Biochemical parameters, dynamic tensiometry and circulating nucleic acids for cattle blood analysis: a review

Total protein content

Refractometry and spectrophotometry are the relatively simple and traditional tools to measure the “total protein” content (i.e., all proteins and popypeptides) in serum, whereas the numerious modifications of the electrophoretic technique are used to evaluate albumin and globulin fractions (Constable, Trefz & Stämpfli, 2019; Kaneko, Harvey & Bruss, 2008; Zaitsev, 2016). The particular data of the “total protein” content in cow’s blood are summarized in the Table 1. In general, the “total protein” content in cow’s blood is in the range of 60–75 g/l for adult animals and significantly increasing (up to 80–89 g/l) for lactating cows (Alberghina et al., 2011; Kholod & Ermolaev, 1988; Voronina, 2017; Zaitsev, 2016) depending on the age, breed, diet, particular lactation period, etc. (Table 1). Any changes of the blood protein values (significantly higher or lower the reference data, mentioned above) is an important key to dysproteinemia diagnosis (Maximov et al., 2012). Hyperproteinemia (increased “total protein” content) is due to dehydration or inflammation, whereas hypoproteinemia (decreased “total protein” content) is caused mainly by insufficient amount of adequate protein in nutrition for animals, diarrhea, etc. (Constable, Trefz & Stämpfli, 2019; Kaneko, Harvey & Bruss, 2008; Zaitsev, 2016). Anemia, problems in feeding of herds or specific animals, some their parasites should be evaluated first, then the problems with the gastrointestinal or urinary tracts should be suspected (Maximov et al., 2012).

Albumin to globulins ratio, fibginogen and “acute phase proteins”

It is obvious that serum is the “blood plasma after coagulation”, that is, without fibrinogen (Schaller et al., 2008; Zaitsev, 2016). That is why, a fibrinogen level is easy to estimate using the difference in the “total protein” values in plasma and serum of the same blood samples. There is not only increasing content of some usual proteins, such as fibrinogen, found by inflammation, but also appearance of special so-called “acute phase proteins” is observed (Constable, Trefz & Stämpfli, 2019; Maximov et al., 2012). Appearance of the “acute phase proteins”, such as C-reactive protein (CRP), cryoglobulins, etc., in blood can be considered as inflammation markers both for humans and animals (Kholod & Ermolaev, 1988; Maximov et al., 2012; Zaitsev & Konopatov, 2005; Zaitsev, 2017). The fibrinogen and globulin levels increased by inflammation for different times, but both are important to evaluate because WBC count level not always accurately points to inflammation for some animals. In general, for cattle a left shift in the complete blood cell count can occurs early and correlates with a degree of inflammation (Constable, Trefz & Stämpfli, 2019; Kaneko, Harvey & Bruss, 2008; Zaitsev, 2016).

The total values and content of the protein fractions are among the most important “integral” biochemical parameters for description of the methabolic activity not only in blood and other tissues, but in the animal organism as a whole. Depending on the age, lactation period, supplements to the basic diet, etc., the albumin/globulin (A/G) ratios of cow’s blood are summarized in the Table 2. In general, the A/G ratios of cow’s blood (Table 1) are in the range of 0.66–0.90 (Alberghina et al., 2011; Kholod & Ermolaev, 1988; Voronina, 2017; Zaitsev, 2016; Xuan, Loc & Ngu, 2018). So, the average value around 0.8 seems to be reasonable reference for cattle A/G ratio. The values of some important blood enzymes will be presented and discussed below.

Serum enzymes

Enzyme blood tests start to be very popular nowadays (Constable, Trefz & Stämpfli, 2019; Kaneko, Harvey & Bruss, 2008; Zaitsev, 2016). The most important and useful enzymes in the case of animal blood tests are the following: lactate-dehydrogenase (LDH), aspartate transaminase (AST), alanine transaminase (ALT), gamma-glutamyltransferase (GGT), etc. (Schaller et al., 2008; Zaitsev, 2010; Zaitsev, 2016, 2017). It is important to highlight that their results should be “interpreted with caution”, because of the huge variation in the reference values even for healthy animals (Vozianov et al., 1999; Zaitsev, 2016; Voronina, 2017).

For example, normal AST values for cows are in the range of 78–132 U/L (Kaneko, Harvey & Bruss, 2008) or 183–2,667 nkat/L (Kholod & Ermolaev, 1988) depending on the age, sex, breed, feeding, etc. (Table 2). In any case the values about 19.3–37.7 U/L (Xuan, Loc & Ngu, 2018) looks strange, but may be explained by variety of supplements to the basic diet (grass, rice straw and rice bran) in the case of Brahman crossbreed cattle (Au Giang Province, Vietnam). In general, ALT values in cattle blood are lower (as compared to AST) and in the range of 11–40 U/L (Kaneko, Harvey & Bruss, 2008) or 450–700 nkat/L (Hazipov & Askarova, 2003) depending on the age, sex, breed, feeding, etc. (Table 2). Our data for adult cattle are the following: AST 62–82 U/L for cows or 67-98 U/L for bulls; ALT 32–36 U/L for cows or 23–29 U/L for bulls at standard farm feeding in Russia during last decades (Zaitsev, 2016, Voronina, 2017). These results are in a reasonable agreement with the majority of the obtained data (Constable, Trefz & Stämpfli, 2019; Kaneko, Harvey & Bruss, 2008; Xuan, Loc & Ngu, 2018), but differ to the data obtained by researchers from Belorussia (Kholod & Ermolaev, 1988) and Tatarstan (Hazipov & Askarova, 2003), which used another analytical approaches. Nevertheless, a pronounced increase in the AST or ALT values (in 4–8 times) is a clear evidence of heart (muscle) or liver disorder even before the clinical evidence. An increase in the ALT level is faster in the “light” cases, whereas, an increase in the AST level is faster in the much more serious cases (Zaitsev, 2016, Voronina, 2017).

In general, GGT values in cattle blood are in the range of 6.1–17.4 U/L (Kaneko, Harvey & Bruss, 2008) or 450–700 nkat/L (Hazipov & Askarova, 2003) depending on the age, sex, breed, feeding, etc. (Table 2). Our data for adult cattle are the following: 28–44 U/L for cows and 30–50 U/L for bulls at standard farm feeding in Russia (Zaitsev, 2016; Voronina, 2017). An increasing GGT content in blood can be connected with some liver diseases such as “cholestasis and hepatocellular membrane damage” in cattle (Constable et al., 2016; Zaitsev, 2017). It is important to highlight that the increased GGT values in blood observed much longer as compared to the increased values of ALT, ACT or some other enzymes. The elevated levels of the abovementioned enzymes in blood indicated that the liver should be further investigated (Maximov, Goudin & Lysov, 2010; Maximov et al., 2012; Zaitsev, 2017). The ALT or ACT isoenzyme profile can be especially useful for specific diagnosis, but it rather complicated and expansive for cattle and used mainly for dogs and cats (Zaitsev, Maksimov & Bardyukova, 2008). Any “malfunction” (mutation, overproduction, underproduction or deletion) of a single enzyme can also indicate a genetic disease. Thus, the general control of enzyme activity is essential for cattle diagnostics (Zaitsev & Konopatov, 2005; Zaitsev, 2016, 2017).

Low molecular serum components

The level of the hemoglobin degradation products, such as total bilirubin, is an important indicator for some pathological conditions (e.g., massive hemolysis of erythrocytes in malaria, obstruction of the bile ducts or other liver diseases) (Constable et al., 2016; Zaitsev, 2017). The reference values of the total bilirubin for cattle (summarized in the Table 3) are in the range of 0–16 µM/L (McSherry et al., 1984; Kholod & Ermolaev, 1988; Kaneko, Harvey & Bruss, 2008; Zaitsev, 2016). The enormous high upper limit of the total bilirubin values in cattle blood (up to 29.67 µM/L) published by Vietnam scientists (Xuan, Loc & Ngu, 2018) may be explained by variety of supplements to the basic diet (grass, rice straw and rice bran) in the case of Brahman crossbreed cattle (Au Giang Province, Vietnam) (Table 3). Moreover, cattle hyperbilirubinemia is well described in the paper of Canadian vets (McSherry et al., 1984). If the level of the hemoglobin degradation product (such as total bilirubin) is abnormal, then it is important to measure the values of the “indirect” (unconjugated) and “direct” (conjugated) bilirubin separately (McSherry et al., 1984; Zaitsev, 2016; Xuan, Loc & Ngu, 2018).

The large deviations in the reference values of the total lipids, triglycerides, fatty acids, cholesterol and phospholipids are discussed in the following reviews and papers (Guédon et al., 1999; Adewuyi, Gruys & Van Eerdenburg, 2005; Zaitsev, 2016).

It is strange that enormous low values of the glucose (from 0.57 mM/L to 1.83 mM/L) in cattle blood published without discussions in the following paper (Xuan, Loc & Ngu, 2018) (Table 4). It may be explained by variety of supplements to the basic diet (grass, rice straw and rice bran) in the case of Brahman crossbreed cattle (Au Giang Province, Vietnam). In contrast, the reasonable glucose values in the cattle blood can be considered as reference: from 2.86 mM/L to 5.66 mM/L (Kholod & Ermolaev, 1988), 2.50–4.16 mM/L (Kaneko, Harvey & Bruss, 2008) (Table 4).

Major inorganic cations and anions

The reference values of the major cations (sodium, potassium, calcium, magnesium) and anions (chlorides, total bicarbonates or CO₂ index, phosphates, etc.) are another essential part of the “serum chemistry profile” of any human or animal (Zaitsev & Konopatov, 2005; Zaitsev, 2016, 2017). The reference values of the major cations and anions for cattle blood are not always presented in the biochemical papers. That is why the authors just showed the most reasonable values here: 140–150 (Na⁺), 4.3–5.8 (K⁺), 2.2–2.8 (Ca²⁺), 0.8–1.2 (Mg²⁺), 105–120 (Cl⁻) mM/L (Kholod & Ermolaev, 1988; Zaitsev, 2016), 17–29 (HCO₃⁻) mM/L (Kaneko, Harvey & Bruss, 2008; Zaitsev, 2016). Metabolic alkalosis (at high CO₂ level), hypochloremia (low chlorides level) and hypokalemia (low potassium level) are the common abnormalities in adult cattle “with gastrointestinal disease” (Zaitsev & Konopatov, 2005; Zaitsev, 2016, 2017). Hyponatremia (low sodium level) and hypochloremia (low chlorides level) are usually occurs together with diarrhea. In the case of acidosis, a hyperkalemia (high potassium level) can be observed, but the blood potassium level is rarely increased essentially and rapidly (Zaitsev & Konopatov, 2005; Zaitsev, 2016, 2017). From one side, a little hypocalcemia (middle low potassium level) can be easily recognized during physical examination of sick cattle without additional biochemical tests, but on the other side, blood biochemistry analysis might be helpful in the most cases of the cation or anion problems (Zaitsev & Konopatov, 2005; Zaitsev, 2016, 2017). In this respect it is noteworthy that hypercalcemia is rare observed, even by general animal treatment using calcium (Zaitsev & Konopatov, 2005; Zaitsev, 2016, 2017). It is important to highlight that the abnormal electrolyte blood parameters are mainly caused by animal nutrition problems.

The methods and equipment of the measurements of dynamic tensiometry parameters of biological liquids

There are numerious methods and devises for the measurements of dynamic tensiometry parameters of biological liquids (Miller & Fainerman, 1998; Hubbard, 2002; Somasundaran, 2015). The one of the most convenient for blood study is the tensiometer BPA-1P (so-called “Maximum Bubble Pressure Tensiometer”). The function principle of the tensiometer BPA-1P and its new generations is based on the maximum pressure measurements in the bubble method (Miller & Fainerman, 1998; Kazakov et al., 2000; ; Khilko, 2014; Voronina, 2017; Zaitsev et al., 2004, 2011a; Zaitsev, 2016, 2018).

The significant advantages of the BPA and relative devices are the following: a small volume of sample, high analysis speed, full automation of the measurement process, the computer processing of the information received (Zaitsev, 2016). The air from the compressor enters the capillary, which is lowered into the test liquid. The maximal pressure in the system is determined (Fig. 1) and used to calculate the surface tension (Zaitsev, 2016).

The pressure required for the separation of the air bubble from the capillary tip, that drops at the air-liquid interfaces, is directly proportional to the surface tension (σ). To overcome the wetting phenomenon in the capillary (dipped into liquid) an excessive air pressure is required. The maximum pressure that occurs during the formation of the air bubble during blowing depends on the capillary radius (Fig. 1 “min” at the left insert). At the moment when the bubble takes the form of a hemisphere capillary radius is equal to the radius of curvature and pressure reaches the maximum value (Fig. 1 “max” at the middle insert). With further bubble growth the curvature radius increases again, which reduces the pressure inside the bubble (Fig. 1 “min” at the right insert). The division of the interval between the bubbles into the so-called “dead time” and the surface “lifetime” is based on the existence of a critical point, depending on the air flow pressure (Zaitsev, 2016). Comparison of the data obtained by BPA-1P with those by other well-known methods (oscillating jet, drop volume, dynamic, capillary, etc.) (Miller & Fainerman, 1998; Kazakov et al., 2000; Khilko, 2014; Voronina, 2017; Zaitsev et al., 2004, 2011b; Zaitsev, 2016, 2018) showed a good agreement between the results.

Hanging drop method is used for measuring the surface tension by PAT-1 device (“Topfen-Blasen-Profiltensiometer”) (Fig. 2). Its advantages include a small volume of liquid to be analyzed, a wide range of life-time measurements of the drop, that is, from 10 to 10,000 s (Zaitsev, 2016).

The experimental error of the measurement of surface tension by the method of hanging drop is about 0.1 mN/m. The main parameter of the droplet hanging on the capillary tip is its volume. The larger a volume of the drop, the more it is different from a spherical shape (Zaitsev, 2016).

Dynamic tensiometry parameters of cattle serum

The dependance of the dynamic surface tension parameters (Zaitsev, 2016) of the serum on the qualitative and quantitative composition has been described (Zarudnaya et al., 2010; Zaitsev et al., 2011b; Zaitsev, 2010, 2018). The dependance of the DT parameters (complete quantitative data set) of the serum of some animals (Fig. 3) have been obtained recently (Table 5) mainly by our group (Zarudnaya et al., 2010; Zaitsev et al., 2011a; Zaitsev, 2010, 2016, 2017) in contrast to some detached (single surface tension data) that has been described previously (Zaitsev, 2016; Voronina, 2017; Zaitsev, Fedorova & Maximov, 2019).

The DT values of the cattle serum (there were from 10 to 15 animals in each age-group) were reported (Zaitsev, 2016; Voronina, 2017). The dynamic tensiometry parameters were obtained from dependences of surface tension (σ) vs. time (t), so-called tensiogram (Fig. 3), at the particular points: t → 0 (σ₀), t = 0.1 s (σ₁), t = 1 s (σ₂) and t = 10 s (σ₃); or as the initial and final tilts of the tensiogram (λ₀ and λ₁ values, respectively).

All DT parameters, measured in cattle serum, undergo pronounced changes with animal age. For example, significant changes observed for the initial tilt of the tensiogram (λ₀), that is, this value for 12 months animals decreased by 47% as compared to the heifers of 6 months age. In addition, the onset of 12–18 months of animal age led to adaptive changes in the parameters of surface tensions, so σ₀–σ₃ values increased or decreased by 3–5% as compared to the heifers of 6 month’s age (that in the frame of the experimental errors). By the time of physiological maturity (18 months), the values λ₀ and λ₁ are increased by 48% and 28%, respectively.

The onset of pregnancy is accompanied by numerous changes in the cow body, especially by the nervous system and endocrine glands. For example, a number of biologically active substances contained in the blood varies significantly during pregnancy, that leads to changes in the blood serum DT values. For cows at 2 or 6 months of pregnancy, the following major changes occurred: λ₁ decreased by 24% or 15% and the value of λ₀ increases by 20% or 25%, respectively, as compared to heifers of 18 month’s age. For not pregnant cows, the following major changes occurred: λ₁ decreased by 35% and the value of λ₀ increases by 10% as compared to heifers of 18 month’s age. In contrast to such significant changes in the λ₁ and λ₀ parameters, the changes in the σ₀–σ₃ values observed by 2–6% only, as compared with the pregnant cows (Table 5).

These data were obtained by MBP and hanging drop methods (using BPA-1P and PAT-1). The average equilibrium DT values (σ_∞ at time → ∞) for male cattle were the following: young calves 46.92 ± 2.54 mN/m, at the middle age 47.03 ± 2.58 mN/m, adult bulls 46.62 ± 1.39 mN/m [81]. The average equilibrium DT values of the curve tilt (λ_∞ at time → ∞) for male cattle were the following: young calves 0.31 ± 0.19 mN∙m⁻¹s^−1/2, middle aged 0.29 ± 0.12 mN∙m⁻¹s^−1/2, adult bulls 0.15 ± 0.05 mN∙m⁻¹s^−1/2 (Zaitsev, 2016).

Thus, there are significant changes in the cattle blood system occur due to age, sex, physiological state, which are “reflected” in the changes of the DT parameters.

Correlations between dynamic tensiometry and biochemical parameters of cattle serum

There are various correlations between DT and biochemical parameters of cattle serum are found recently (Zaitsev, 2016, 2017). A large amount of strong positive (21 items) and negative (13 items) or middle positive (7 items) and negative (5 items) correlations for heifer (6 months) were found (Table 6). The strong and middle (no matter positive or negative) correlations are promising for the further practical applications and only these correlations will be discussed below. There are positive correlations of σ₁ with the level of proteins, triglycerides, cholesterol, glucose, calcium, sodium, and a negative correlation with the level of urea, potassium and chloride for heifers (Table 6). The σ₂ values have a positive correlation with the level of albumin, triglycerides, cholesterol, glucose and sodium, and negative correlation with the level of total protein, urea, and chlorides. The σ₃ value rises with increasing levels of urea, potassium and chloride, and decreases with increasing levels of albumin, triglycerides, cholesterol, glucose, calcium and sodium. The λ₀ values have a positive correlation with majority of the biochemical parameters studied, whereas a negative correlation with urea, potassium and chlorides are found. The λ₁ values have a positive correlation with the level of total protein, triglycerides, cholesterol, glucose, calcium, sodium, and a negative correlation with the level of urea, potassium and chloride (Table 6).

There are medium correlations between dynamic surface tension parameters and biochemical indicators of blood serum for heifer aged 1.5 years (Table 7). The tensiogram tilts rise with increasing concentration of total protein and total cholesterol in the serum, and decrease with increasing potassium concentration (Table 7). The σ₁ value has a positive correlation with the level of sodium and inorganic phosphorus, and a negative correlation with the level of total protein, total cholesterol, and chlorides. The σ₃ value has a negative correlation with the level of protein, lipids and chlorides, and a positive correlation with the level of urea, calcium and phosphorus. The λ₀ value has a positive correlation with the levels of albumin, triglyceride, glucose and serum chlorides for heifers and negative correlation with the level of urea, total calcium, phosphorus and sodium. The λ₁ value has a positive correlation with the level of cholesterol, urea, glucose, calcium and potassium, and a negative correlation with the level of total calcium and inorganic phosphorus (Table 7).

Results of the correlation analysis of pregnant cow blood serum (Table 8) can be summarized as follows: the σ₁ value has negative correlation with the level of albumin, triglycerides, cholesterol, glucose, and chlorides and a positive correlation with the level of calcium, phosphorus and potassium; the σ₂ and σ₃ parameters have a negative correlation with the level of total protein, lipid and chloride in the serum of lactating cows and a positive correlation with the level of urea, calcium and potassium (for σ₃). The levels of urea, calcium, phosphorus and potassium have the greatest impact on the λ₀ value. The λ₁ value has a positive correlation with the level of protein, triglycerides, cholesterol, and chlorides and negative correlation with the level of urea and total calcium (Table 8).

There is a positive correlation of σ₁ with urea, chloride, albumin; and a negative correlation with the level of glucose (only σ₀), calcium, cholesterol, total protein (only σ₁), σ₃ has a positive correlation with the level of glucose, calcium, cholesterol and negative correlation with the level of urea, chlorides, albumin for bovine (Table 9). The λ₀ value has a positive correlation with the level of protein, urea, chloride, cholesterol and negative correlation with triglycerides, glucose and cations (potassium, sodium). There is a negative correlation for λ₁ value (Table 9). Thus, the DT parameters depend on both quantitative and qualitative changes in the cow blood because of the particular physiological state (pregnancy, lactation) (Zarudnaya et al., 2010, Zaitsev, 2016, 2017; Voronina, 2017).