The Impact of Breed, Testing Time and Metabolic Profile on the Variation of Copper Concentration in Sheep Blood Serum

Copper (Cu) is an essential element for life and is required as a co-factor in hundreds of enzymatic reactions involved in red blood cell production, energy generation, hormone formation, collagen synthesis and protection against oxidative damage (Kulkarni et al., 2012; Adams et al., 2018). Ruminants are probably more susceptible to Cu toxicosis than other species. To avoid Cu toxicosis, sheep should be grazed in copper-deficient pastures, which in addition may contain antagonistic minerals, namely sulphur (S), molybdenum (Mo) and iron (Fe). As ruminants have poor homeostatic control over Cu absorption, they have developed certain mechanisms for storing excess Cu in the liver by decreasing Cu excretion in the bile. However, when exposed to Cu concentrations above physiological requirements, ruminants do not modulate Cu excretion in the bile and excessive hepatic Cu accumulation occurs (Lopez-Alonso and Miranda, 2020). Sheep are more susceptible to Cu toxicity than cows or goats - they have a very limited capacity to excrete the Cu via the bile duct, as a result of a low capacity to accumulate the Cu bound to metallothioneins in the liver. There are also differences among sheep breeds in Cu tolerance; yet, these have not been traced to variations in the expression of particular copper-binding proteins. The Suffolk and the Merino breeds are particularly at risk.
       
Haematological and biochemical analyses in farm animals have been extensively discussed in the study often pointing to a specific differential diagnosis or suggesting a treatment and prognosis (Arfuso et al., 2016). The aim of the study was to analyse the relationship between the Cu concentration and other blood parameter values having taken into consideration their physiological limits based on the variation of copper concentration in the three different sheep breeds.

Location, animals and study design
 
The research was performed according to the Law of the Republic of Lithuania No. 8-500 on Protection, Keeping and Use of Animals, dated 06/11/1997 (Valstybės žinios (Official Gazette) No. 108 dated 28/11/1997) and orders of the State Veterinary Service of the Republic of Lithuania on Breeding, Care and Transportation of Laboratory Animals (No. 4-361, dated 31/12/1998) and use of Laboratory Animals for Scientific Tests (No. 4-16, dated 18/01/1999). The study’s approval number was PK014606.
       
The study was conducted on the sheep farm in the Eastern region of Europe, Lithuania, from April 2018 to May 2019. The average ambient temperature during the study year was 10.50 ±1°C, the relative humidity was 77.60 ±2%. Sixty sheep, were selected according to the following criteria: 3.5±0.3 years old, not pregnant during the entire investigation period, clinically healthy, exhibiting the following clinical parameters of an average rectal temperature of 38.5±0.2°C, rumen contractions three to four times per two minutes during the precise clinical examination. The farm conducts the monitoring of infectious diseases. Routine deworming is performed twice a year. The tested sheep were divided into the following three groups: the Suffolk (n = 20), the Merino (n = 20) and the Lithuanian blackhead (n = 20). Average body weight of the sheep was 35±5 kg. All sheep were kept following the loose housing system and were fed at the same time, the same ration, balanced according to their physiological needs. The grazing season was from April to October. The pastures were predominantly grasses and legumes. In addition, in summer, all sheep on the farms at all times had ad libitum access to the complete mineral mixture blocks -”Sheep Rockes” (Tithebarn, UK) (sodium 38%, magnesium 1000 mg/kg, calcium 1%, phosphorus 1%, cobalt 100 mg/kg, iodine 50 mg/kg, manganese 100 mg/kg, selenium 20 mg/kg and zinc 120 mg/kg). In winter, the ewes were kept in barns and fed the grain mixture (300 g/day), grass hay (Timothy grass, red clover, white clover, alfalfa, ryegrass and quack grass). Water was provided ad libitum.
 
Analytical procedures
 
The study was conducted from April 2018 to May 2019. Blood samples were collected between 0700 to 0800 hrs after overnight fasting by jugular venipuncture, using vacuum tubes without any anticoagulant. Sixty blood samples were taken once per month on the same day from the identical animals. In the study, 780 blood samples were tested (13 months x 60 animals). Further research was performed in the Lithuanian University of Health Science, Veterinary Academy. The samples were collected in 8°C temperature containers and transported immediately to the laboratory for analysis. Within an hour, the blood samples were centrifuged for 10 min. at 3500 rpm and blood serum was collected and stored in -20°C environment. The blood serum biochemical parameters, such as copper (Cu), zinc (Zn), iron (Fe), urea, glucose (Gl), aspartate aminotransferase (AST), gamma glutamyl transferase (GGT), total bilirubin (TBIL), albumin (Alb), creatinine (CREA) and lactate dehydrogenase (LDH) were measured using commercial kits on the Selectra Junior analyser (AC Dieren, The Netherlands, 2006). Cortisol level was measured using the automated analyser TOSOH® AIA-360 (South San Francisco, CA), performing a comparable fluorescent enzyme immunoassay, which is run in small, single-use test cups that contain all necessary reagents. Accuracy and performance data for human and canine cortisol, including analyte recovery and dilutional studies, have been previously evaluated (Higgs et al., 2014). Daily checks, calibration curves and maintenance procedures were carried out according to the System Operator’s Manual. For blood haematology sixty blood samples were taken once per month on the same day from the identical animals at the same time like for biochemistry in a vacuum tube system containing 10% EDTA. The values determined were erythrocytes (RBC), haemoglobin (HGB), haematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), leukocytes (WBC) and platelet count (PLT). Haematological parameters were measured using the MS4-5 VET automatic analyser (Melet Schloesing Laboratories, France 2012).
 
Data analysis and statistics
 
Blood parameters were grouped by physiological limits into three classes, where the middle class refers to the norm (Class 2, Cu values 46-140) and the two extreme ones denote, respectively, the values under or above the physiological limits (Class 1, Cu values <46; Class 3, Cu values ≥141) (Table 1). In the analyses, the breed, the month of study and blood parameters were considered as categorical variables. The level of significance was P<0.05 for all tests.
 

       
Data were analysed using the software IBM SPSS Statistics (version 25.0, IBM, Munich, Germany). The descriptive statistics of data were presented as mean ± standard error of the mean of a sample. The normality of all data recorded in the study was assessed by Shapiro-Wilk normality test. A multiple comparison (Bonferroni test) was applied to compare the Cu value among the breeds. The GLM repeated measures technique (general linear model) was used to assess the changes in serum Cu concentration (μg/dL). The difference between the proportions was assessed using the two-proportion z-test procedure. The Chi Square (c²) statistics was used to determine the relationship between the following categorical variables: Cu concentration among the breeds and Cu concentration in respect to other blood parameter values having taken into consideration their physiological limits. To analyse the factors contributing to the Cu level in the blood of sheep, multivariable logistic regression models were used, applying a backward stepwise logistic model to eliminate all non-significant explanatory variables. Categorical variables (the breed, the month of study and certain blood parameters) were continuously removed from the models according to the significance of the Wald criterion. Finally, two independent variables were used in the model.

The impact of breed on Cu level in blood serum
 
In sheep, breed is known to have a strong impact on the susceptibility to Cu disorders. Certain breeds have been classified as tolerant or resistant in terms of Cu toxicity. As a result, it is possible to improve the resistance of sheep to Cu deficiency and excess by appropriate cross breeding and selection programs (Stevanovic et al., 2015). In recent years, copper toxicity of sheep has been increasing with the intensification of sheep industry, increased housing of ewes and use of breeds susceptible to Cu poisoning, such as the Texel and the Merino (Anchordoquy et al., 2017). The results of data analysis showed a large variation (Cv = 64.03%) in the concentration of Cu in blood samples (n = 780). The value of Cu according to physiological limits (46-140 µg/dl) was found to be normal in 427 samples and in 353 samples (24.4%) it was higher (≥141 µg/dl). Most samples (80.1%) with normal Cu concentration were found in the Lithuanian blackhead sheep. Some samples (with normal Cu concentration) were found in the Suffolk sheep (by 4.2% less) and for the Merino breed it was by - 10.6% less compared to the Lithuanian blackhead sheep (Fig 1). The highest average value of Cu concentration (above the norm) was found in the Merino breed. Two breeds (the Merino and the Suffolk) of the three used in the present research may be relatively susceptive to Cu content. These genetic differences in Cu metabolism may be related to the efficiency of dietary absorption, biliary excretion of endogenous Cu or even the amount of feed intake (Stevanovic et al., 2015).
 

 
Impact of season on the changes in the blood serum Cu levels
       
The highest serum Cu concentration was observed in January (147.1 ± 12.20 μg/dl) and the lowest - in May (97.6 ± 18.85 μg/dl) in 2019 in the Lithuanian blackhead sheep breed (P<0.001) (Fig 2). Having assessed the changes in Cu concentration in sheep breeds according to the physiological limits of this blood parameter, it was found that most blood samples showed the values that met the physiological limits were collected in the first month of the study, i.e. in April 2018 (90-100%) and in the last two months of the study, i.e. in April and May 2019 (80.1-100%) (Fig 2). The lowest number of blood samples with Cu values meeting the physiological limits was collected from the Lithuanian blackhead sheep at the end of the grazing period, i.e. in October 2018 (26.3% of the samples) and in the Merino breed (10%) and the Suffolk breed (31.3%) in January 2018. The increase in Cu concentration can be related with stress, mineral deficiencies, ambient temperature and possibly higher humidity in these months (Ramirez et al., 2001).
 

 
Relationship between Cu concentration and blood parameters
 
Table 2 represents the percentage distribution of blood samples according to the concentration of Cu and blood parameters. For the sheep with normal Cu concentration, the percentage of blood samples that corresponded to the recommended norm was found to be from 60.9% (by Fe level) to 82.3% (by CREA level). The Chi Square (c²) test of independence demonstrated the relationship between the Cu concentration and the concentrations of blood Fe, CREA and the activity of GGT (P ≤0.001), MCH and MCHC (P = 0.005), HGB (P = 0.006), Gl (P = 0.028), PLT (P = 0.029) and ALB (P = 0.041). The analyses showed that the increase in Cu above the normal range was associated with a significant decrease in PLT below the normal range. This proves the fact that Cu has a direct effect on iron metabolism and thus, it indirectly affects the biosynthesis of haemoglobin (Cybulski et al., 2009). When the concentrations of Cu exceed the limit values, it enters the secretory pathway to bind to metallothionein in the Golgi body and is stored in the lysosomes, which thus protect the cell from free Cu. When the concentration of Cu in the blood increases, the liver can take a large amount of Cu from the blood and if the Cu level exceeds the tolerance limit, the activities of various enzymes can be inhibited, which can cause liver cell denaturation necrosis and Cu excretion dysfunction (Suttle et al., 2002). When the Cu level in the liver is excessive, a large amount of it enters into the bloodstream and then - into the red blood cells and the intracellular Cu concentration continues to rise so that the concentration of glutathione in red blood cells is reduced, erythrocyte fragility is increased and haemolysis occurs (Shen et al., 2010).
 

       
Blood glucose values are also known to be related to genetic predisposition (Durak et al., 2015). Glucose concentrations may alter with the secretion of catecholamines and may also increase or decrease secondarily. Lowest concentration of glucose and albumin of the examined animals may be a logical consequence of any nutritional deficiencies (Durak et al., 2015). An alternative hypothesis is that the concentration of blood creatinine might have been related with the trace elements and liver enzymes. This effect would be induced by the reduction in the glomerular filtration rate (Yokus et al., 2006). Compared to the reference values the average activity of GGT in the samples of sheep blood serum was slightly elevated, in 19.6% of samples, its normal range was observed in 33.3% and the decreased value was found in 75.0%. The increased activity of this enzyme in clinically healthy sheep can be considered as a consequence of the intensification of metabolic processes and may be related to Cu content (Stevanovic et al., 2015).
       
The concentration of cortisol can be different for each individual because some animals secrete more stress hormones than others. Of all domestic animals, sheep are the most sensitive to the effects of stress (Behera et al., 2018). Based on the results of this study, it can be stated that the increase in the amount of Cu in the blood serum does not have a significant effect on the increase in the amount of cortisol, there is no relationship between serum Cu and plasma cortisol concentrations.

Based on the results of this study, it can be concluded that copper metabolism in the Merino breed is poorer compared to that observed in other two of the studied breeds. The lowest number of blood samples with copper levels that met the physiological limits was collected from the Lithuanian blackhead sheep at the end of the grazing period and from the Merino and the Suffolk breeds – in the coldest month of the year. The increase in Cu concentration could be associated with stress due to altered dietary conditions, changes in ambient temperature, mineral deficiencies, especially antagonists. The concentration of Cu in the blood serum of sheep was associated with blood Fe, CREA and the activity of GGT, MCH and MCHC, HGB, Gl, PLT and ALB. This proves the fact that Cu has a direct effect on Fe metabolism. When the concentration of Cu in the blood increases, the liver can take a large amount of Cu from the blood and the activities of various enzymes can be inhibited.