Introduction

The Wumeng prairie is located in the Yunnan-Guizhou Plateau of Southwest China, where the three provinces of Guizhou, Yunnan, and Sichuan meet, and is an important pasture for Guizhou black goats. Goat farming is vital to the production system in the Wumeng prairie in the Western China [1, 2]. In recent years, Guizhou black goats in the Wumeng prairie area have been affected by a locally nutritional and metabolic disease associated with copper (Cu) deficiency, which is characterized by pica, emaciation, anemia, dyskinesia, and neurological disorders such as ataxia; animals are also prone to falls and fractures. The incidence is estimated at 15–25% and the mortality may reach 60%. Similar syndromes have been observed in yaks [3] and sheep [4]. The local farmers suffer seriously from this problem.

Nano-copper (Nano-Cu) is a common artificial material, which has been widely used in the production of intrauterine devices, anti-osteoporosis, anti-aging nano-drugs, lubricant additives, high-efficiency catalysts, and animal feed additives, bringing technological revolution to the development of medicine, industry, and animal husbandry [5,6,7,8,9,10]. Nano-Cu has the advantages of small particle size and large specific surface area. In addition to the nutritional characteristics of traditional trace element additives, it also has the advantages of small dosage, high bioavailability, and improving animal immune function [11, 12]. Cu is an important mineral for animals, and plays an essential roles in growth and development [13, 14]. Cu is a component of superoxide dismutase (SOD). SOD has the ability to scavenge free radicals [15, 16]. The antioxidant system is the defense system for scavenging free radicals, comprising non-enzymatic and enzymatic systems. The non-enzymatic system includes mainly vitamin E, vitamin C, cysteine, glutathione (GSH), Cu, iron (Fe), zinc (Zn), and selenium (Se). The enzymatic system consists of SOD, glutathione peroxidase (GSH-Px), catalase (CAT), and other antioxidant enzymes [17,18,19]. The anti-oxidation effect is to remove excess free radicals, maintain the balance between oxidation and anti-oxidation and retain the healthy physiological state of animals. The antioxidant function of animals has an essential relationship with many mineral elements.

The purpose of this research was to explore effects of nano-Cu on blood parameters and antioxidant function in animals and to provide a reference for further study prevention and treatment in Cu deprivation disease in Guizhou black goats.

Material and Methods

Experimental Range

The Cu-deficient ranges were located in Weining County in the Chinese Yunnan-Guizhou Plateau (27°36´–28°26´N, 103°39´–104°47´E), with an average elevation of 2200 m above sea level, annual precipitation of 960 mm, and average atmospheric temperature of 10–12 °C. The grassland vegetation is mainly Puccinellia (Chinam poensis ohuji), Siberian Nitraria (Nitraria sibirica Pall), floriated astragalus (Astragalus floridus), poly-branched astragals (Astragalus polycladus), falcate whin (Oxytropis falcate), ewenki automomous banner (Elymus nutans), common leymus (Leymus secalinus), and june grass (Koeleria cristata).

Experimental Animals

Sixty Cu-deficient goats, aged 1 year, from Caohai farm in Weining County, were selected. All animals had pica, anemia, depressed appetite, growth retardation, rough coat, and emaciation. Thirty healthy Guizhou black goats were selected from Liangshuigou farm in Weining County, where the diseases had not been reported. All of these animals were judged to be in good health after clinical examination, and they were used as the reference group. All Cu-deficient animals were randomly divided into 2 groups. Group 1 (nano-Cu groups) was supplied with nano-Cu (2 g/goat, once/week). Group 2 [copper sulfate (CuSO4) groups] was supplied with CuSO4 (2 g/goat, once/week).

Collected Sample

Ten soil samples were collected from the surface layer (0 to 20 cm) in each pasture, marking sampling points. Ten samples of mixed herbage were collected from each pasture. In order to reduce soil pollution, forage were cut 0.1 cm above ground level. Samples of hair were taken from necks of Guizhou black goats. Each sample was washed with acetone, rinsed five times with deionized water, and then kept on a silica gel in a desiccator until analyses. Samples of blood were obtained from the jugular vein using a vacuum blood collection tube without anticoagulant and a vacuum blood collection tube containing 1% sodium heparin as an anticoagulant. Samples of liver were taken. Platelets and coagulation were measured 1 day before operation, and the puncture points were located by ultrasound. Vitamin K (10 mg/mL) at a dose of 1 mL/goat was administered by intramuscular injection 15 min before the operation, and the animal was then anesthetized using a local anesthetic agent, typically 3 mL/kg 1% lidocaine. A small incision was made on the right side of the abdomen and a biopsy needle was inserted. During breath holding, insert the needle into the liver, quickly remove the tissue sample, and then place the bandage over the incision. The collected blood and liver samples were returned to the laboratory at a low temperature (4–8 °C) for further processing and analysis within 8 h.

Analysis of Sample

The samples of soil were heated in a microwave in a mixture of nitric acid (HNO3), hydrofluoric acid (HF), and perchloric acid (HClO4) (5:2:5) mixture to dissolve the sample. The forage and animal tissues were dissolved in HNO3 and HClO4 (4:1) by microwave heating.

The concentrations of mineral elements in soil, forage, and animal tissue were determined by inductively coupled plasma atomic emission spectrometry (ICP-AES). The elements analyzed were Cu, Zn, Fe, Se, molybdenum (Mo), cobalt (Co), fluorine (F), and manganese (Mn).

Levels of hemoglobin (Hb), packed cell volume (PCV), red blood cell (RBC), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobinconcentration (MCHC), and white blood cell (WBC) were determined using an automated hematology analyzer (SF-3000, Sysmex-Toa Medical Electronic, Kobe, Japan). Ceruloplasmin (Cp), lactate dehydrogenase (LDH), aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (AKP), creatinine (CRT), cholesterol (Chol), blood urea nitrogen (BUN), GSH-Px, SOD, CAT, malondialdehyde (MDA), total antioxidant capacity (T-AOC), sodium (Na), potassium (K), magnesium (Mg), calcium (Ca), and inorganic phosphorus (IP) levels in serum were determined using an automated biochemical analyzer (Olympus AU 640, Olympus Optical Co., Tokyo, Japan).

Statistical Analyses

Experimental data were analyzed by using the statistical package (SPSS, version 23.0, Inc., Chicago, Illinois, USA). Data are presented as means ± S.D., and values were considered to be statistically significant at P < 0.01.

Results

Mineral Contents in Soil and Forage

As shown in Table 1, the Cu contents in the soil and forage from the Cu-deprived ranges were significantly lower than those in healthy areas (P < 0.01). No significant difference was found in the concentrations of other elements.

Table 1 Mineral contents in soil and forage in studied pastures (μg/g)
Full size table

Effect of Cu Deficiency on Mineral Contents in Animal Tissues

The concentrations of mineral elements in the blood, hair, and liver are given in Table 2. The Cu concentrations in the blood, hair, and liver from Cu-deprived Guizhou black goats were significantly lower than those in healthy animals (P < 0.01), while the concentration of Fe was significantly higher than that in Guizhou black goats (P < 0.01).

Table 2 Mineral contents in tissues in Guizhou black goats (μg/g)
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Effect of Nano-Cu on Mineral Contents in Blood

The concentrations of mineral elements in blood are shown in Table 3, nano-Cu supplementation for up to 10 days, the Cu content in blood significantly increased, and the concentration of Fe significantly decreased (P < 0.01). After 20 days of continuous supplement, Cu and Fe content in blood are basically stable. The effect of CuSO4 group was significantly lower than that in nano-Cu group (P < 0.01). It can be found that supplementing nano-Cu is the best way to solve Cu deprivation in Guizhou black goats.

Table 3 Effect of nano-Cu on mineral contents in blood (μg/g)
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Effect of Nano-Cu on Physiological Parameters

As shown in Table 4, compared with the Cu-deprived Guizhou black goats, Hb, RBC, and PCV levels in experimental groups were significantly increased (P < 0.01). There was no significant difference between the Nano-Cu and CuSO4 groups.

Table 4 Hematological values in Guizhou black goats
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Effect of Nano-Cu on Biochemical Parameters

As shown in Table 5, compared with the Cu-deprived Guizhou black goats, serum Cp levels in experimental groups were significantly increased, while serum LDH, AKP, AST, ALT, and CRT were significantly decreased (P < 0.01). There was no significant difference between the Nano-Cu and CuSO4 groups. Compared with the Cu-deprived animals, serum SOD, GSH-PX, and CAT activities in nano-Cu and CuSO4 groups were significantly higher, while serum MDA levels were significantly lower (P < 0.01). There was a significant difference between the Nano-Cu and CuSO4 groups (P < 0.01), and nano-Cu had the best effect.

Table 5 Serum biochemical parameters in Guizhou black goats
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Discussion

Mineral nutrition plays an important role in the evolution, growth, and reproduction of animals [20]. Plant growth cannot be separated from the soil, which provides most of the nutrients needed by herbage [21, 22]. Thus, a better understanding of the mineral characteristics in soil, forage, and animal ecosystem can not only clarify the distribution of mineral elements in soil and forage but also improve our understanding of the health status in Guizhou black goats [23,24,25]. We analyzed the mineral concentrations of the soil and forage and found that the content of Cu was significantly lower and Fe concentration was significantly higher in affected compared with healthy pasture (P < 0.01). Cu deprivation depends not only on the total Cu concentration in the diet but also on other factors which affect Cu absorption and utilization [26]. Li et al. reported that Cu deficiency in Tibetan sheep was induced by S and Fe overdose [27]. The influence of Fe on the absorption and utilization of Cu is mainly due to antagonism between Fe and Cu. As Fe competes with Cu for intestinal protein binding sites, it consequently reduces the rate of Cu absorption [28]. Consequently, many researchers believe that the best ratio of Fe to Cu is 10:1 and excessive Fe will lead to Cu deprivation, consistent with the results of our study.

Cu is an essential trace element for humans and animals. It is present in various enzymes, cofactors, and reactive proteins and plays an important role in its activity, such as Cp, cytochrome oxidase, SOD, and lysyl oxidase. Severe deprivation results in obvious symptoms and Cu deficiency-related diseases of grazing ruminants have been widely reported in the reference. Shen reported a disease of Tibetan gazelle in Qinghai lake watershed of China, which was related to Cu deprivation caused by high S concentration in forage, this disease locally referred to as “shakeback disease” because of an unsteady gait [29]. Yuan et al. reported an illness in Guizhou semi-fine wool sheep related to Cu deficiency by high Mo and S contents in herbage in Guizhou province, China [30]. The main signs of such disorders included pica, emaciation, infertility, growth retardation, ataxia, dyskinesia, and abnormalities in bone development. And these diseases were prevented and cured by oral administration of CuSO4. Compared with those disorders above, this sickness in the study occurred in different animals, region, nutrition deficiency, and characteristics. This was the first report of this disease and was controlled by nano-Cu in the Guizhou black goats.

Nano-Cu particles have an extremely active role in biological systems entering the stomach due to their nanoscale and huge specific surface area. The absorption effect of nano-Cu and CuSO4 on Cu-deprived Guizhou black goats was studied, and it was proved that nano-Cu had better absorption effect. RBC is the main carriers of oxygen in the blood [31]. RBC contains Hb, which can combine with oxygen, so that the cells can transport the external oxygen absorbed by the alveoli to the tissue for metabolism [32]. Hb concentrations for healthy animals are 130–150 g/L. Lower contents may be linked to Cu deprivation. And in our study, the Hb, RBC, and PCV from Cu-deprived Guizhou black goats were significantly increased and returned to normal levels by oral administration of nano-Cu or CuSO4. The Cp content was significantly lower in affected compared with healthy Guizhou black goats (P < 0.01). Under normal condition, most of the Cu in serum is present as Cp, which plays an essential role in promoting the rate of Fe saturation of transferrin and in the utilization of Fe by bone marrow. So Cu deficiency not only markedly reduces the concentration of Cp, but it causes anemia, which type varies between and within species. In rats, lambs, rabbits, and pigs, the anemia is hypochromic, but in chickens and dogs, it is normochromic and normocytic. In this study, the microcytic anemia was observed in Guizhou black goats. Serum AST, ALT, and AKP activity can reflect the health of the liver, and LDH activity can reflect the degree of liver and muscle damage [33,34,35,36]. The activity of AST and ALT in serum in affected Guizhou black goats was significantly reduced, suggesting that the liver cells of Cu-deficient goats may be damaged. Increased serum AKP, LDH, and other enzyme activities suggest that muscle and kidney cells may also be damaged.

GSH-Px is an important peroxidase that is widely distributed in animals, and which can catalyze the reduction of reduced glutathione to hydrogen peroxide and thus help to protect the integrity of the cell membrane structure and function [37, 38]. SOD is one of the main antioxidant enzymes, which can remove excess free radicals in the body and reduce the degree of nucleic acid damage [39,40,41]. CAT is another important antioxidant enzyme, which can catalyze the decomposition of H2O2, thereby playing an antioxidant role. When lipid peroxidation occurs in the body, MDA will eventually form, and excessive MDA will damage the cell membrane structure, resulting in cell loss of function and even apoptosis. Therefore, changes in MDA content cannot only reflect the degree of lipid peroxidation in the body but also reflect the degree of damage to the body. The higher the MDA content, the greater the damage to the body [42]. In the current study, nano-Cu supplementation significantly increased the activities of GSH-Px, CAT, SOD, and significantly decreased the MDA concentration in serum, which could in turn reduce the formation of lipid peroxides and improve the resistance of the organism to oxidative damage. T-AOC can comprehensively evaluate the antioxidant function of the body [43]. Cu deprivation affects the antioxidant function, and causes various diseases in Guizhou black goats. Nano-Cu supplementation can improve the antioxidant function, reduce the production of lipid peroxides, protect the integrity of the cell membrane structure and function, and improve the functions of various tissues and organs in Guizhou black goats.

Conclusion

According to results, Cu deficiency in Guizhou black goats is mainly caused by low Cu concentrations in the soil and forage. Cu deprivation in forage in natural ranges will not only influenced the Cu content in the blood but also severely disrupted the blood parameters and antioxidant function in Guizhou black goats.