THE ATTEMPT TO DETERMINE CORRELATION BETWEEN MILK PRODUCTION AND ERYTHROCYTE CELL MEMBRANE PROTEIN CONCENTRATION

Janusz Rydlewski¹, Wojciech Zawadzki², Albert Czerski³, Mariusz Grządziel⁴, Jerzy Rudnicki^5
1 Department of Internal and Parasitic Diseases with Horse, Dog and Cat Disease Clinic, Wrocław University of Environmental and Life Sciences, Poland
² Department of Animal Physiology and Biostructure, Faculty of Veterinary Medicine, Wrocław University of Environmental and Life Sciences, Poland
³ Department of Animal Physiology, Wrocław University of Environmental and Life Sciences, Poland
⁴ Department of Mathematics, Wrocław University of Environmental and Life Sciences, Poland
⁵ 2^nd Department of General and Oncological Surgery, Wroclaw Medical University, Poland

ABSTRACT

The research was carried out on 20 clinically healthy dairy cows characterized by high and low milk production. Blood analysis was performed in order to determine the differences in erythrocyte cell membrane protein concentration in comparison with milk productivity. Statistically significant correlation was found between concentration of erythrocyte membrane protein and milk production in the electrophoresis test of ghost erythrocyte cell membrane protein. In the high milk production animal group, the mean concentration of total spectrin in cell membranes is higher on average by 11.23% (p≤0.001), ancirin 2.1 by 8.37% (p≤0.01) and ancirin 2.3 by 13.47% (p≤0.01) than in the low milk production animal group respectively. No significant differences were observed in the concentration [%] of other erythrocyte cell membrane proteins isolated during electrophoresis.

Key words: erythrocyte, cell membrane protein, dairy cow, milk production.

INTRODUCTION

Dairy cow milk production depends on many factors. The most important ones are the following: nutritional-zoohygienic conditions in the cow-shed and genetic traits. It is well known that animals with ensured optimal living conditions are able to develop fully their production potential programmed in their genes [9].Unfortunately, in spite of identical animal feeding and identical zoohygienic conditions in the cow-shed not all animals reach high milk production. The differences occurring in animal milk production in the cow-shed are related to genetic traits of a particular individual, recorded in the genetic material (DNA). It limits the cow-shed profits and increases the price of the final product, i.e. milk. High financial outlays are also incurred to make a suitable choice of herd individuals and to conduct rational selection of individuals. Out of all breeding costs incurred, 2–3% of the costs are constituted by expenditures incurred for efficiency control, out of which 53.9% of the production costs are constituted by milk production control costs [7, 9, 10, 11, 12]. On the market, no test is available for fast and cheap determination of animal production capacity (milk productivity) before the animal reaches the productive age. Therefore, the authors have decided to investigate whether there is any correlation between erythrocyte protein concentration [%] and dairy cow milk production. The existence of such correlation would create a possibility of fast and cheap control of animal production capacity before it reaches maturity. It would also advance the breeding progress and facilitate animal selection for the basic herd. The occurrence of RBC cytoskeleton proteins and their concentration are strictly determined by genes [6]. Erythrocyte cell membrane includes a number of proteins, which totally constitute approximately 50% of its chemical composition [17]. The proteins belong to the most thoroughly recognized membrane proteins, particularly human erythrocyte membrane proteins. RBS cytoskeleton membrane includes a number of proteins such as: spectins (spectin 1.0 and spectin 2.0), ancirin (ancirin 2.1, ancirin 2.2, ancirin 2.3), anion transporting proteins (protein 2.6, protein 3.0, protein 4.1, protein 4.2), protein 4.9 (denatin), protein 5.0 (actin), protein 6.0 (glyceraldehyde-3-phosphat dehydrogenase) and protein 7.0 [15]. Peripheral proteins participate in the construction of cell cytoskeleton and are responsible for the maintenance of double-layer lipid membrane [18]. They also play an important role in the maintenance of the shape and flexibility of erythrocyte ensuring free and reversible change of erythrocyte shape during its way through small capillaries.

MATERIALS AND METHODS

The research was conducted on 20 clinically healthy dairy cows of Holstein-Friesland breed (HF) or Polish Black and White breed with HF cows’ blood admixture. The animals originated from 2 private cow-sheds of Lower Silesia Province. They were kept in free stand cow-sheds and fed according to the accepted dietary rules. 10 individuals from each shed were selected for the investigation, out of which 5 individuals were characterized by the highest milk production in a given shed, and another 5 – with the lowest milk production. The animals were qualified to a given group on the basis of the milk production reading from the cow-shed book kept by the attendants. The animals were divided on the basis of obtained production results into 2 experimental groups: with high milk production (10 animals, over 9.000 litres during lactation) and with low milk production (10 animals, below 7 litres during lactation). The blood for testing was drawn from jugular vein with an injection needle of 1.6 mm diameter into a syringe of 10 ml volume filled with anticoagulant (3.8% sodium citrate in voluminal ratio of 1 vol. of anticoagulant to 9 vol. of blood). The drawn blood was analyzed with regard to the differences in the quantity of peripheral proteins and integral erythrocyte membranes. In order to do it, erythrocyte membranes “erythrocyte ghosts” were extracted from the collected blood according to Dodge et al. method [8]. The obtained erythrocyte ghosts were then subject to electrophoresis test, applying electrophoresis under denaturizing conditions, in 7% polyacrylamide gel SDS according to Laemmli method [16]. Gel staining to prove the protein presence was carried out in Coomasie R250 solution (methanol/acetic acid/H₂O in 188:60:360 relation). Scanning of the obtained polyacrylamide gels and analysis of the surface area occupied by particular protein bands was conducted by means of BioGene computer software 1-D, according to the instruction included in “Operating Manual”. The gels were scanned with the application of SNAPSCAN 1236 scanner manufactured by Agfa.

In the obtained results, the mean erythrocyte protein content (in experimental animal groups) was compared to the mean animal milk production. The obtained values of erythrocyte proteins were presented in per cents. The data were compared according to Shapiro-Wilk method. The differences between the examined parameters were recognized as statistically significant at the confidence level of p<0.05.

RESULTS

A statistically significant difference in cow milk production was found in the examined animal groups of high and low milk production, which is presented in table 1. The mean animal milk production per 350 days of lactation is higher by 69% in the animal group of high milk production in comparison to the animal group of low milk production (p≤0.001). Furthermore, there is correlation between animal high milk production and HF cow gene level [%]. HF level is on average higher by 28% (p≤0.001) in the high milk production animal group. As for the mean milk fat and the mean milk protein concentration, they are similar in both groups and do not show characteristics of statistical significance.

Table 1. Mean animal productivity in high and low milk production animal group

Animal groups	Mean level of overall milk production recalculated into 305 days of lactation [L]	Mean milk fat [g/100ml]	Mean milk protein concentration [g/100ml]	[%] Mean level of HF [%]
High milk production animal group	10237.8±976***	3.85±0.48	3.41±0.39	95.3±8.28***
Low milk production animal group	6057±1323	3.96±0.49	3.43±0.17	74.2±19.07

***p ≤ 0.001

In the electrophoresis test of erythrocyte proteins, statistically significant correlation was observed between the animal milk production and erythrocyte protein content in the isolated erythrocyte cell membranes. In the animal group of high milk production, the mean content of total spectin in erythrocyte cell membranes is on average higher by 11.23% (p≤0.001) in comparison to the animal group of low milk production. A higher concentration of ancirin 2.1 (by 8.37%, p≤0.01) and ancirin 2.3 (by 13.47%, p≤0.01), in comparison to the animal group of low milk production, was also observed. No differences with regard to the concentration of the remaining erythrocyte proteins were observed between the animal group of high and low milk production (table 2, fig.1).

Table 2. Protein composition [%] of erythrocyte cell membrane in high and low production animal group. In the table the statistical differences in composition of erythrocyte cell membrane protein between high and low milk production animal group are marked

Cell membrane protein

Spectynin [%]

Ancirin [%]

Protein [%]

Animal groups

całk. Total

1.0

2.0

całk. total

2.1

2.2

2.3

BPA

4.1

4.2

4.9

6.0

7.0

Aktyna Actin [%]

High milk production animal group

19.17 ±1.95***

7.62 ±1.54

10.54 ±2.18

13.98 ±1.49

7.89 ±1.19**

3.83 ±1.61

3.2 ±0.77*

18.99 ±1.28

9.71 ±1.22

7.68 ±1.68

0.45 ±0.31

8.52 ±1.5

8.19 ±0.76

14.48 ±2.81

Low milk production animal group

17.35 ±1.38

7.77 ±0.66

9.57 ±0.99

13.48 ±1.57

7.28 ±1.32

4.17 ±1.87

2.82 ±0.56

17.12 ±2.48

10.32 ±1.54

7.28 ±1.64

0.41 ±0.29

9.44 ±2.86

8.62 ±0.72

15.68 ±3.74

***p ≤ 0.001 **p ≤ 0.01 *p ≤ 0.05

Fig. 1. The electrophoresis test of ghost erythrocyte cell membrane protein in dairy cows. A – High milk production animal, B – Low milk production animal

DISCUSSION

Usually no great differences are found with regard to the concentration change and molecular mass of erythrocyte cytoskeleton proteins [14]. The variability in the concentration of erythrocyte membrane proteins, especially concerning spectins (spectin 1.0 and spectin 2.0) and ancirin (protein 2.1, 2.2 and 2.3) was described in mice [3, 4, 5, 13, 19, 20], while the lack of protein 6.0 - in rats and mice [1, 2]. In the available literature, no studies can be found devoted to the cytoskeleton protein concentration in cows. There are no studies, either, confirming the existence of differences in protein concentration between particular individuals or cow breeds. It seems, however, that such differences exist both between species and breeds. According to Witfield et al. [20], the determined differences in molecular mass and concentration of RBC (red blood cell) cytoskeleton proteins are species dependant, which is also confirmed by the results obtained by Guerra-Shinohara [14]. On the basis of the obtained results it is possible to state that there are no differences with regard to the presence of RBC cytoskeleton proteins in the examined cows. Identical proteins of erythrocyte membrane are found in all individuals. There are however, fluctuations of the content of particular membrane proteins, which can be proved on the basis of electrophoresis results, these fluctuations, however, are small and statistically insignificant except for spectrins and ancirins (ancirin 2.1 and 2.2). The fact of finding correlation between animal milk production and content of total spectin, ancirin 2.1 and ancirin 2.3 is surprising. The concentration of these proteins is statistically higher in animals characterized by higher milk production than in individuals with lower milk production. It may be partly connected with the breed-related difference between individuals, since lower milk production individuals have a lower level of HF genes. Higher milk production of individuals with a higher level of HF cow traits is not surprising, for HF breed cows are characterized by high milk production and are regarded as typical dairy cow breed. Both spectrin and ancirins belong to peripheral proteins supporting the maintenance of erythrocyte shape and flexibility. As for spectin, it is the main protein of cytoskeleton and has at least four binding points, out of which one is a binding point of ancirin 2.1[17]. This fact proves a close functional dependence between spectrin and ancirin.

It would be necessary to conduct experiments on a higher number of animals (population), considering several cow breeds in order to confirm the results of this study. Apart from confirming the results, this would also allow determining the variability limits in the erythrocyte protein level (total spectin, ancirin 2.1 and ancirin 2.3) between low and high milk production individuals. The existence of such correlation would significantly advance the breeding progress and would enable sooner animal selection for the production herd.

CONCLUSIONS

In dairy cows characterized by high milk production, a higher concentration of total spectin, ancirin 2.1 and ancirin 2.3 was found in erythrocyte membranes in comparison to erythrocyte proteins isolated from low milk production animals.

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Janusz Rydlewski
Department of Internal and Parasitic Diseases with Horse, Dog and Cat Disease Clinic,
Wrocław University of Environmental and Life Sciences, Poland
pl. Grunwaldzki 47, 50-366 Wrocław, Poland

Wojciech Zawadzki
Department of Animal Physiology and Biostructure, Faculty of Veterinary Medicine, Wrocław University of Environmental and Life Sciences, Poland
C.K. Norwida 31
50-375 Wrocław
Poland
Phone: +48 71 320 5401
email: wojciech.zawadzki@up.wroc.pl

Albert Czerski
Department of Animal Physiology,
Wrocław University of Environmental and Life Sciences, Poland
C. K. Norwida 31, 50-375 Wrocław, Poland

Mariusz Grządziel
Department of Mathematics,
Wrocław University of Environmental and Life Sciences, Poland
Grunwaldzka 53, 50-357 Wrocław, Poland

Jerzy Rudnicki
2^nd Department of General and Oncological Surgery,
Wroclaw Medical University, Poland
M. Skłodowskiej-Curie 66, 50-369 Wrocław, Poland

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