0.6 9.1 ± 0.5 9.9 ± 0.7 0.57
2 9.7 ± 0.4 9.3 ± 0.3 9.2 ± 0.3 9.0 ± 0.4 0.97
6 9.4 ± 0.4 8.9 ± 0.3 8.4 ± 0.3 9.1 ± 0.4 0.41
12 10.0 ± 0.3 9.7 ± 0.3 9.4 ± 0.4 9.4 ± 0.4 0.95
24 10.3 ± 0.5 10.2 ± 0.4 9.6 ± 0.4 10.4 ± 0.4 0.51
48 10.0 ± 0.5 10.0 ± 0.4 9.4 ± 0.4 9.8 ± 0.5 0.72
72 9.7 ± 0.6 9.3 ± 0.5 9.0 ± 0.5 9.5 ± 0.6 0.74
144 9.4 ± 0.4 8.6 ± 0.3 9.3 ± 0.3 8.7 ± 0.4 0.75
1The P-value for the treatment × time interaction was P = 0.36.


View Full Table | Close Full ViewTable 4.

The percentage of granulocyte cells that underwent respiratory burst (Burst%; least squares means ± SEM) of bulls castrated by banding (BAND), burdizzo (BURD), or left untreated (CON), or injected with cortisol (CORT)

 
–2 4.4 ± 1.5 4.4 ± 1.6 6.1 ± 1.6 6.7 ± 1.5 0.63
2 7.9 ± 1.0 6.1 ± 1.0 6.2 ± 0.9 5.7 ± 0.9 0.83
6 6.3 ± 1.1 6.0 ± 1.1 6.3 ± 1.0 6.6 ± 1.1 0.65
12 9.8 ± 1.5 7.8 ± 1.5 7.2 ± 1.6 5.3 ± 1.6 0.88
24 3.6 ± 1.2 4.9 ± 1.1 4.9 ± 1.0 4.6 ± 1.1 0.64
48 5.0 ± 1.2 4.6 ± 1.2 6.7 ± 1.1 7.1 ± 1.2 0.20
72 4.6 ± 1.1a 5.4 ± 1.1a 8.2 ± 1.0b 5.3 ± 1.1ab 0.07
144 4.2 ± 1.0 4.6 ± 1.0 5.4 ± 0.9 4.6 ± 1.0 0.74
a,bLeast squares means within a row that do not have common superscripts differ (P < 0.05); the P-value for the treatment × time interaction was P = 0.09.


View Full Table | Close Full ViewTable 5.

The mean fluorescence intensity (BurstMean) of granulocyte cells that underwent respiratory burst (least squares means ± SEM) of bulls castrated by banding (BAND), burdizzo (BURD), or left untreated (CON), or injected with cortisol (CORT)1

 
–2 2.6 ± 0.2 2.2 ± 0.1 2.3 ± 0.1 2.7 ± 0.2 0.15
2 2.7 ± 0.1 2.7 ± 0.2 2.5 ± 0.1 2.5 ± 0.2 0.30
6 2.6 ± 0.1 2.7 ± 0.2 2.6 ± 0.1 2.5 ± 0.2 0.76
12 2.6 ± 0.1 2.8 ± 0.1 2.6 ± 0.2 2.4 ± 0.2 0.84
24 2.7 ± 0.5 2.4 ± 0.5 2.8 ± 0.4 3.6 ± 0.4 0.47
48 2.5 ± 0.1 2.9 ± 0.2 2.6 ± 0.1 3.0 ± 0.2 0.13
72 2.4 ± 0.1 2.9 ± 0.2 2.7 ± 0.1 2.7 ± 0.2 0.59
144 2.4 ± 0.1 2.5 ± 0.2 2.5 ± 0.1 2.8 ± 0.2 0.14
1The P-value for the treatment × time interaction was P = 0.03.


View Full Table | Close Full ViewTable 6.

The percentage of granulocyte-positive cells in leukocytes (least squares means ± SEM) of bulls castrated by banding (BAND), burdizzo (BURD), or left untreated (CON), or injected with cortisol (CORT)

 
–2 28.4 ± 3.73 26.8 ± 4.19 33.0 ± 6.37 24.8 ± 4.11 0.65
2 25.3 ± 3.05b 36.9 ± 4.53ab 48.3 ± 3.70a 26.9 ± 6.16b 0.004
6 30.5 ± 5.39 36.9 ± 6.51 35.6 ± 4.74 37.7 ± 4.14 0.77
12 26.9 ± 5.21 30.5 ± 2.95 36.5 ± 7.43 25.4 ± 4.63 0.46
24 26.0 ± 6.76a 26.3 ± 3.61a 45.5 ± 6.63b 23.5 ± 5.08a 0.03
48 32.7 ± 5.53ab 28.9 ± 3.66a 47.4 ± 6.88b 29.3 ± 5.56a 0.08
72 27.1 ± 5.61a 26.7 ± 6.11a 43.3 ± 5.80b 20.8 ± 2.60a 0.03
144 24.0 ± 2.95ab 35.3 ± 5.50a 38.6 ± 6.38a 20.4 ± 4.28b 0.04
a,bLeast squares means within a row that do not have common superscripts differ (P < 0.05); the P-value for the treatment × time interaction was P = 0.03.


View Full Table | Close Full ViewTable 7.

The mean fluorescence intensity of granulocyte-positive neutrophils (least squares means ± SEM) of bulls castrated by banding (BAND), burdizzo (BURD), or left untreated (CON), or injected with cortisol (CORT)

 
–2 7.1 ± 0.63 5.9 ± 0.64 7.3 ± 0.55 7.3 ± 0.59 0.31
2 6.0 ± 0.50b 4.8 ± 0.18a 6.2 ± 0.31b 5.5 ± 0.37ab 0.04
6 6.4 ± 0.82 5.2 ± 0.62 6.6 ± 0.57 6.6 ± 0.37 0.31
12 6.5 ± 0.50 5.9 ± 0.48 6.3 ± 0.64 6.7 ± 0.51 0.71
24 7.2 ± 0.98 5.8 ± 0.43 6.8 ± 0.55 6.7 ± 0.50 0.50
48 6.6 ± 0.58b 4.3 ± 0.51a 6.2 ± 0.52b 6.6 ± 0.49b 0.01
72 6.1 ± 0.87 5.5 ± 0.62 5.65 ± 0.75 6.2 ± 0.42 0.85
144 7.4 ± 0.53b 6.5 ± 0.42ab 6.0 ± 0.47a 7.5 ± 0.44b 0.07
a,bLeast squares means within a row that do not have common superscripts differ (P < 0.05); the P-value for the treatment × time interaction was P = 0.04.


View Full Table | Close Full ViewTable 8.

The percentage of CD62-L-positive neutrophils (least squares means ± SEM) of bulls castrated by banding (BAND), burdizzo (BURD), or left untreated (CON), or injected with cortisol (CORT)1

 
–2 92.0 ± 1.7 94.5 ± 1.8 92.4 ± 1.7 92.4 ± 1.7 0.74
2 89.3 ± 5.7 93.6 ± 5.8 96.8 ± 5.6 83.6 ± 5.6 0.35
6 78.8 ± 6.1 85.3 ± 6.1 79.4 ± 6.0 91.6 ± 6.0 0.44
12 89.4 ± 6.0 89.4 ± 6.1 81.0 ± 6.4 89.5 ± 6.0 0.65
24 75.8 ± 7.3 88.4 ± 7.5 89.1 ± 7.9 86.6 ± 7.3 0.47
48 93.0 ± 5.9 93.5 ± 5.9 95.1 ± 5.8 83.6 ± 5.8 0.48
72 81.2 ± 6.5 82.1 ± 6.6 90.9 ± 6.5 81.7 ± 6.4 0.65
144 92.0 ± 7.3 82.5 ± 6.9 86.8 ± 6.9 80.6 ± 6.9 0.79
1The P-value for the treatment × time interaction was P = 0.21.


View Full Table | Close Full ViewTable 9.

The mean fluorescence intensity of CD62-L-positive neutrophils (least squares means ± SEM) of bulls castrated by banding (BAND), burdizzo (BURD), or left untreated (CON), or injected with cortisol (CORT)1

 
–2 3.8 ± 0.3 4.0 ± 0.3 3.9 ± 0.2 4.3 ± 0.3 0.66
2 4.1 ± 0.2 4.1 ± 0.3 4.2 ± 0.2 3.9 ± 0.1 0.98
6 4.3 ± 0.3 4.2 ± 0.2 4.1 ± 0.2 3.9 ± 0.2 0.97
12 4.6 ± 0.3 4.1 ± 0.3 4.5 ± 0.4 4.2 ± 0.3 0.89
24 4.8 ± 0.4 4.8 ± 0.4 4.1 ± 0.5 4.5 ± 0.5 0.62
48 4.5 ± 0.4 3.8 ± 0.3 4.4 ± 0.3 3.8 ± 0.4 0.69
72 4.9 ± 0.5 4.3 ± 0.4 4.2 ± 0.4 4.6 ± 0.5 0.77
144 5.3 ± 0.5 4.1 ± 0.4 5.2 ± 0.4 4.5 ± 0.4 0.29
1The P-value for the treatment × time interaction was P = 0.34.


View Full Table | Close Full ViewTable 10.

The mean serum IL-8 concentration (least squares means ± SEM; pg/mL) of bulls banding castrated (BAND), burdizzo castrated (BURD), left untreated (CON), or injected with cortisol (CORT)

 
–2 78.4 ± 11.18a 55.0 ± 12.85a 44.6 ± 8.52b 78.9 ± 14.96a
2 79.9 ± 12.46b 49.7 ± 13.91ab 41.4 ± 11.53a 77.6 ± 11.53b
6 70.5 ± 7.20 52.3 ± 10.79 48.9 ± 13.65 70.0 ± 13.46
12 63.1 ± 11.39 42.5 ± 8.91 52.0 ± 13.70 54.0 ± 14.11
24 55.3 ± 17.90 60.1 ± 9.64 44.0 ± 13.36 62.1 ± 16.44
48 53.5 ± 9.67ab 65.0 ± 16.19ab 43.0 ± 16.37a 88.7 ± 8.74b
72 70.8 ± 8.68 61.7 ± 10.11 61.9 ± 17.04 72.7 ± 9.90
144 96.2 ± 16.03 67.4 ± 17.85 86.9 ± 19.95 80.9 ± 11.01
a,bLeast squares means within a row that do not have common superscripts differ (P < 0.05); the P-value for the treatment × time interaction was P = 0.01.

 

References

Footnotes