Impact of pre-slaughter stress on hematological and biochemical parameters in non-ambulatory non-injured pigs
Article information
Abstract
In Brazil, more than 57 million pigs are transported annually to slaughterhouses, and approximately 0.02% (approximately 11,500 pigs) experience severe fatigue, indicating compromised animal welfare. This study assessed the hematological and biochemical parameters in pigs during the pre-slaughter period, comparing animals with non-ambulatory, non-injured (NANI) syndrome with those without the condition upon arrival at the slaughterhouse. Blood samples were collected from 71 pigs (36 NANI and 35 non-NANI) to assess the indicators of stress. Both groups showed mean values for albumin, creatine phosphokinase (CK)-total, CK-MB, cortisol, fibrinogen, and leukocyte, segmented leukocyte, and erythrocyte counts above the established physiological reference ranges. The NANI pigs, however, exhibited significantly higher levels of CK-total (109,573.3 vs. 37,103.33 U/L), cortisol (11.83 vs. 8.94 mcg/dL), and lactate (15.48 vs. 11.85 mg/dL), as well as increased erythrocyte-related parameters (15.23 vs. 13.96 g/dL for hemoglobin; 44.93% vs. 41.96% for hematocrit), lymphocytes (39.5% vs. 31.61%), fibrinogen (677.78 vs. 444.44 mg/dL), and segmented cells (59.95% vs. 46.78%). Conversely, non-NANI pigs showed higher glucose levels (101.22 vs. 79.33 mg/dL). These findings suggest that pre-slaughter handling procedures, including loading, transport, and unloading, induce considerable physiological stress, particularly in pigs affected by NANI syndrome.
Introduction
Transporting pigs to slaughterhouses is a complex and stressful process for the animals. Factors such as fasting, loading conditions, animal density, journey duration, and environmental conditions, including temperature, humidity, and thermal variations, contribute to this stress [1]. This stress negatively impacts animal welfare [2] and meat quality, affecting pH, color, and water-holding capacity [3].
Loading pigs for transport to a slaughterhouse is the most critical phase, often causing heart rates to surge to 160 beats per minute and increasing salivary, blood cortisol, and lactate levels [4,5]. Poor management during this stage can worsen injuries and clinical signs and symptoms, including reluctance to walk, lameness, or decubitus in non-ambulatory injured pigs. Non-ambulatory non-injured (NANI) pigs show fatigue and inertia, leading to sternal decubitus, and possibly death during transport [3,6].
The hematological and biochemical parameters are effective tools for identifying and quantifying stress in animals [4,7]. Key measures include the serum cortisol concentration, serum creatine phosphokinase (CK) activity, and plasma lactate levels [4,5,8]. In addition, measuring the serum albumin concentration, performing blood counts, and determining the serum CK-MB activity can provide insights into dehydration [9], acute or chronic anemia [10], and cardiac health [11].
Given the limitations of current laboratory tools for assessing stress associated with pre-slaughter handling, particularly in identifying NANI or non-NANI (asymptomatic pigs), and the need to minimize such stress through proper welfare practices, this study compared the hematological and biochemical parameters in NANI pigs with those of non-NANI pigs showing no clinical signs of fatigue at unloading. This comparison aims to improve the understanding of the metabolic changes associated with these conditions and to identify strategies to reduce stress.
Materials and Methods
Ethics statements
The Ethics Committee for the Use of Animals at the State University of Londrina approved all research procedures in this study (protocol number: 18199.2018.92).
Animals, facilities, and management
The experiments were conducted at a slaughterhouse certified by the Federal Inspection Service, located in the northern region of Paraná, Brazil (23°24'54.8" S 51°04'73.1" W).
Seventy-one commercial pigs were assessed, with 35 and 36 classified as non-NANI and NANI, respectively. The NANI group comprised 14 surgically castrated males, 9 immunocastrated males, and 13 females, whereas the non-NANI group contained 14 surgically castrated males, 9 immunocastrated males, and 12 females. The study used a completely randomized 2 × 3 factorial design (NANI and non-NANI: surgically castrated, immunocastrated, and female).
Pigs exhibiting clinical signs such as wheezing, skin discoloration, muscle tremors, movement paralysis, and abnormal vocalization were classified as NANI [12]. Pigs without these signs were categorized as non-NANI and considered asymptomatic. At unloading, the non-NANI pigs were selected after the NANI pigs were identified in the batch, ensuring both groups came from the same truck. The NANI pigs were then placed in separate resting pens from the non-NANI pigs.
The pigs in this study came from commercial farms with standardized feeding protocols, management practices, and loading facility layouts. They had an average live weight of 121 kg (± 13.51 kg) and were transported 460 km to the slaughterhouse, a journey that took approximately 8 hours, followed by a 3-hour rest at the slaughterhouse. The trucks used were three-story models with a total area of 112.5 m2 and a live-weight density of 220 kg/m2.
The pigs were marked with a stick (AgroMark; Agro-Pecuária Industrial Ltd., Brazil) for tracking until they entered the restrainer, where blood samples were collected.
Animal management, blood collection, and analysis
All pre-slaughter handling procedures complied with Brazilian legislation [13,14] on the Technical Regulation for Pre-Slaughter Handling and Humane Slaughter, as well as the stunning methods authorized by the Ministry of Agriculture, Livestock, and Supply.
Pigs were housed in resting pens at a density of 0.6 m2 per animal for 3 hours before slaughter, with unrestricted access to water. The non-NANI pigs were moved to the stunning facility in groups of 10 to 15, guided through a single-file chute using rattle paddles and handling boards. Electric prods were used where needed, such as when pigs hesitated or refused to move. The NANI pigs were transported to emergency slaughter in a wheelbarrow.
Before exsanguination, the pigs were stunned using electronarcosis. A 250 V electrical current was applied bilaterally behind the ears (temporal fossae), and an additional 120 V was delivered to the chest (between the 4th and 5th left intercostal spaces), at a 1 A current intensity and a 50 Hz frequency for 5 seconds.
The pigs were bled by incising the major vessels. Blood was collected into 4 mL Firslab (FirstLab, Brazil) vacuum tubes containing fluoride and ethylenediaminetetraacetic acid (EDTA) for lactate and glucose analysis; EDTA for blood counts; and a clot activator for albumin, cortisol, creatine phosphokinase (CK-total), and CK-MB analysis.
Whole blood samples were analyzed for the hematologic parameters using a POC H-100 iV Diff automated veterinary hematology device (Sysmex, Poch IV diff, Japan). The measurements included hematocrit, blood cell count, mean corpuscular volume, mean corpuscular hemoglobin concentration, and red blood cell distribution width. The platelet count (thrombogram) and leukogram variables (neutrophils, neutrophil lineage, lymphocytes, eosinophils, basophils, and monocytes) were assessed on a Romanowsky-stained blood smear under an optical microscope. The total plasma protein and fibrinogen levels were determined by refractometry and heat precipitation, respectively.
The serum samples were analyzed on a Siemens Dimension Xpand Plus automated biochemical analyzer (Siemens Healthcare Diagnostics Inc., Germany) using commercial kits for each analyte: Siemens/Dimension CKI Flex and Dimension MBI Flex methods for CK-Total and CK-MB, respectively, and the Albumin-pp reagent (Analisa; Gold Analisa Diagnóstica, Brazil) for albumin. The plasma samples were assessed for glucose and lactate using Siemens/Dimension Gluc Ver Flex and Dimension LA methods, respectively.
Statistical analysis
The data were analyzed using a 2 × 3 factorial design (NANI and non-NANI; females: surgically castrated and immunocastrated). All variables showed a normal distribution (Shapiro-Wilk test). The results were analyzed using analysis of variance in R (R Foundation for Statistical Computing, Austria). The isolated effects were examined for variables without interaction effects. Comparisons between sexes and the interactions between variables were conducted using Tukey’s test (p < 0.05).
Results
The immunocastrated pigs had a final average weight of 129.73 kg, significantly higher (Table 1, p = 0.026) than that of the surgically castrated males (115.01 kg). Female pigs weighed an intermediate 121.39 kg, not differing from the male groups (p = 0.02). The non-NANI and NANI pigs had similar average weights (p = 0.61) (Table 1).
Body weight, hematological, and biochemical variables of non-ambulatory, non-injured (NANI) and non-NANI pigs
Most serum biochemical responses showed no treatment × sex interaction, except for CK-MB activity (Fig. 1) and erythrocyte counts (Fig. 2). The albumin levels were similar between sexes (Table 1), but the NANI pigs tended to have higher albumin levels than the non-NANI pigs (p = 0.058).
Mean values (± standard error) of the interaction between non-ambulatory, non-injured (NANI) and non-NANI pigs and sex class for creatine phosphokinase-MB (CK-MB). Bars with different uppercase letters indicate a significant difference between the non-NANI and NANI pigs within the same sex. Bars with different lowercase letters indicate a significant difference between sexes within the same health status (p-value = 0.0003, 2-way ANOVA + Tukey’s test) (n = 14 NANI surgically castrated males; n = 9 NANI immunocastrated males; n = 13 NANI females; n = 14 non-NANI surgically castrated males; n = 9 non-NANI immunocastrated males; n = 12 non-NANI females).
Mean values (± standard error) of the interaction between non-ambulatory, non-injured (NANI) and non-NANI pigs and sex class for hematological variable erythrocytes. Bars with different uppercase letters indicate a significant difference between the non-NANI and NANI pigs within the same sex. Bars with different lowercase letters indicate a significant difference between sexes within the same health status (p-value = 0.0238, 2-way ANOVA + Tukey’s test) (n = 14 NANI surgically castrated males; n = 9 NANI immunocastrated males; n = 13 NANI females; n = 14 non-NANI surgically castrated males; n = 9 non-NANI immunocastrated males; n = 12 non-NANI females).
The total creatine kinase (CK-total) activity was similar regardless of sex. NANI pigs, however, had significantly higher CK-total concentrations (109,573.3 U/L) than non-NANI pigs (37,103.33 U/L) (Table 1), indicating muscle damage associated with their non-ambulatory status. The CK-MB activity was substantially higher in the NANI females (14,066.67 U/L), with no significant differences between the non-NANI immunocastrated and surgically castrated males (Fig. 2).
The cortisol levels were significantly higher in the NANI pigs (11.83 µg/dL) than in the non-NANI animals (8.94 µg/dL) (p = 0.03), indicating an acute stress response. The serum glucose concentrations were low in the NANI pigs (79.33 mg/dL) and surgically castrated males and females (80.33 mg/dL), whereas the immunocastrated males maintained normal levels (105.83 mg/dL) (Table 1). The lactate concentrations were also elevated in the NANI pigs (15.48 mg/dL) compared to the non-NANI pigs (11.85 mg/dL), suggesting a shift toward anaerobic glycolysis in compromised animals.
The hematological evaluations showed that the NANI pigs had higher hemoglobin (15.23 g/dL) and hematocrit (44.93%) than the non-NANI pigs (13.96 g/dL and 41.96%, respectively), indicating hemoconcentration caused by dehydration and stress-induced splenic contraction (Table 1). All groups exhibited leukocytosis (mean 23,727.78/µL), with NANI pigs showing increased lymphocytosis (39.5%), which is indicative of physiological stress and inflammation. The fibrinogen levels were higher in the NANI pigs (677.78 mg/dL) than in the non-NANI pigs (444.44 mg/dL). In addition, the NANI females had higher erythrocyte counts (9.54 × 106/mm3), consistent with erythrocytosis (Fig. 2).
Discussion
Immunocastration has shown promising results as an alternative to surgical castration because castration performed without anesthesia or analgesia compromises animal welfare by causing pain, stress, and discomfort. This method effectively suppresses the testicular function in the weeks preceding slaughter, reduces boar taint, and increases the slaughter weight and carcass quality [15–17], explaining the observed differences relative to the other experimental groups. Nevertheless, increased animal activity and the higher prevalence of body lesions before the onset of effective immunization may adversely affect animal welfare [18].
On average, the pigs in the experiment had albumin levels above the normal physiological range for the species (1.80–3.30 g/dL) [19]. Elevated albumin, particularly in NANI pigs, indicates moderate dehydration from prolonged fasting, heat exposure, and limited water intake before slaughter. Albumin is a key indicator of the plasma volume contraction, rising when fluid loss exceeds replacement [20]. This dehydration is supported by higher hemoglobin and hematocrit values, likely due to splenic contraction induced by catecholamines [3,21]. These hematological changes are consistent with stress-induced sympathetic activation, which aims to enhance oxygen delivery to the peripheral tissues. Urrea et al. [22] reported that pigs transported at high densities rested more and drank significantly less water than those transported at lower densities, suggesting that fatigued pigs prefer to recover from transport stress by lying down rather than drinking.
Recent studies have shown that pigs subjected to intense pre-slaughter stress from prolonged transport, thermal challenges, limited space, and rough handling exhibit significant increases in stress biomarkers, including cortisol, lactate, CK, and acute-phase proteins [2,5,23]. These findings support these results because the rise in CK-total and CK-MB in NANI pigs indicates severe muscle fatigue and microlesions, confirming that the NANI status is physiologically distinct. Similar CK elevations have been observed in pigs subjected to prolonged transport or intense exertion during loading [5], highlighting muscular disruption as a key pathophysiological mechanism in fatigued animals.
Elevated CK-MB activity in NANI females may indicate myocardial stress or hypoxic injury caused by tachycardia and dyspnea during transport. Research has shown that swine females exhibit higher cortisol and CK-MB levels in response to heat and physical stress, likely due to differences in hypothalamic–pituitary–adrenal axis regulation [24]. This suggests that females may be more susceptible to cardiovascular changes under prolonged stress.
Elevated CK-MB levels in NANI pigs may reflect the cardiac factors associated with NANI syndrome symptoms, such as tachycardia, dyspnea, and stress, leading to myocardial changes that compensate for systemic alterations.
Increased serum cortisol and lactate levels in NANI pigs indicate acute stress. Cortisol mediates the metabolic adaptation to stress by mobilizing glucose and promoting gluconeogenesis [25]. The decrease in blood glucose in NANI animals suggests depleted hepatic glycogen stores, which drive anaerobic metabolism and lactic acidosis [2]. This metabolic shift results from physical fatigue, dehydration, and low energy reserves. Similar responses, including hypoglycemia and hyperlactatemia, have been observed in pigs during long-distance transport and under high temperature conditions [5].
Immunocastrated males tend to eat more than females and non-castrated males after their second immunization, 3 to 4 weeks before slaughter [26]. This increased feed intake may explain the higher glycemic levels observed despite pre-slaughter fasting.
Leukocytosis and lymphocytosis in NANI pigs indicate acute stress and inflammation. Stress mobilizes leukocytes from lymphoid organs into the circulation, a process mediated by catecholamines [27,28]. Elevated fibrinogen levels further support this because fibrinogen is an acute-phase protein that increases during inflammation or tissue injury [29]. Recent studies have suggested that fibrinogen is a valuable biomarker for assessing stress-induced inflammation in pigs [2,23].
The erythrogram results indicate hemoconcentration and increased oxygen demand. The higher erythrocyte counts in NANI females may reflect the stronger splenic contraction response or sex-dependent hormonal influences on erythropoiesis [24]. Although compensatory, these responses can elevate the blood viscosity and strain the cardiovascular system, increasing the risk of circulatory collapse in fatigued animals.
Pre-slaughter stress is a multifactorial condition influenced by the transport duration, stocking density, microclimate, fasting, and handling practices, all of which activate physiological, metabolic, and inflammatory pathways that compromise animal welfare and may negatively affect the carcass and meat quality [1,2]. In pigs affected by NANI syndrome, hematological and biochemical alterations (increased CK, CK-MB, cortisol, fibrinogen, lactate, hemoglobin, and hematocrit, together with reduced glucose concentrations) underscore the complex and integrated nature of the stress response. These changes reflect the interactions between metabolic, endocrine, and inflammatory systems and are significantly influenced by the transport duration, ambient temperature, and individual animal condition. Consequently, implementing welfare-oriented strategies during transport and lairage, such as reducing journey times, providing adequate rest, and optimizing microclimatic conditions, are essential for mitigating pre-slaughter stress and improving animal welfare outcomes.
Notes
The authors declare no conflict of interest.
Author’s Contributions
Conceptualization: Bridi AM, Correia ER; Data curation: Bridi AM, Correia ER, Carvalho RH, Ferreira NA, Flaiban KKMC, da Silva CA; Formal analysis: Bridi AM, Carvalho RH, Correia ER, Ferreira NA, Flaiban KKMC, da Silva CA; Funding acquisition: Bridi AM; Investigation: Bridi AM, Correia ER, Ferreira NA, Flaiban KKMC, da Silva CA; Methodology: Bridi AM, Correia ER, Flaiban KKMC, da Silva CA; Project administration: Bridi AM; Resources: Bridi AM; Supervision: Bridi AM, Flaiban KKMC, da Silva CA; Original draft: Correia ER; Writing - review and editing: Bridi AM, Carvalho RH, Flaiban KKMC, da Silva CA.
Data Availability Statement
The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.