Electronic Journal of Polish Agricultural Universities (EJPAU) founded by all Polish Agriculture Universities presents original papers and review articles relevant to all aspects of agricultural sciences. It is target for persons working both in science and industry,regulatory agencies or teaching in agricultural sector. Covered by IFIS Publishing (Food Science and Technology Abstracts), ELSEVIER Science - Food Science and Technology Program, CAS USA (Chemical Abstracts), CABI Publishing UK and ALPSP (Association of Learned and Professional Society Publisher - full membership). Presented in the Master List of Thomson ISI.
2013
Volume 16
Issue 3
Topic:
Animal Husbandry
ELECTRONIC
JOURNAL OF
POLISH
AGRICULTURAL
UNIVERSITIES
Czerniawska-Pi徠kowska E. , Szewczuk M. , Choci這wicz E. , Cioch B. 2013. THE COMPARISON OF YIELD, NUTRITIVE VALUE AND TECHNOLOGICAL USEFULNESS OF MILK FROM HOLSTEIN-FRIESIAN COWS OF BLACK-AND-WHITE STRAIN DEPENDING ON THE IGFI/SnaBI AND IGF1R/HinfI POLYMORPHISMS, EJPAU 16(3), #05.
Available Online: http://www.ejpau.media.pl/volume16/issue3/art-05.html

THE COMPARISON OF YIELD, NUTRITIVE VALUE AND TECHNOLOGICAL USEFULNESS OF MILK FROM HOLSTEIN-FRIESIAN COWS OF BLACK-AND-WHITE STRAIN DEPENDING ON THE IGFI/SNABI AND IGF1R/HINFI POLYMORPHISMS

Ewa Czerniawska-Pi徠kowska, Ma貪orzata Szewczuk, Ewa Choci這wicz, Barbara Cioch
Department of Ruminant Science, West Pomeranian University of Technology in Szczecin

 

ABSTRACT

The aim of this study was to determine the frequency of the polymorphic variants of the IGFI/SnaBI and IGF1R/HinfI genes in the analyzed herd of Holstein-Friesian cows of Black-and-White strain and to compare the yield, concentration of protein fraction components as well as selected technological parameters of milk. For the IGFI/SnaBI polymorphism in the studied herd of dairy cows, the higher frequency of allele A (0.660) was found compared with allele B (0.340). In the analyzed group of cows, the BB individuals were not found. For the IGF1R/HinfI polymorphism, the AB heterozygotes (52%) predominated over the BB homozygotes (48%). No cows with the AA genotype were found. In the studied population, a higher frequency of allele B (0.740) than that of allele A (0.260) was observed. The cows with the IGFI/SnaBI AB genotype were characterized by higher milk, fat and protein yield than were cows with the AA genotype, whereas the AA genotype was associated with a higher protein (P ≤ 0.05), fat and lactose content in milk. On the other hand, the analyzed IGF1R/HinfI polymorphism affected milk, FCM, fat and protein yield as well as percentage of fat (P ≤ 0.05), protein and lactose in milk, favoring the BB genotype. The analysis of the selected technological parameters of milk in HF cattle with regard to the IGFI/SnaBI and IGF1R/HinfI polymorphisms showed that the milk from cows with the IGF1R/HinfI BB genotype was characterized by a higher casein and solids-not-fat content compared with the remaining analyzed cow genotypes (AB and AA).

Key words: milk, Holstein-Friesian breed, technological parameters, IGFI/SnaBI and IGF1R/HinfI.

INTRODUCTION

Nutritive quality of milk depends on genetic and environmental factors. Cow’s milk and dairy products are one of the main sources of animal protein in the human diet. Bioactive peptides, whose main source is milk proteins, besides the main nutritive function, have also a regulatory function in the human organism, which is the basis for regarding them as bioactive food ingredients [Meisel 2001]. Such properties are characteristic of, among other things, some casein fractions and lactoferrin. Milk proteins also determine the processing usefulness of milk and affect the way of this processing.

The aim of this study was to determine the frequency of the polymorphic variants of the IGFI/SnaBI and IGF1R/HinfI genes in the analyzed herd of Holstein-Friesian cows of Black-and-White strain and to compare the yield, concentration of protein fraction components as well as selected technological parameters of milk.

MATERIALS AND METHODS

The study was performed in the West Pomeranian Province. The research material consisted of 50 Holstein-Friesian cows (HF) of Black-and-White strain. The cows selected from the herd were managed in a confinement system and stayed under the same environmental conditions. Peripheral blood was taken from the external jugular vein to the test-tubes containing EDTA as an anticoagulant. The MasterPureTM Genomic DNA Purification Kit (Epicentre Technologies) was used for DNA isolation. The polymorphism of the IGF1 and IGF1 receptor genes was identified by means of PCR-RFLP. The amplification of the IGF1 gene promoter fragment was performed using primer sequences designed by Ge et al. [2001], whereas the insulin-like growth factor 1 receptor gene fragment (IGF1R intron 4) within which the polymorphic site deposited in the NCBI data base (Accession No. rs41960620) is located was amplified with the original primer sequences designed using Primer3 software (Table 1).

Table 1. Primer sequences used for the amplification of selected gene fragments
Gene
Primer
Primer sequence
Fragment
IGF1 / SnaBI
IGF677F
5’-ATTACAAAGCTGCCTGCCCC-3’
250 bp
IGF897R
5’-ACCTTACCCGTATGAAAGGAATATACGT-3’
IGF1R / HinfI
IGF1RF
5’- CTGGATATGTCCGCCTTAGC - 3’
231 bp
IGF1RR
5’- ACAGCTCTTGTGTCCCTGGT – 3’

PCR was performed in the reaction mixture containing the DNA template, thermostable enzyme – DNA Taq polymerase (FermentasTM), buffer with (NH4)2SO4 (Fermentas™) and MgCl2 (Fermentas™), nucleotide mix, pair of primer sequences (Oligo, IBB PAN, Warsaw). The whole reaction mixture was adjusted to a final volume of 20 μl with the nuclease-free deionized water (Epicentre TechnologiesTM). The following reaction thermal profile was applied: preliminary denaturation of DNA template at 94ºC for 5 min, followed by 33 cycles: denaturation of the double-stranded DNA template at 94ºC for 50 s, annealing of the primers to single-stranded template at 59.5ºC (Ta) for 60 s, synthesis of complementary strands (polymerase activity) at 72ºC for 50 s and final elongation at 72ºC for 7 min. The reaction was carried out in the BiometraTM thermocycler. A specific DNA product of 250 bp was obtained for the gene encoding insulin-like growth factor I, whereas the products of 231 bp were obtained for the IGF-I receptor gene. Amplification products were digested with restriction enzymes. The IGF1 gene fragment was digested with 5 units of Eco105I (SnaBI) (10 U/µl, TAC↓GTA) (Fermentas™) at 37ºC for 3 h. In the case of the IGF1R gene fragment, the PCR product was digested for 3 h at 37ºC with 5 units of the HinfI restriction enzyme (10 U/µl, G/ANTC) (Fermentas™). In order to verify the obtained results, 10 µl of the product was separated electrophoretically in 2% agarose gels (Basica Prona Agarose™) with ethidium bromide. The gels were subjected to electric field in the presence of 1 x TBE buffer, and then analyzed under UV light (312 nm) in the Vilber Lourmat™ transilluminator. The length of the obtained products was compared with the pUC19/MspI molecular mass marker (Fermentas™).

In the collected milk samples, the cytological quality of milk was first determined with the Somacount 150 (Bentley) apparatus. All milk samples containing over 400,000 cells per ml3 (somatic cell count), which did not comply with the standards for the class Extra, were eliminated. Moreover, the milk chemical composition was determined, i.e. fat, total protein, lactose, dry matter, solids-not-fat with the MilkoScan FT120 (Foss Electric) apparatus. Bioactive milk constituents were determined comprehensively. The content of whey proteins such as: β-lactoglobulin (β-LG), α-lactoalbumin (α-LA), lactoferrin (Lf), lactoperoxidase (Lp), lysozyme (Lz) and bovine serum albumin (BSA) was determined with HPLC and Supelcosil LC-380 column according to the method by Kuczyńska et al. [2011].

Milk yield analysis was performed using the A4 method. It was based on the data from milk recording including milk, fat and protein yield (in kg) as well as fat and protein percentage in milk. To compare the obtained results, 4% fat corrected milk (FCM) yield was also calculated.

The data were analyzed statistically. Means and standard deviation were calculated. The significance (P ≤ 0.05) of differences between the mean values of the analyzed traits with regard to genotypes (IGFI/SnaBI and IGF1R/HinfI) was determined with one-way analysis of variance and the Duncan’s multiple range test using Statistica®10 PL software.

RESULTS

In the analyzed population of Holstein-Friesian (HF) cows (Table 2), two IGFI/SnaBI genotypes, i.e. AA and AB determined by two alleles (IGFI/SnaBI A and IGFI/SnaBI B) were identified. In the analyzed group of cows, the heterozygous (AB) animals predominated (68%). A higher frequency of allele A (0.660) was found compared with allele B (0.340). For the IGF1R/HinfI polymorphism, two alleles (A and B) were identified. The following allele frequencies were observed: allele A – 0.260 and allele B – 0.740. In the studied herd, the AB heterozygotes predominated (52%) over the BB homozygotes (48%), whereas the AA genotype was not found.

Table 2. Genotype and allele frequencies for the IGFI/SnaBI and IGF1R/HinfI polymorphisms
Number
IGFI/SnaBI genotype
Alleles
AA
AB
BB
A
B
n
16
34
frequency
0.436
0.449
0.116
0.660
0.340
IGF1R/HinfI genotype
Alleles
AA
AB
BB
A
B
n
26
24
frequency
0.068
0.385
0.548
0.260
0.740

The results presented in Table 3 indicate that the IGFI/SnaBI AB genotype was more advantageous in terms of milk (8407 kg), fat (334.94 kg), protein (271.15 kg) and FCM (8387 kg) yield compared with the AA genotype.

Table 3. Milk performance of cows with regard to the genotypes for the IGFI/SnaBI and IGF1R/HinfI polymorphisms
Analyzed traits
Genotype
IGFI/SnaBI
IGF1R/HinfI
AA
AB
AB
BB
± S
± S
± S
± S
Milk [kg]
7786 ± 1034
8407 ± 1253
8188 ± 1098
8230 ± 1349
Fat [kg]
329.94 ± 41.75
334.94 ± 63.35
319.04 ± 34.10
348.83 ± 71.84
Protein [kg]
259.81 ± 31.06
271.15 ± 36.24
266.15 ± 32.55
269.00 ± 37.67
Fat [%]
4.27 ± 0.49
3.99 ± 0.49
3.92a ± 0.36
4.25a ± 0.59
Protein [%]
3.35b ± 0.20
3.24b ± 0.15
3.26 ± 0.17
3.29 ± 0.18
FCM [kg]
8063 ± 945
8387 ± 1361
8061 ± 882
8525 ± 1525
Lactose [%]
4.88 ± 0.17
4.87 ± 0.20
4.91 ± 0.21
4.83 ± 0.16
Urea [mg/l]
193 ± 58.36
171 ± 58.84
185 ± 63.58
171 ± 54.09
SCC [x 1000 in 1 ml3]
105 ± 89.35
140 ± 99.3
127 ± 95.84
126 ± 98.90
abc – Means within columns followed by small letters are significantly different at (P ≤ 0.05).

In the present study, a higher fat (by 0.28%), protein (by 0.11%) and lactose (by 0.01%) content was found in the milk from AA cows. An analysis of the data concerning milk performance traits with regard to the IGF1R/HinfI gene polymorphism in Polish Holstein-Friesian cows shows the trend towards higher milk, fat, protein and FCM yield in the BB homozygotes. It should also be emphasized that the milk from these cows was characterized by a higher fat (0.33%) and protein (0.03%) content. The differences were statistically significant at P≤ 0.05. A higher lactose percentage was found (Table 3) in milk from the AB heterozygous cows (4.91%). The IGFI/SnaBI AA genotype was characterized by a higher content of urea (193 mg/l) and a lower somatic cell count (SCC – 105 x 1000 in 1 ml3).

In the studied herd, milk density (Table 4) was identical and amounted to 1.031 (g/ cm3). In the present study, the highest dry matter content for the IGFI/SnaBI and IGF1R/HinfI polymorphisms was found in cows with the AA (13.25%) and BB (13.24%) genotypes, respectively. In the present study, the FFA content ranged from 0.17 mmol/100 g of fat for the IGFI/SnaBI AA genotype to 0.19 mmol/100 g of fat for IGF1R/Hinf BB genotype. The highest casein content (Table 4) was found in milk from cows with the IGF1R/HinfI BB genotype (2.89%) and the lowest one in AB cows (2.83%). For the IGFI/SnaBI polymorphism, these values were similar for the analyzed genotypes (2.85% and 2.86% for the AA and AB genotypes, respectively). The cows with the IGFI/SnaBI AB genotype were characterized by higher milk, fat and protein yield than were cows with the AA genotype, whereas the AA genotype was associated with a higher protein (P ≤ 0.05), fat and lactose content in milk. On the other hand, the analyzed IGF1R/HinfI polymorphism affected milk, FCM, fat and protein yield as well as percentage of fat (P ≤ 0.05), protein and lactose in milk, favoring the BB genotype. The analysis of the selected technological parameters of milk in HF cattle with regard to the IGFI/SnaBI and IGF1R/HinfI polymorphisms showed that the milk from cows with the IGF1R/HinfI BB genotype was characterized by a higher casein, potential acidity, citric acid  and solids-not-fat content compared with the remaining analyzed cow genotypes. The IGFI/SnaBI AB and IGF1R/HinfI AB genotypes were characterized by a slightly lower freezing point (0.558°C).

Table 4. Parameters of technological usefulness of milk with regard to the genotypes for the IGFI/SnaBI and IGF1R/HinfI polymorphisms
Analyzed traits
Genotype
IGFI/SnaBI
IGF1R/HinfI
AA
AB
AB
BB
± S
± S
± S
± S
Density [g /cm3]
1.031 ± 0.001
1.031 ± 0.002
1.031 ± 0.001
1.031 ± 0.001
Dry matter [%]
13.25 ± 1.16
13.14 ± 0.83
13.12 ± 0.95
13.24 ± 0.94
Solids-not-fats [g/100 g milk]
8.99 ± 0.39
8.99 ± 0.35
8.97 ± 0.38
9.01 ± 0.35
Potential acidity [°SH]
6.90 ± 0.98
6.97 ± 0.97
6.70 ± 1.05
7.21 ± 0.80
Citric acid [g /100 g milk]
0.12 ± 0.02
0.12 ± 0.02
0.12 ± 0.02
0.13 ± 0.02
FFA[mmol/100 g fat]
0.17 ± 0.07
0.18 ± 0.04
0.18 ± 0.06
0.19 ± 0.06
Freezing point [°C]*
0.557 ± 0.025
0.558 ± 0.022
0.558 ± 0.024
0.557 ± 0.022
Casein [%]
2.85 ± 0.28
2.86 ± 0.25
2.83 ± 0.25
2.89 ± 0.26
* – absolute value

Based on the results presented in Table 5, it was found that the milk from cows with the IGF1R/HinfI BB genotype was characterized by a higher content of β-lactoglobulin (β – LG – 4.28 g/l), α – lactoalbumin (α – LA – 1.57 g/l), lactoferrin (Lf – 0.26 g/l), and lysozyme (Lz – 38.08 μg/l). The IGF1R/HinfI AB genotype was was characterized by the lowest content of Lz (28.46 μg/l). The differences were statistically significant at P≤ 0.05. The milk from cows with the IGFI/SnaBI AA genotype was characterized by a lower content of  β – LG (4.03 g/l), α – LA (1.30 g/l), Lp (0.18 mg/l), Lf (0.23 g/l). The IGF1R/HinfI AB genotype was characterized by a higher content of bovine serum albumin (BSA – 0.47 g/l) and the IGF1R/HinfI BB genotype a lower (BSA – 0.43 g/l).

Table 5. The content of bioactive milk proteins with regard to the genotypes for the IGFI/SnaBI and IGF1R/HinfI polymorphisms
Whey protein
Genotype
IGFI/SnaBI
IGF1R/HinfI
AA
AB
AB
BB
± S
± S
± S
± S
β-LG [g /l]
4.03 ± 0.84
4.31 ± 1.07
4.15 ± 0.91
4.28 ± 1.11
α-LA [g/l]
1.32 ± 0.41
1.51 ± 0.52
1.34 ± 0.39
1.57 ± 0.57
Lf [g /l]
0.23 ± 0.08
0.25 ± 0.09
0.23 ± 0.09
0.26 ± 0.09
BSA [g/l]
0.45 ± 0.18
0.45 ± 0.25
0.47 ± 0.19
0.43 ± 0.26
Lp [mg/l]
0.18 ± 0.07
0.22 ± 0.14
0.20 ± 0.10
0.22 ± 0.15
Lz [μg/l]
35.73 ± 20.97
31.83 ± 10.60
28.46a ± 11.72
38.08a ± 15.99
abc – Means within columns followed by small letters are significantly different at P ≤ 0.05.

DISCUSSION

In the study by Szewczuk et al. [2011], out of the four analyzed cattle breeds (Holstein-Friesian of Black-and-White strain, Hereford, Red Angus and Limousin), the highest frequency of the IGF1R/HinfI AB genotype (52% of the analyzed population) was recorded in the Hereford breed. In the case of Holstein-Friesian breed, the cited authors found the highest frequency in the BB homozygotes (0.4950), a similar frequency for the AB genotype (0.4555), and the lowest frequency for the AA genotype (0.0495). Similarly, as in the present study (Table 2), the authors observed a higher frequency of allele B (0.7228) compared with allele A (0.2772). For the IGFI/SnaBI polymorphism in the Polish Holstein-Frisian breed, Szewczuk et al. [2012] showed that the AB genotype was the most common (0.5622). The authors found a higher frequency of allele A (0.5448) compared with allele B (0.4552), which was confirmed in the present study (Table 2).

Szewczuk et al. [2012] similarly found the highest milk yield in cows with the AB genotype. On the other hand, the milk from BB cows was characterized by the highest fat yield (307 kg) as well as protein (3.38%) and fat (4.14%) content according to the cited authors.

Urea content in milk in the analyzed population remained within the physiological range 150–300 mg/l (Table 3). Similar values (163–277 mg/l) were obtained by Kuczyńska et al. [2011], who analyzed various models of cow feeding on organic farms.

Technological usefulness of milk is mainly determined by its density, which reflects the content of basic chemical constituents, solids-not-fat, acidity, citric acid and casein content [Kuczyńska 2011].

There is a lack of other literature data on the association between the technological usefulness of milk and the IGFI/SnaBI and IGF1R/HinfI polymorphisms. An increase in the level of free fatty acids (FFA) in milk causes the formation of undesirable odors and aftertaste in dairy products. Milk acidity is the basic criterion of its freshness evaluation and determines its technological usefulness. According to the Polish Norm [1999], the titratable acidity of raw milk should be 6.0–7.5°SH. In the present study, these values remained within the normal range.

Cheng et al. [2008] report that the lactoferrin concentration in milk may be affected by many different factors, among other things, health state of cows, yield and even the number of the analyzed samples. The same lactoperoxidase content in the present study (Lp – 0.22 mg/l) was recorded in the milk from cows with the IGFI/SnaBI AB and IGF1R/HinfI BB genotypes. Taking into consideration the novelty of the present work, the research should be continued on a larger population of animals.

An effect of the IGFI/SnaBI and IGF1R/HinfI polymorphisms on the content of individual protein fraction components has not been fully understood so far. Due to the lack of available literature on this subject, the comparison of results was impossible. The experiment described in this study was aimed at supplementing the knowledge in this field.

REFERENCES

  1. Cheng J.B., Wang J.Q., Bu D.P., Liu G.L., Zhang C.G., Wei H.Y., Zhou L.Y., Wang J.Z., 2008. Factors affecting the lactoferrin concentration in bovine milk. J. Dairy Sci., 91 (3), 970–976.
  2. Ge W., Davis M.E., Hines H.C., Irvin K.M., Simmen R.C.M., 2001. Association of genetic marker with blood serum insulin-like growth factor-I concentration and growth traits in Angus cattle. J. Animal Sci., 79, 1757–1762.
  3. Kuczyńska B., 2011. Składniki bioaktywne i parametry technologiczne mleka produkowanego w gospodarstwach ekologicznych i konwencjonalnych [Bioactive components and technological parameters of milk produced on organic and conventional farms]. Wyd. SGGW, Warsaw (in Polish).
  4. Kuczyńska B., Nałęcz-Tarwacka T., Puppel K., Gołębiewski M., Grodzki H., Slósarz J., 2011. Zawartość bioaktywnych składników mleka w zależności od modelu żywienia krów w certyfikowanych gospodarstwach ekologicznych [The content of bioactive components in milk depending on cow feeding model in certified ecological farms]. J. Res. Appl. Agric. Engineering, 56 (4), 7–13 (in Polish).
  5. Meisel H., 2001. Bioactive peptides from milk proteins: a perspective for consumers and producers. Australian J. Dairy. Technol., 56, 83–92.
  6. Polish Norm PN-A-86002:1999. Raw milk.
  7. Szewczuk M., Wilkowiecki P., Zych S., Czerniawska-Piątkowska E., Rzewucka-Wójcik E., Wójcik J., 2011. Identification of the polymorphism in intron 4 of the bovine gene coding for insulin-like growth factor I receptor (IGF1R/HinfI). Conf. „The role of tradition in animal husbandry and breeding in the age of globalization”. LXXVI Sci. Conf. of Polish Society of Animal Production. Poznań (14-16.09.2011), 80.
  8. Szewczuk M., Zych S., Czerniawska-Piątkowska E., Wójcik J., 2012. Association between IGF1R/i16/TaqI and IGF/SnaBI polymorphism and milk production traits in Polish Holstein-Friesian cows. Anim. Sci. Pap. Rep., 30 (1), 13–24.

Accepted for print: 23.08.2013


Ewa Czerniawska-Pi徠kowska
Department of Ruminant Science, West Pomeranian University of Technology in Szczecin
Doktora Judyma 10
71-460 Szczecin
Poland
email: Ewa.Czerniawska-Piatkowska@zut.edu.pl

Ma貪orzata Szewczuk
Department of Ruminant Science, West Pomeranian University of Technology in Szczecin
Doktora Judyma 10
71-460 Szczecin
Poland

Ewa Choci這wicz
Department of Ruminant Science, West Pomeranian University of Technology in Szczecin
Doktora Judyma 10
71-460 Szczecin
Poland

Barbara Cioch
Department of Ruminant Science, West Pomeranian University of Technology in Szczecin
Doktora Judyma 10
71-460 Szczecin
Poland

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