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LITHUANIAN UNIVERSITY OF HEALTH SCIENCES
VETERINARY ACADEMY
Dissertation has been prepared at the Lithuanian Veterinary Academy,
K. Janušauskas Laboratory of Animal Genetics during the period of 2005–
2010.
Scientific Supervisor –
Prof. Dr. Ilona Teodora MICEIKIENö (Lithuanian University of Health
Sciences Veterinary Academy, Biomedical Sciences, Zootechny – 13 B).
Zootechny Science Councile:
Chairman –
Prof. Habil. Dr. Romas GRUŽAUSKAS (Lithuanian University of
Health Sciences Veterinary Academy, Biomedical Sciences, Zootechny –
13 B).
Natalija Krasnopiorova
CATTLE GROWTH HORMONE, LEPTIN AND
MYOSTATIN GENE POLYMORPHISM AND
INFLUENCE TO FARM TRAITS IN CATTLE BRED
IN LITHUANIA
Doctoral Dissertation Summary
Biomedical Sciences, Zootechny (13 B)
Members:
Dr. Natalija BURBULIS (Lithuanian University of Agriculture, Biomedical Sciences, Agronomy – 06 B);
Dr. Sigita KERZIENö (Lithuanian University of Health Sciences Veterinary Academy, Biomedical Sciences, Zootechny – 13 B).
Assoc. Prof. Dr. Sigut÷ KUUSIENö (Lithuanian Forest Research Institute, Biomedical Sciences, Biology – 01 B);
Dr. Nijol÷ PEČIULAITIENö (Lithuanian University of Health Sciences
Veterinary Academy, Biomedical Sciences, Zootechny – 13 B);
Opponents:
Prof. Dr. Vida JUOZAITIENö (Lithuanian University of Health
Sciences Veterinary Academy, Biomedical Sciences, Zootechny – 13 B);
Prof. Habil. Dr. Aniolas SRUOGA (Vytautas Magnus University, Biomedical Sciences, Biology – 01 B).
Dissertation will be defended at the open session of the Research Council of Lithuanian University of Health Sciences Veterinary Academy Dr.
S. Jankausko auditorium 11 am LT on 23 of December 2010.
Address: Tilž÷s 18, LT-47118 Kaunas, Lithuania.
The summary of doctoral dissertation was sent out on the 23th of November 2010 according to the confirmed address list.
Kaunas, 2010
The doctoral dissertation is available at the library of Veterinary Academy Lithuanian University of Health Sciences.
LIETUVOS SVEIKATOS MOKSLŲ UNIVERSITETO
VETERINARIJOS AKADEMIJA
Disertacija rengta 2005–2010 metais Lietuvos veterinarijos akademijoje
K. Janušausko gyvūnų genetikos laboratorijoje.
Mokslinio darbo vadov÷ –
Prof. dr. Ilona Teodora MICEIKIENö (Lietuvos sveikatos mokslų universiteto Veterinarijos akademija, biomedicinos mokslai, zootechnika –
13 B).
Zootechnikos mokslo krypties taryba:
Pirmininkas –
Prof. habil. dr. Romas GRUŽAUSKAS (Lietuvos sveikatos mokslų universiteto Veterinarijos akademija, biomedicinos mokslai, zootechnika –
13 B).
Natalija Krasnopiorova
GALVIJŲ AUGIMO HORMONO, LEPTINO IR
MIOSTATINO GENŲ ĮVAIROVö BEI ĮTAKA ŪKINöMS
SAVYBöMS LIETUVOJE VEISIAMŲ GALVIJŲ TARPE
Nariai:
Dr. Natalija BURBULIS (Lietuvos žem÷s ūkio universitetas, biomedicinos mokslai, agronomija – 06 B);
Dr. Sigita KERZIENö (Lietuvos sveikatos mokslų universiteto Veterinarijos akademija, biomedicinos mokslai, zootechnika – 13 B).
Doc. dr. Sigut÷ KUUSIENö (Lietuvos miškų institutas, biomedicinos
mokslai, biologija – 01 B);
Dr. Nijol÷ PEČIULAITIENö (Lietuvos sveikatos mokslų universiteto
Veterinarijos akademija, biomedicinos mokslai, zootechnika – 13 B);
Daktaro disertacijos santrauka
Biomedicinos mokslai, zootechnika (13 B)
Oponentai:
Prof. dr. Vida JUOZAITIENö (Lietuvos sveikatos mokslų universitetas
Veterinarijos akademija, biomedicinos mokslai, zootechnika – 13 B);
Prof. habil. dr. Aniolas SRUOGA (Vytauto Didžiojo universitetas, biomedicinos mokslai, biologija – 01 B).
Disertacija bus ginama viešame Zootechnikos mokslo krypties tarybos
pos÷dyje, kuris įvyks 2010 m. gruodžio m÷n. 23 d. 11 val. Lietuvos sveikatos mokslų universiteto Veterinarijos akademijos Dr. S. Jankausko auditorijoje.
Adresas: Tilž÷s 18, LT-47118, Kaunas, Lietuva.
Kaunas, 2010
Disertacijos santrauka išsiųsta 2010 m. lapkričio m÷n. 23 d. pagal patvirtintą adresų sąrašą.
Disertaciją galima peržiūr÷ti LSMU Veterinarijos akademijos bibliotekoje.
INTRODUCTION
Aim of the present study
To investigate polymorphism of growth hormone, leptin and myostatin
genes in cattle grown in Lithuania and their influence to farm traits.
It is known that the productivity of livestock and other farming characteristics, such as meat yield, growth rate, milk production, milk composition,
and others are inherited from generation to generation, and their formation
and functional characteristics are determined by genes. Currently, with the
help of new molecular methods, genetic evaluation of cattle is carried out
directly analysing the DNA, which contains all the genetic information of
animal’s inherited characteristics. These methods allow for detailed investigation of bovine genome, and determination as which genes and how operate and influence meat and milk quantity and quality, what genetic factors
influence cattle health, genetic diversity and genetic relationships among
breeds (Blott et al., 1998).
Specific synthetic markers are used for cattle genotyping that recognize
and determine such characteristics as meat yield, milk yield, but less is
known of genes that are related to reproductive performance. Specific genetic markers, if used in a targeted way in animal genotyping, significantly
increase the efficiency of selection because they can help to carry out the
selection by one or more features, which are manifested due to the occurrence of one or more bonded genes. A method for the determination of such
locus of quantitative traits is called Marker Assisted Selection (MAS) (Lien
et al., 1993).
There is a number of genes discovered that affect the meat and milk production. This is GH (growth hormone), GHR (growth hormone receptor),
STAT5 (signal vector and transcription activator), MSTN (myostatin), LEP
(leptin), IGF1 (insulin growth factor), PIT1 (pituitrin transcription factor),
PREF1 (preadipocyte factor), casein and lactoglobuline genes. Research
shows that a new gene identification technology allows investigating the
genotype of the animal and determining the genes encoding the productivity
and quality traits, and use them in the selection process as genetic markers.
Use of genetic markers for livestock breeding offers the opportunity to
evaluate the animal from inside and make full use of the inherent farming
characteristics. Genetic markers can be used both for the identification of a
single gene and a gene group of a decisive trait or traits group. Another advantage for the use of genetic markers in breeding is that this method of
livestock valuation is reliable, allows in a young age to identify the genes
controlling the selective and technological value of livestock, to assess the
genetic variability and genetic defects of farm animals, and enables to avoid
the manifestation of unwanted traits on time. Use of genetic markers in selection can greatly accelerate the breeding process, improve the quality of
agricultural production, reduce its production costs and make production
more competitive in foreign markets.
Practical importance
The polymorphisms of the bovine growth hormone and leptin gene
found and their influence on the characteristics of farming such as milk production, composition and fattening indicators enable them to be used as
biomarkers in the selection process thus accelerating it through the molecular technologies, increasing the breeding value of livestock and improving
their farming characteristics and quality of products.
5
6
Tasks of the present study
1. Genotype polymorphisms in cattle GH (growth hormone), LEP
(leptin) and MSTN (myostatin) genes.
2. Estimate allele and genotype frequencies in GH, LEP and MSTN
locuses in cattle grown in Lithuania.
3. Investigate influence of one nucleotide polymorphisms (VNPs) in
GH and LEP genes for cattle milk yield, milk composition and weight gain.
4. Investigate influence of VNP in GH gene for cattle semen quality.
Novelty of the present study
In this study first time in Lithuanian cattle population (11 breeds and 3
crossbreeds) are presented SNPs polymorphisms in candidate genes GH,
LEP, MSTN and their influence on farm traits.
MATERIAL AND METHODS
Selected animals
In different stages of this research in total were genotyped 359 animals
belonging to Lithuanian Black and White (LBW), Old type Lithuanian
Black and White (O-LBW), Lithuanian Red (LR), Danish Black and White
(DBW), Danish Red (DR), Holstein (H), Hereford (He), Limousin (Li),
Simmental (Si), Sharolais (Sa), Angus (An) and crossbreds of Lithuanian
Black and White with Belgian Blue, Sharolais and Limousins. 270 animals
were genotyped for myostatin gene polimorphisms, 128 cows for GH and
LEP gene polymorphisms investigating influence to milk traits, 163 bulls for
GH and LEP gene polymorphisms investigating influence to beef traits, 33
bulls for GH gene polymorphisms to investigate influence to semen quality.
The data of productivity of animals were obtained from Record Processing Center SE “Kaimo verslo pletros ir informacijos centras”, daily gain
records from SE “Silutes veislininkyste”, semen quality records from SE
“Panevezio veislininkyste”.
Fat, kg ijklm = µ + GHi + LEPj + breedk + lactationl +
lactationlenghreg + eijklm
Research methods
Blood samples to vacuum test-tubes were collected from jugular vein
(Venoject, Belgium) with EDTA (K3). DNA was extracted using standard
phenol-chloroform purification method (Miller et al, 1988). Hair root samples for DNA genotyping were extracted using method recieved from Van
Haeringen laboratory (Holland) and deep – frozen sperm using standard
phenol-chloroform purification method according Dr. J. Kantanen MTT
(Finland).
The method of polymerase chain reaction (PCR) and restriction length
polymorphism (RFLP) (Sakai et al., 1988) were used to genotype GH, LEP
and MSTN polymorphisms. All PCR reactions were performed using Applied Biosystems 2700 Thermal Cycler. The PCR based detection of polymorphisms was carried out using primers’ sequencies, polymerase chain
reaction conditions and restriction endonucleases according to for growth
hormone gene (Sadeghi et al., 2005), for leptin gene (Choudhary et al.,
2005) and for myostatin gene (Smith et al., 2000; Stasio and Rolando,
2005), techniques and procedures used at the Janusauskas Laboratory of
Animal Genetics, Lithuanian Veterinary Academy. Visualization of the different GH, LEP and MSTN genetic types was carried out by agarase gel
electroforesis after staining the gels with ethidium bromide, using Heliorab
video documentation system.
Protein, kg ijklm = µ + GHi + LEPj + breedk + lactationl +
lactationlenghreg + eijklm
Statistical analysis
Observed number of alleles, genotypes, observed and expected heterozigosity for each locus and each breed, average heterozigosity over all loci
were used to assess the genetic variability of studied populations. The Hardy
Weinberg equilibrium was performed to evaluate the population differentiation.
Associations between different polymorphic sites and milk yield, milk
composition and weight daily gain in GH and LEP genes of Lithuanian cattle were analysed using single factorial and multifactioral analysis of variance (ANOVA) with R package (Gentlemen and Ihaka, 1997). Following General Linear Models were used for calculations of effects:
Milk yield, kg ijklm = µ + GHi + LEPj + breedk + lactationl +
lactationlenghreg + eijklm
7
Fat, % ijklmn = µ = + GHi + LEPj + breedk + lactationl + eijklm
Protein, %ijklmn = µ = + GHi + LEPj + breedk + lactationl + eijklm
Average milk yield per day, kg ijklm = µ = + GHi + LEPj +
breedk + lactationl + eijklm
Daily gain, g ijklm = µ = + GHi + LEPj + breedk + bulll + eijklm
RESULTS AND DISCUSSION
In recent years, there is a significant increase in interest in the beef and
dairy cattle breeding based on genetic technologies. Quantitative traits, such
as animal growth rate, feed conversion efficiency, carcass and internal fat
mass, or such as milk, fat and protein content and some technological properties of milk are controlled by multiple loci in the chromosomes. Genes
candidates are selected on the basis of known relationships between physiological and biochemical processes and properties. Sequence alterations may
be used as genetic markers (Beckmann and Soller, 1983).
Distribution of alleles’ and genotypes’ frequencies in single nucleotide
polymorphisms between growth hormone (GH) and leptin (LEP) genes
The polymorphism of the growth hormone and leptin gene were studied
in 10 dairy and beef cattle breeds. Frequencies of alleles and genotypes of
the growth hormone GH and leptin LEP genes for each tested breed were
calculated. According to our findings and other scientists, the growth hormone GH gene showed two alleles A and B, with A allele predominant in all
breeds (Sabour et al., 1997; Lucy et al., 1993; Kemenes et al., 1999; Vukasinovic et al., 1999). In our findings the frequency of allele A of the
growth hormone GH gene varied from (0.563) in Simmental breed to
(0.900) in Hereford breed. Selection is carried out in Europe in respect of
the allele B and specific herds of cattle are formed. Much attention is paid to
the allele B because it is associated with improvement of milk processing
characteristics (Strzalkowska et al., 2002; Lien et al., 1999; McLean et al.,
1984) and meat properties (Dario et al., 2004). The highest frequency of GH
8
B allele we found in Danish Red and Simmental cattle, the lowest in Hereford. Our data correlate with other scientists testing results. High frequency
of GH B allele was identified in Slovak Simmental (Chrenek et al., 1991)
and Jerry (Lucy et al., 1993) breeds, low frequency in Hereford breed (Vukasinovic et al., 1999). According to our results, two alleles A and B of the
leptin gene were found. The frequency of allele A of leptin LEP gene in the
investigated cattle ranged from (0.700) in the Danish Red breed to (0.875) in
Simmental breed. Comparing our findings with literature data, similar results of studies were obtained. These results strongly suggest that selection
forces could act against the LEP gene allele B or the selection environment
was more favourable to allele A. Our research results show that cattle breeds
bred in Lithuania usually have the same LEP alleles, as the European cattle
breeds. No statistically significant differences were found between dairy and
beef cattle breeds for the growth hormone and leptin genes’ allele frequencies (Table 1).
Table 1. Frequencies of growth hormone GH and leptin LEP gene alleles in 10 cattle breeds bred in Lithuania
1 lentel÷. Augimo hormono GH ir leptino LEP genų alelių dažniai 10
Lietuvoje veisiamų galvijų veisl÷se
Breed / Veisl÷
DBW / DJ
DR / DZ
H
LBW-OT / SG-LJ
LBW / LJ
LR / LZ
Milk breeds /
Pienin÷s veisl÷s
HE
SI
SA
LI
Beef breeds /
M÷sin÷s veisl÷s
Tested group /
Tirta grup÷
Frequency of GH
gene alelles /
GH geno aleliai
A
B
0.692
0.308
0.600
0.400
0.692
0.308
0.722
0.278
0.823
0.177
0.739
0.261
Frequency of LEP
gene alleles /
LEP geno aleliai
A
B
0.705
0.295
0.700
0.300
0.808
0.192
0.778
0.222
0.855
0.145
0.717
0.283
0.751
0.249
0.787
0.213
0.900
0.563
0.850
0.639
0.100
0.438
0.150
0.361
0.850
0.875
0.775
0.861
0.150
0.125
0.225
0.139
0.750
0.250
0.824
0.176
0.751
0.249
0.799
0.201
9
Table 2. Frequencies of growth hormone GH and leptin LEP gene
genotypes in 10 cattle breeds bred in Lithuania
2 lentel÷. Augimo hormono GH ir leptino LEP genų genotipų dažniai
10 Lietuvoje veisiamų galvijų veisl÷se
Breed /
Veisl÷
DBW / DJ
DR / DZ
H
LBW-OT / SG-LJ
LBW / LJ
LR / LZ
Milk breeds /
Pienin÷s veisl÷s
HE
SI
SA
LI
Beef breeds /
M÷sin÷s veisl÷s
Tested group /
Tirta grup÷
GH genotypes /
GH genotipai
AA
AB
BB
0.487 0.410 0.103
0.450 0.300 0.250
0.462 0.462 0.077
0.444 0.556
0.731 0.183 0.086
0.565 0.348 0.087
LEP genotypes /
LEP genotipai
AA
AB
BB
0.590 0.231 0.179
0.550 0.300 0.150
0.692 0.231 0.077
0.667 0.222 0.111
0.742 0.226 0.032
0.522 0.391 0.087
0.604
0.294
0.102
0.660
0.254
0.086
0.900
0.500
0.750
0.583
0.125
0.200
0.111
0.100
0.375
0.050
0.306
0.700
0.750
0.550
0.750
0.300
0.250
0.450
0.222
0.028
0.681
0.138
0.181
0.660
0.330
0.011
0.629
0.244
0.127
0.660
0.278
0.062
Three different genotypes – AA, AB and BB – were found in both the
growth hormone GH and leptin LEP genes loci. The AA genotype of the
growth hormone GH gene in the lowest frequency (0.444) was found in the
old genotype Lithuanian Black and White cattle breed, the highest (0.900) in
Hereford breed of cattle. Meanwhile, the GH genotype AB with lowest frequency of (0.111) was found in Limousin cattle breed, the highest (0.556) in
the old genotype Lithuanian Black and White cattle breed. In Hereford cattle
breed, GH AB genotype was not identified. The genotype BB of the growth
hormone GH was found the highest frequency (0.375) in Simmental breed.
In the old Lithuanian genotype Black and White cattle breed, GH BB genotype was not found. The genotype AA of leptin gene ranged from (0.522) to
(0.750) frequencies. Meanwhile, genotype LEP AB ranged from (0.222) in
Limousin and in the old genotype Lithuanian Black and White cattle breeds
to (0.450) in Charolais cattle breed. LEP BB genotype was not found in
10
Hereford, Simmental and Charolais breeds of cattle. According to researcher
Bonvillani (2000) the LEP gene genotype BB was not detected in Argentinian Holstein cattle; Simmental and Hereford cattle breeds showed the highest frequency of genotype AA as well as in our study (Table 2).
All the tested breeds had lower observed heterozygosity than expected
value with the exception of two breeds: old genotype Lithuanian Black and
White cattle and Charolais, where the observed heterozygosity was higher
than the expected, but the differences were not statistically significant. Statistically significant deviation from Hardy-Weinberg equilibrium's law and
little observed heterozygosity was found in the Lithuanian Black and White
and Limousine breeds. After grouping the stock into dairy and beef breeds,
both groups showed a statistically significant deviation from Hardy Weinberg equilibrium with the downward heterozygosity trend, with 7.23
(p<0.007) and 14.6 respectively (p<0.001) (Table 3).
Table 3. Mean expected and observed heterozigosity in 10 cattle
breeds bred in Lithuania
3 lentel÷. Vidutinis teorinis ir faktinis heterozigotiškumas 10 Lietuvoje veisiamų galvijų veisl÷se
Breed / Veisl÷
DBW / DJ
DR / DZ
H
LBW-OT / SG-LJ
LBW / LJ
LR / LZ
Milk breeds /
Pienin÷s veisl÷s
HE
SI
SA
LI
Beef breeds /
M÷sin÷s veisl÷s
Tested group /
Tirta grup÷
Observed
Expected
Heterozygosity / Heterozygosity /
Faktinis Hetero- Teorinis Heterozigotiškumas
zigotiškumas
0.321
0.421
0.300
0.450
0.346
0.368
0.389
0.373
0.204
0.270
0.370
0.396
χ2 – test
(p – meaning) /
(p – reikšm÷)
3.24 (0.072)
2.04 (0.154)
0.31 (0.580)
0.93 (0.335)
3.98 (0.046)
0.1 (0.756)
0.274
0.355
7.23 (0.007)
0.150
0.188
0.325
0.167
0.218
0.355
0.302
0.350
1.88 (0.170)
2.23 (0.136)
1.65 (0.199)
9.62 (0.002)
0.234
0.332
14.6 (0.001)
0.261
0.348
14.84 (0.0001)
11
The assessment of genetic diversity in the loci of growth hormone and
leptin gene showed that taking the whole investigated group of animals, the
observed heterozygosity was found to be less than expected, but the deviation from Hardy - Weinberg equilibrium's law was not statistically significant (p>0.05). Less genetic diversity of the growth hormone gene locus in
both dairy cattle and beef group was found than theoretically expected,
while in the leptin gene locus in beef group a statistically significantly
higher heterozygosity was found than expeceted (Table 4).
Table 4. Mean expected and observed heterozigosity in growth hormone and leptin gene locus
4 lentel÷. Vidutinis teorinis ir faktinis heterozigotiškumas augimo
hormono ir leptino genų lokusuose
Breed /
Veisl÷
Milk
breeds /
Pienin÷s
veisl÷s
Beef
breeds /
M÷sin÷s
veisl÷s
Tested
group /
Tirta
grup÷
Growth hormone gene /
Augimo hormono genas
χ2 – test
H ExH ObH Ob(p – measerved / pected /
served /
ning) /
Faktinis Teorinis
Faktinis
(p – reikšm÷)
Leptin gene /
Leptino genas
χ2 – test
H Ex(p – meapected /
ning) /
Teorinis
(p–reikšm÷)
0.294
0.374
8.87(0.012)
0.254
0.335
11.7(0.003)
0.138
0.375
37.5(0.000)
0.330
0.289
1.83(0.401)
0.244
0.374
35.2(0.000)
0.278
0.321
5.18(0.075)
Association study between different SNPs in GH and LEP genes and
milk productivity traits
In the investigated cow group, the mean amount of milk and milk component content obtained was 6193.5 ± 124.35 kg of milk over lactation, 4.46
± 0.03% milk fat, 276.2 ± 5.74 kg of milk fat, 3.41 ± 0.02% milk protein,
210.6 ± 4.25 kg of milk protein, 27.8 ± 0.31 kg of milk on average over day,
8021.6 ± 164.04 kg of base milk over lactation, an average of 35.7 ± 0.36 kg
of base milk over day.
In order to highlight the effect of the growth hormone and leptin gene on
12
milk yield and milk components and to eliminate other factors, a multifactor
analysis was performed. Linear mixed model was applied for the analysis of
individual factors such as growth hormone GH and leptin LEP gene, effects
of breed, lactation and combined factors on milk yield and milk components. Breed and lactation significantly influenced all indicators of the milk
yield and composition. Breed influenced 2.3% of the milk yield diversity,
6.9% fat percent and 6.1% percent protein diversity. Lactation affected 1.4%
of milk yield diversity, 1.6 % fat kg and 2.5% protein kg diversity. The
growth hormone, the gene significantly influenced 2.1% of the milk yield
diversity, 1.9% milk fat percentage and 0.1% milk protein kg diversity.
Leptin gene significantly influenced 1.7% of the milk yield diversity, 1.7%
milk fat kg and 1.6% protein kg (Table 5).
Table 5. Influence of genetic factors to milk yield and composition
5 lentel÷. Genetinių veiksnių įtaka pieno kiekiui ir sud÷čiai
Genetic
factors /
Genetiniai
veiksniai
GH gene /
GH genas
LEP gene /
LEP genas
Lactation dur. /
Laktacijos
trukm÷
Breed /
Veisl÷
Lactation /
Laktacija
Breed x lact. /
Veisl÷ x
laktacija
Breed x GH /
Veisl÷ x GH
Lact x GH /
Lakt.x GH
Breed x Lep /
Veisl÷ x LEP
Lact x Lep /
Lakt.xLEP
Milk
Protein, Protein,
Number of per lactation, Fat, % / Fat, kg /
kg /
%/
classes /
kg /
Riebalai, Riebalai,
Baltymai, Baltymai,
%
kg
Klasių sk. Pieno per
%
kg
laktaciją, kg
The differences between breeds and in the breeds between lactations
were statistically significant, therefore differences between the genotypes
are presented in the charts after the evaluation of lactation and breed influence.
kg
7300
7100
6900
6700
6500
6844,5
6300
6100
5900
5700
-759,5
*
-823,9
**
AA
AB
5500
3
2.1*
1.9*
0.1
0.9
0.1*
3
1.7***
0.2
1.7***
0.5*
1.6***
* - p<0,05; ** - p<0,01; *** - p<0,001
Cov
71.4***
–
71.7***
–
78.0***
Fig. 1. Comparison of milk yield according GH genotypes
1 pav. GH genotipų palyginimas pagal pieno kiekį
5
2.3***
6.9***
1.1**
6.1***
2.4***
4
1.4***
0.2
1.6***
5.2***
2.5***
12
1.9**
5.2*
2.6***
3.8
1.2**
12
0.5
4.5**
0.5
1.1
0.5
12
0.4
0.3
0.5
1.2
0.2
15
1.1*
3.1
0.7
3.1*
0.9**
12
0.5
0.2
0.3
0.4
0.4
It was found that the cows of BB genotype of growth hormone gene
yielded 823.9 kg (p <0.01) more milk over lactation than the cows with AA
genotype and 759.5 kg (p<0.05) than the cows with AB genotype (Figure1),
they had lower milk fat content 0.37% (p<0.05) compared to AB and 0.48%
compared to AA (p<0.001) genotype cows (Figure 2) and their milk protein
content was 0.07% less than for AB and 0.10% less than of AA genotype
cows (Figure 3). Our data correlate with scientists Khatami (2005) and Zhou
(2005) who found a link between the GH allele B with dairy properties.
Similar trends for milk yield were found in the research of Lucy et al. (1993)
and Lee et al. (1996). On the other hand, scientists Grochowska and
Zwierzchowski (2000) found a greater allele A role in milk production in
Holstein cattle. Meanwhile, researchers Van der Welf et al. (1996) in their
study could not find a link in the GH1 locus to dairy properties.
*P<0.05; **P<0.01; ***P<0.001
13
BB
14
kg
6900
%
4,8
6700
4,6
4,4
6500
6300
+0,48
***
6100
+0,37
*
+639,9
***
+670,6
*
AB
BB
5900
4,2
5700
5837,7
4,1
5500
4
AA
AA
AB
BB
* - p<0,05; ** - p<0,01; *** - p<0,001
* - p<0,05; ** - p<0,01; *** - p<0,001
Fig. 2. Comparison of fat amount (%) in milk according GH genotypes
2 pav. GH genotipų palyginimas pagal pieno riebumą (%)
%
3,5
Fig. 4. Comparison of milk yield (in lactation) according LEP genotypes
4 pav. LEP genotipų palyginimas pagal pieno kiekį (per laktaciją)
%
4,8
4,7
3,4
+0,10
4,6
+0,07
4,5
3,3
3,4
4,4
3,2
+0,12
+0,17
AB
BB
4,48
4,3
AA
AB
BB
* - p<0,05; ** - p<0,01; *** - p<0,001
Fig. 3. Comparison of protein amount (%) in milk according GH genotypes
3 pav. GH genotipų palyginimas pagal pieno baltymingumą (%)
15
AA
* - p<0,05; ** - p<0,01; *** - p<0,001
Fig. 5. Comparison of fat amount (%) in milk according LEP genotypes
5 pav. LEP genotipų palyginimas pagal pieno riebumą (%)
16
Table 6. Influence of genetic factors to cattle daily weight gain
6 lentel÷. Genetinių veiksnių įtaka galvijų priesvoriui
%
3,7
Number of classes /
Klasių sk.
Daily gain 120-215 days, g /
Priesvor.per parą 120-215 d., g
Daily gain 215-310 days, g /
Priesvor.per parą 215-310 d., g
Daily gain 310- 405 days, g /
Priesvor.per parą 310- 405 d., g
Daily gain 405-500 days, g /
Priesvor.per parą 405-500 d., g
Average daily gain 120 -500 days, g /
Vidutinis priesvoris 120 -500 d., g
Weight on 500 day., kg /
Svoris 500 d., g
3,6
3
2.4*
1.2
2.3
1.2
0.9
0.2
3
4.2*
4.4*
0.2
1.9
0.2
0.004
3,5
+0,13
*
3,4
3,45
-0,02
3,3
Genetic
factors /
Genetiniai
veiksniai
3,2
AA
AB
BB
* - p<0,05; ** - p<0,01; *** - p<0,001
Fig. 6. Comparison of protein amount (%) in milk according LEP
genotypes
6 pav. LEP genotipų palyginimas pagal pieno baltymingumą (%)
It was found that the leptin gene, genotype AA cows yielded an average
of 639.9 kg (p <0.001) less milk per lactation than cows with genotype AB
and 670.6 kg (p<0.05) than cows with BB genotype (Figure 4), and they had
0.12% lower fat content than genotype AB cows and 0.17% lower than
genotype BB cows (Figure 5). Nevertheless, the milk of genotype AA cows
had higher protein content by 0.02% than that of AB genotype cows, but
0.13% less than of genotype BB cows (p<0.05) (Figure 6).
Association study between different SNPs in GH and LEP genes and fattening traits
In order to highlight the influence of the growth hormone and leptin gene
and eliminate other factors contributing to one of the cattle fattening indicators – the weight gain, a multifactor analysis was performed. Linear mixed
model was applied for the analysis of individual factors such as growth
hormone GH and leptin LEP genes, effects of breed, sire, and combined
factors on the weight gain of animals. The breed and the sire statistically
significantly affected weight gain rates in all the fattening periods. Breed
influenced by an average 16.4% and the sire – 17.6% weight gain characteristics. Both growth hormone and leptin genes statistically significantly influenced weight gain indicators only during the first half of fattening.
Growth hormone gene influenced the 2.4% rate of weight gain per day, and
leptin gene 4.2% (Table 6).
17
GH gene /
GH genas
Lep gene /
LEP genas
Breed /
Veisl÷
Sire /
Bulius
Breed x GH/
Veisl÷ x GH
Sire x GH /
Bulius x GH
Breed x Lep/
Veisl÷ x LEP
Sire x Lep /
Bulius x LEP
6
18.2*** 15.9*** 17.3*** 14.2*** 10.8*** 18.6***
18
26.8*** 25.2***
5.4
13.2*
16.5** 12.8***
16
2.1
3.8
7.8
3.3
4.5
20.4***
40
13.4***
6.5
5.3
2.8
9.6
14.1***
15
1.8
1.8
2.1
3.5
2.7
7.9*
35
1.6
2.2
2.4
2.4
1.0
3.0
*P<0.05; **P<0.01; ***P<0.001
The differences between breeds and in the breeds between sires were statistically significant, therefore differences between the genotypes are presented in the charts after the evaluation of sire and breed influence
18
g
1400
g
1400
AverageAverage
daily gain
daily gain
Vidutinis priesvoris, g
AA
1200
Average
dailydaily
gain gain
Average
Vidutinis priesvoris, g
AA
1200
AB
1000
AB
1000
BB
600
800
650
700
750
800
600
BB
650
800
700
750
800
850
600
AA
AB
BB
AA
400
AB
BB
400
200
200
I quarter
/ I ketv.
I quarter
II quarter
/ II ketv.
II quarter
III quarter
/ III ketv.
III quarter
IV quarter
/ IV ketv.
IV quarter
I quarter
/ I ketv.
I quarter
II quarterII/quarter
II ketv.
III quarterIII/ quarter
III ketv.
IV quarter
/ IV ketv.
IV quarter
Fig. 7. Daily weight gain in different fattening periods according GH
genotypes
7 pav. Priesvoriai skirtingais pen÷jimosi laikotarpiais pagal GH
genotipus
Fig. 8. Daily weight gain in different fattening periods according
LEP genotypes
8 pav. Priesvoriai skirtingais pen÷jimosi laikotarpiais pagal LEP
genotipus
The influence of bovine GH gene polymorphism to meat properties was
studied among Italian Podolian cattle. Animals with BB genotype showed a
lower growth rate than individuals with two other genotypes (Dario et al.,
2005). According to the research of Zwierzchowski et al. (2001), it was reported that the bull-calves with genotype BB had higher daily weight gain
than bull-calves with other genotypes. Di Stasio et al. (2002) found no relationship between the GH polymorphism and meat production characteristics
of Piedmont cattle. According to our data bull-calves with the growth hormone GH genotype AA had 68.9 kg greater weight gain than those with
genotype AB and 44.7 kg greater weight gain than those with genotype BB
(Figure 7).
A comparison of different indicators of the average daily weight gain in
accordance with LEP leptin AA, AB and BB genotypes showed that bullcalves with leptin LEP gene AB had 32.8 kg greater weight gain than bullcalves with genotype AA and 6 kg greater weight gain than those with genotype BB (Figure 8).
Association study between different SNPs in GH gene and semen quality
To assess the GH gene influence on semen quality, database of the following phenotypic characteristics has been formed - semen volume, semen
concentration, semen mobility. Bulls of the GH gene, genotype BB had better semen characteristics than the bulls with genotypes AA or AB, but statistically significant effect was found only for the semen mobility index. Bulls
with GH genotype BB matured faster in comparison with AB and BB genotype bulls. BB genotype bulls had a better exterior score, which statistically
significantly exceeded the exterior evaluation of the genotype AA bulls (Table 7). There is also scientific evidence that growth hormone may affect
sexual development and testicular function (Lin T., 1996).
19
20
Table 7. Influence of growth hormone gene to semen quality
7 lentel÷. Augimo hormono geno įtaka galvijų spermos kokybei
Genotype /
Genotipas
Semen volume, ml /
Spermos tūris, ml
Semen concentration, mlrd/ml
Spermos koncentracija
Semen mobility, scores /
Spermos judrumas
Weight at 3 month age, kg /
Svoris 3 m÷n.
Weight at 6 month age, kg /
Svoris 6 m÷n.
Weight at 9 month age, kg /
Svoris 9 m÷n.
Evaluation of exterior, scores/
Eksterjero vertinimas
AA
AB
BB
4.22±0.27
4.07±0.19
4.30 ±0.4
0.89±0.05
0.98±0.06
0.98±0.05
11.77±1.30
8.93±0.68 a
14.06±1.74 b
117.94±3.39 114.89±3.56
116.5±7.68
226.83±7.63 227.56±6.49 243.17±18.42
424.33±10.43 429.56±6.23 445.17±17.89
13.46±0.15 a
13.62±0.15
14.08±0.12 b
a,b – averages, (p≤0.05)
Distribution of alleles’ and genotypes’ frequencies in two cattle myostatin gene mutations – MSTN1 and MSTN2
The study of 270 different breeds of animals, belonging to Lithuanian
Black and White (LBW), Lithuanian Red (LR), Hereford (He), Limousin
(Li), Simmental (Si), Sharolais (Sa), Angus (An) and crossbreds of Lithuanian Black and White with Belgian Blue, Sharolais and Limousins took place in order to identify the bovine myostatin gene mutation MSTN1 (11 bp
deletion, at 821 bp exon 3) and MSTN2 (single nucleotide change, G→A, in
the 938th base pair in exon 3). The study in the investigated group of animals
resulted in the identification of two MSTN1 alleles: L1 - wild-type, and
MSTN1–a mutated allele and one MSTN2 allele–L2. The MSTN2 locus is
monomorphous in the investigated group of cattle regardless the breed. Neither heterozygous animals, carriers of a mutated gene, nor homozygous
animals having two copies of the modified gene were found.
The wild-type allele L1 of the myostatin gene was found with the frequency of 0.95, while the mutant allele MSTN1 – with the frequency of
0.05. A total of 89% of the animals tested had a homozygous wild
L1/L1genotype, 11% had a heterozygous L1/MSTN1 genotype and were the
carriers of the double-muscle allele. Animals, having two mutated myostatin
21
alleles with the genotype MSTN1/MSTN1 were not found in the test group.
It was found that 100% of Charolais, Limousine, Simmental, Angus,
Lithuanian Black and White, Lithuanian Red, Lithuanian Black and White
crossbreeds with Charolais and Limousine cattle had a wild, i.e. unchanged
myostatin gene genotype L1/L1, and the Lithuanian Black and White crossbreeds with the Belgian Blue all had one copy of the mutated myostatin
gene and had the genotype L1/MSTN1. Since the myostatin gene mutation
(11 bp deletion) was not found in Lithuanian Black and White cattle breed,
the crossbreeds inherited this altered allele from the parental side, i.e. Belgian Blue sires. Examination of the phenotype showed that all animals had
partly expressed double-muscled phenotype characteristics.
Table 8. Frequencies of myostatin MSTN1 gene alleles and genotypes
in cattle bred in Lithuania
8 lentel÷. Galvijų miostatino MSTN1 genotipų ir alelių dažniai tirtoje
Lietuvoje veisiamų galvijų grup÷je
Alelles /
Aleliai
L1
MSTN1
Frequencies /
Dažniai
0.95
0.05
Genotypes /
Genotipai
L1/L1
L1/ MSTN1
MSTN1/ MSTN1
Frequencies /
Dažniai
0.89
0.11
0
Our results correlate with those of other researchers. Grobet with coworkers examined the prevalence of seven mutations of the myostatin gene
in eleven breeds and found that different myostatin gene mutations are specific to specific breeds - the F 94 L mutation was specific to the Limousine
breed, 414 C-T mutation was found in all breeds studied with low frequency, but was characteristic to Galloway and Salers breeds, the mutations
of 7-ins 10 nt 419, the mutation Q 204 X, E 226 X and C 313 Y were only
found in Limousine breed, but they did not determine the double-muscled
phenotype except for F 94 L, the mutation 11 base pair deletion was found
in Belgian Blues like in our study and in the South Devon cattle breeds
(Grobet et al.,1998). Understanding of double muscling phenotype and creation of specific diagnostic PCR tests for miostatin gene polymorphism identification gives possiblities to farmers to perform genotypic selection according their needs.
22
CONCLUTIONS
1. In the investigated group of cattle, the growth hormone gene allele A
was found with the frequency of 0.751, allele B with the frequency of 0.249.
Allele A was found with the maximum frequency (0.900) in Hereford breed,
and the allele B (0.438) in Simmental breed. Leptin gene allele A was found
with the frequency of 0.799, and the allele B was found with the frequency
of 0.201. Allele A was found with the maximum frequency (0.875) in Simmental breed, and B allele B (0,300) in the Danish Red breed. Growth hormone allele frequencies between dairy and beef cattle breeds did not differ.The frequency of leptin gene allele B in dairy cattle breeds group was
higher than in the group of beef cattle breeds.
2. In the investigated group of cattle the growth hormone gene genotype
AA was found in 62.9% of the cattle, heterozygous AB genotype in 24.4%,
BB genotype in 12.7%. Genotype AA, with the highest frequency of 90%
was found in Hereford cattle, genotype AB in 55.6% of Lithuanian Black
and White and genotype BB in 37.5% of the old Lithuanian Black and
White genotype. Leptin gene genotype AA was found in 66.0% animals,
heterozygous genotype AB in 27.8%, BB genotype in 6.2%. Genotype AA
with the highest frequency was found in Simmental and Limousin cattle,
genotype AB in the Lithuania's Red and genotype BB in Danish Red cows.
Genotype BB was not found on all in Hereford, Simmental and Charolais
breeds.
3. Examination of the 11 base pairs deletion mutation, MSTN1 myostatin gene’s wild L allele was found with the frequency of 0.89, whereas
the mutant allele MSTN1 with 0.11. As many as 85% of the tested animals
had a homozygous wild L/L genotype, 15% had a heterozygous L/MSTN1
genotype and were the carriers of a double-muscled allele. Animals, having
two changed myostatin alleles with genotype MSTN1/MSTN1 were not
found in the analysed group. It was found that 100% of the Lithuanian Black
and White, Limousine, Simmental and Sharolais cattle had a wild, i.e. unchanged myostatin gene genotype L/L, and the Lithuanian Black and White
crosses with the Belgian Blue all had one copy of the mutated myostatin
gene and had the genotype L/MSTN1.
vine milk yield and composition, the largest statistically significant impact
of the growth hormone gene to the average percentage of fat content and
milk per day was determined. It affected around 2 percent of the diversity of
these indicators. The growth hormone’s allele A increases milk fat percentage, and the allele B increases milk volume during lactation. Statistically
significant effect of the leptin gene was found to the protein and fat content
and milk yield during lactation. It influenced around 2.5% of the diversity of
these indicators. Leptin gene’s allele B increases the amount of milk during
lactation.
6. The investigation of genetic factors on the weight gain of animals in
different fattening periods showed that the leptin gene statistically significantly influenced weight gain in the first two fattening quarters. Leptin gene
affected 0.1 to 4.4% of the variety of these indicators. Leptin gene’s allele A
increased weight gain of cattle. The cattle with growth hormone genotype
AA had the greatest weight gain, but the differences were not statistically
significant.
7. Cattle with growth hormone genotype BB had the best semen quality
and exterior evaluation.
8. Cattle growth hormone GH, leptin LEP, myostatin MSTN1 genes
were found polymorphic, and it allows them to be used as genetic biomarkers for selection. The myostatin gene MSTN2 was found monomorphous in the investigated group of animals.
PROPOSALS
The polymorphisms of the cattle growth hormone and leptin gene found
and their influence on the characteristics of farming such as milk production, composition and fattening indicators enable them to be used as biomarkers in the selection process thus accelerating it through the molecular
technologies, increasing the breeding value of livestock and improving their
farming characteristics and quality of products.
4. After the examination of the single nucleotide mutation in myostatin
gene MSTN2 all animals were found to be similar under the genotype L/L,
with the wild-type myostatin gene allele. Mutated allele was not found in the
investigated group of animals.
5. The investigation of effects of genetic factors on the indicators of bo23
24
LIST OF PUBLICATIONS ON DISSERTATION TOPIC
Scientific papers in journals listed by ISI:
1. Kupstait÷ D., Indriulyt÷ R., Krasnopiorova N., Baltrenait÷ L.,
Miceikien÷ I. Augimo hormono geno polimorfizmo įtaka bulių reprodukcinems savybems // Veterinarija ir zootechnika. ISSN 1392 - 2130. 2008. T.
42 (64). P. 48-50.
Scientific papers in International reviewed journals:
1. Krasnopiorova N., Baltr÷nait÷ L., Navakauskait÷ G., Morkūnien÷ K.
Miceikien÷ I. Leptin gene polymorphism in cattle bred in Lithuania. Proceedings of the 15th Baltic Animal Breeding Conference. Riga, Latvia.
2010. P.21-24.
2. Natalija Krasnopiorova, Kristina Morkūnien÷, Lina Baltr÷nait÷,
Paulius Morkūnas, Ilona Miceikien÷. Lietuvoje veisiamų galvijų miostatino
geno polimorfizmo tyrimai. Žem÷s ūkio mokslai. 2010. Nr. 3-4. (Straipsnis
priimtas publikacijai).
Conference thesis:
1. Krasnopiorova N., Kupstait÷ D., Indriulyt÷ R., Miceikien÷ I. Influence of growth hormone gene polymorphism to cattle reproduction trait.
The 59th Annual Meeting of the European Association for Animal Production. Vilnius. 2008. P. 79
REZIUMö
Darbo tikslas – ištirti augimo hormono, leptino ir miostatino genų genetinių tipų paplitimą Lietuvoje veisiamų galvijų veisl÷se ir nustatyti jų įtaką
ūkin÷ms savyb÷ms.
Darbo uždaviniai:
1. Genotipuoti galvijų GH (augimo hormono), LEP (leptino) ir MSTN
(miostatino) genų polimorfizmus.
2. Nustatyti GH, LEP ir MSTN genų alelių ir genotipų dažnius
Lietuvoje veisiamų galvijų tarpe.
3. Įvertinti genotipuotų vieno nukleotido polimorfizmų (VNP) GH ir
LEP genuose įtaką pieno kiekiui, sud÷čiai ir galvijų priesvoriui.
4. Įvertinti GH geno polimorfinių vietų įtaką galvijų spermos kokybei
bei eksterjerui.
Darbo naujumas:
Pirmą kartą Lietuvoje auginamų galvijų populiacijoje (11 veislių ir 3
mišrūnų) buvo ištirti vieno nukleotido polimorfizmai GH, LEP ir MSTN
genuose kandidatuose bei nustatyta jų įtaka ūkin÷ms savyb÷ms.
Darbo praktin÷ reikšm÷:
Rasti galvijų augimo hormono ir leptino genų polimorfizmai bei jų įtaka
tokioms galvijų ūkin÷ms savyb÷ms kaip pieno kiekis, sud÷tis bei pen÷jimosi
rodikliai sudaro prielaidas juos naudoti kaip biožymenis selekcijos procese
taip jį paspartinant molekulinių technologijų pagalba, padidinant gyvulių
veislinę vertę bei pagerinant jų ūkines savybes bei produkcijos kokybę.
TYRIMŲ REZULTATAI IR APTARIMAS
Augimo hormono ir leptino geno polimorfizmas tirtas 10 pieninių ir m÷sinių galvijų veislių. Paskaičiuoti augimo hormono GH ir leptino LEP genų
skirtingų variantų dažniai kiekvienoje tirtoje veisl÷je. Nustatyta, kad tiek
augimo hormono GH, tiek leptino LEP genai tur÷jo po du alelius (A ir B)
visose tirtose veisl÷se. Augimo hormono GH geno A alelio dažnis kito nuo
(0,563) Simentalių veisl÷je iki (0,900) Herefordų veisl÷je. Leptino LEP geno A alelio dažnis svyravo tirtoje galvijų populiacijoje nuo (0,700) Danijos
žalųjų veisl÷je iki (0,875) Simentalių veisl÷je. Dažniausiai sutinkamas daugelyje pasaulio galvijų veislių tiek augimo hormono GH A alelis, tiek leptino LEP A alelis buvo rasti aukštu dažniu visose tirtose veisl÷se. Statistiškai
reikšmingų skirtumų tarp pieninių ir m÷sinių galvijų veislių augimo hormono ir leptino geno alelių dažnių nenustatyta (1 lentel÷).
Tiek augimo hormono GH, tiek leptino LEP genų lokusuose buvo nusta-
25
26
tyta po 3 skirtingus genotipus - AA, AB ir BB. Augimo hormono GH geno
AA genotipas mažiausiu dažniu (0,444) rastas senojo genotipo Lietuvos
juodmargių galvijų veisl÷je, didžiausiu (0,900) Herefordų galvijų veisl÷je.
Tuo tarpu AB genotipas mažiausiu dažniu (0,111) Limuzinų galvijų veisl÷je,
didžiausiu (0,556) senojo genotipo Lietuvos juodmargių galvijų veisl÷je.
Herefordų galvijų veisl÷se AB genotipas nebuvo nustatytas. Augimo hormono GH BB genotipas didžiausiu dažniu rastas (0,375) Simentalių veisl÷je.
Senojo genotipo Lietuvos juodmargių galvijų veisl÷je BB genotipas nebuvo
nustatytas. Leptino geno AA genotipas įvairavo nuo (0,522) iki (0,750) dažnio. Tuo tarpu AB genotipas įvairavo nuo (0,222) Limuzinų ir senojo genotipo Lietuvos juodmargių galvijų veisl÷se iki (0,450) Šarolę galvijų veisl÷se.
Herefordų, Simentalių ir Šarolę galvijų veisl÷se BB genotipas nebuvo nustatytas (2 lentel÷).
Visose tirtose veisl÷se rastas mažesnis faktinis heterozigotiškumas nei
teorinis išskyrus dvi veisles: senojo genotipo Lietuvos juodmargių galvijų ir
Šarol÷, kur faktinis heterozigotiškumas buvo didesnis nei teorinis, tačiau
skirtumai nebuvo statistiškai reikšmingi. Statistiškai reikšmingas nuokrypis
nuo Hardžio–Weinbergo pusiausvyros d÷snio bei mažai išreikštas heterozigotiškumas nustatytas Lietuvos juodmargių ir Limuzinų veisl÷se. Sugrupavus veisles į pieninių ir m÷sinių, abiejose veislių grup÷se nustatytas statistiškai reikšmingas nuokrypis nuo Hardžio–Weinbergo pusiausvyros d÷snio
heterozigostiškumo maž÷jimo kryptimi, atitinkamai 7,23 (p<0,007) ir 14,6
(P<0,001) (3 lentel÷).
Įvertinus genetinę įvairovę augimo hormono ir leptino geno lokusuose
nustatyta, kad imant visą tirtų galvijų grupę, faktinis heterozigotiškumas
rastas mažesnis nei teorinis, bet nuokrypis nuo Hardžio–Weinbergo pusiausvyros d÷snio nebuvo statistiškai reikšmingas (P>0,05). Augimo hormono geno lokuse tiek pieninių galvijų grup÷je, tiek m÷sinių rasta mažesn÷
genetin÷ įvairov÷ nei teoriškai planuota, tuo tarpu leptino geno lokuse m÷sinių galvijų grup÷je rastas statistiškai reikšmingai didesnis heterozigotiškumas nei prognozuotas (4 lentel÷).
Pieno kiekio ir pieno sud÷tinių dalių vidurkiams paskaičiuoti panaudoti
Lietuvoje auginamų pieninių veislių - Danijos juodmargių, Danijos žalųjų,
Holšteinų, Lietuvos juodmargių, senojo genotipo Lietuvos juodmargių karvių 2008 - 2009 m. kontrol÷s produktyvumo rodikliai, gauti iš „Žem÷s ūkio
informacijos ir kaimo verslo centro“. Tirtoje karvių grup÷je vidutinis pieno
kiekis ir pieno sud÷tinių dalių kiekis gautas 6193,5±124,35 kg pieno per
laktaciją, 4,46±0,03 proc. pieno riebalų, 276,2±5,74 kg pieno riebalų, 3,41±
0,02 proc. pieno baltymų, 210,6±4,25 kg pieno baltymų, 27,8±0,31 kg vidutiniškai pieno per parą, 8021,6±164,04 kg bazinio pieno per laktacija,
35,7±0,36 kg vidutiniškai bazinio pieno per parą.
Siekiant išryškinti augimo hormono ir leptino genų įtaką pieno kiekiui ir
pieno sud÷tin÷ms dalims bei eliminuoti kitų veiksnių įtaką atlikta daugiafaktorin÷ analiz÷. Pagal tiesinį mišrų modelį buvo ištirta atskirų veiksnių, tokių
kaip augimo hormono GH ir leptino LEP genų, veisl÷s, laktacijos ir kombinuotų veiksnių įtaka pieno kiekiui ir pieno sud÷tin÷ms dalims. Veisl÷ ir laktacija statistiškai reikšmingai įtakojo visus pieno kiekio ir sud÷ties rodiklius.
Veisl÷ įtakojo 2,3 proc. pieno kiekio įvairov÷s, 6,9 proc. riebalų ir 6,1 proc.
baltymų procento įvairov÷s. Laktacija įtakojo 1,4 proc. pieno kiekio įvairov÷s, 0,2 proc. riebalų ir 5,2 proc. baltymų procento įvairov÷s. Augimo hormono genas statistiškai reikšmingai įtakojo 2,1 proc. pieno kiekio įvairov÷s,
1,9 proc. pieno riebalų procento, 0,1 proc. pieno baltymų kiekio. Leptino
genas statistiškai reikšmingai įtakojo 1,7 proc. pieno kiekio įvairov÷s, 1,7
proc. pieno riebalų ir 1,6 proc. pieno baltymų kiekio (5 lentel÷). Skirtumai
tarp veislių ir veisl÷se tarp laktacijų buvo statistiškai reikšmingi, tod÷l grafikuose skirtumai tarp genotipų pateikti įvertinus veisl÷s ir laktacijos įtaką.
Nustatyta, kad GH BB genotipo karv÷s dav÷ daugiau pieno per laktaciją
823,9 kg (P<0,01) negu AB ir 759,5 kg (P<0,05) nei AA genotipą turinčios
karv÷s (1 pav.), tur÷jo mažesnį pieno riebumą 0,37 proc. (P<0,05) negu AB
ir 0,48 proc. negu AA (P<0,001) genotipą turinčios karv÷s (2 pav.) ir mažesnį pieno baltymingumą 0,07 proc. negu AB ir 0,10 proc. negu AA genotipą turinčios karv÷s (3 pav.). Nustatyta, kad leptino geno AA genotipo karv÷s dav÷ vidutiniškai 639,9 kg (P<0,001) mažiau pieno per laktaciją negu
AB ir 670,6 kg (P<0,05) negu BB genotipą turinčios karv÷s (4 pav.), tur÷jo
mažesnį pieno riebumą 0,12 proc. negu AB ir 0,17 proc. negu BB genotipą
turinčios karv÷s (5 pav.), tačiau AA genotipo karvių pienas pasižym÷jo didesniu baltymingumu 0,02 proc. negu AB genotipo karvių, bet mažesniu
0,13 proc. negu BB (P<0,05) genotipą turinčių karvių (6 pav.).
Siekiant išryškinti augimo hormono ir leptino genų įtaką bei eliminuoti
kitų veiksnių įtaką vienam iš galvijų pen÷jimosi rodiklių – priesvoriui, buvo
atlikta daugiafaktorin÷ analiz÷. Pagal tiesinį mišrų modelį ištirta atskirų
veiksnių, tokių kaip augimo hormono GH ir leptino LEP genų, veisl÷s, buliaus ir kombinuotų veiksnių įtaka galvijų priesvoriui. Veisl÷ ir bulius statistiškai reikšmingai įtakojo priesvorio rodiklius visais pen÷jimosi laikotarpiais. Veisl÷ vidutiniškai įtakojo 16,4 proc., o bulius 17,6 proc. priesvorio
rodiklių. Tiek augimo hormono tiek leptino genai statistiškai reikšmingai
įtakojo priesvorio rodiklius tik pirmąjį pen÷jimosi pusmetį. Augimo hormono genas įtakojo 2,4 proc. priesvorio per parą rodiklio, o leptino genas 4,2
proc. (6 lentel÷). Palyginus skirtingų priesvorių rodiklių vidurkius pagal
augimo hormono GH AA, AB ir BB genotipus nustatyta, kad augimo hormono GH AA genotipą turintys buliukai tur÷jo 68,9 kg ir didesnį priesvorį
nei AB ir 44,7 kg didesnį priesvorį nei BB genotipą turintys buliukai. Au-
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gimo hormono genotipas atskiriems pen÷jimosi laikotarpiams statistiškai
reikšmingos įtakos netur÷jo (6 pav.). Palyginus skirtingų priesvorių rodiklių
vidurkius pagal leptino LEP AA, AB ir BB genotipus nustatyta, kad leptino
geno LEP AB genotipo buliukai tur÷jo 32,8 kg didesnį priesvorį nei AA ir 6
kg didesnį priesvorį nei BB genotipo buliukai (7 pav.).
Siekiant įvertinti GH geno įtaką spermos kokybei buvo suformuota sekančių fenotipinių požymių duomenų baz÷: spermos tūris; spermos koncentracija; spermos judrumas. GH geno BB genotipo buliukai tur÷jo geresnius
spermos rodiklius nei AA ar AB genotipų, tačiau statistiškai reikšminga
įtaka nustatyta tik spermos judrumo rodikliui (7 lentel÷).
Ištyrus 270 skirtingų veislių galvijus, siekiant identifikuoti galvijų miostatino geno mutaciją MSTN1 (11 bp deleciją, įvykusią 821 bp vietoje 3 egzone), tirtoje galvijų grup÷je buvo identifikuoti du aleliai: L1–laukinio tipo
ir MSTN1–mutuotas alelis. Siekiant identifikuoti galvijų miostatino geno
mutaciją MSTN2 (vieno nukleotido pasikeitimą, t. y. G→A 938-oje bazių
poroje 3 egzone), visi gyvuliai rasti vienodi pagal genotipą L2/L2, t. y. turintys laukinio tipo miostatino geno alelį dviem kopijomis. MSTN2 genas
yra monomorfiškas tirtoje galvijų grup÷je nepriklausomai nuo veisl÷s. Nei
heterozigotinių galvijų, mutuoto geno nešiotojų, nei homozigotinių galvijų,
turinčių dvi pakitusio geno kopijas, nerasta. Miostatino geno laukinio tipo
alelis L1 buvo rastas 0,95 dažniu, o mutavęs alelis MSTN1–0,05 dažniu. 89
proc. tirtų gyvulių tur÷jo homozigotinį laukinį L1/L1genotipą, 11 proc.heterozigotinį L1/MSTN1 genotipą ir buvo dvigubo raumeningumo alelio
nešiotojai. Gyvulių, turinčių du pakitusius miostatino alelius su genotipų
MSTN1/MSTN1 tirtoje grup÷je nenustat÷me (8 lentel÷). Nustačius, kad tirtoje galvijų grup÷je identifikuoti galvijai turi dviejų tipų genotipus, t. y.
L1/L1-laukinio tipo genotipą ir L1/MSTN1 mutuotą genotipą, buvo atlikta
analiz÷ kaip šie genotipai paplitę atskirose veisl÷se. Nustatyta, kad 100 proc.
Šarol÷, Limuzinai, Simentaliai, Angusai, Lietuvos juodmargiai, Lietuvos
žalieji, Lietuvos juodmargių mišrūnai su Šarol÷ ir su Limuzinais galvijai
tur÷jo laukinį, t. y nepakitusio miostatino geno genotipą L1/L1, o mišrūnai
Lietuvos juodmargių su Belgijos m÷lynaisiais visi tur÷jo po vieną pakitusio
miostatino geno kopiją ir tur÷jo genotipą L1/MSTN1. Kadangi miostatino
geno mutacija (11 bp delecija) Lietuvos juodmargių galvijų veisl÷je nebuvo
nustatyta, šį pakitusį alelį mišrūnai paveld÷jo iš t÷vin÷s pus÷s, t.y. iš Belgijos m÷lynųjų veisl÷s bulių. Ištyrus jų fenotipą buvo nustatyta, kad visi galvijai tur÷jo dalinai išreikštus dvigubo raumeningumo fenotipo požymius. Dvigubo raumeningumo fenotipo supratimas ir miostatino geno aleliams specifinių diagnostinių PGR metodų sukūrimas sudaro sąlygas vykdyti genotipinę gyvulių selekciją galvijų dvigubo raumeningumo geno atžvilgiu pagal
augintojo poreikius.
5. Ištyrus genetinių veiksnių įtaką galvijų pieno kiekio ir sud÷ties rodikliams, nustatyta didžiausia statistiškai reikšminga augimo hormono GH geno
įtaka riebalų procentui ir vidutiniam pieno kiekiui per parą. Jis įtakojo apie 2
proc. šių rodiklių įvairov÷s. Augimo hormono GH geno A alelis didina riebalų procentą piene, o B alelis pieno kiekį per laktaciją. Taip pat nustatyta
statistiškai reikšminga leptino LEP geno įtaka riebalų ir baltymų kiekiui ir
pieno kiekiui per laktaciją. Jis įtakojo apie 2.5 proc. šių rodiklių įvairov÷s.
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IŠVADOS
1. Tirtoje galvijų grup÷je augimo hormono geno A alelis rastas 0,751
dažniu, B alelis - 0,249 dažniu. A alelis didžiausiu dažniu (0,900) rastas
Herefordų veisl÷je, o B alelis (0,438) - Simentalių veisl÷je. Leptino geno A
alelis rastas 0,799 dažniu, o B alelis - 0,201 dažniu. A alelis didžiausiu dažniu (0,875) rastas Simentalių veisl÷je, o B alelis (0,300) Danijos žalųjų veisl÷je. Augimo hormono geno alelių dažniai tarp pieninių ir m÷sinių galvijų
veislių nesiskyr÷, o leptino geno B alelio dažnis pieninių galvijų veislių grup÷je buvo didesnis nei m÷sinių galvijų veislių grup÷je.
2. Tirtoje galvijų grup÷je augimo hormono geno AA genotipą tur÷jo
62,9 proc. galvijų, heterozigotinį AB genotipą 24,4 proc., BB genotipą 12,7
proc. AA genotipą didžiausiu 90 proc. tur÷jo Herefordų veisl÷s galvijai, AB
genotipą 55,6 proc. Lietuvos juodmargiai ir BB genotipą 37,5 proc. seno
genotipo Lietuvos juodmargiai. Leptino geno AA genotipą tur÷jo 66,0 proc.
galvijų, heterozigotinį AB genotipą 27,8 proc., BB genotipą 6,2 proc. AA
genotipą didžiausiu dažniu tur÷jo Simentalių ir Limuzinų veisl÷s galvijai,
AB genotipą Lietuvos žalieji ir BB genotipą Danijos žalieji. BB genotipas
visai nerastas Herefordų, Simentalių ir Šarole veisl÷se.
3. Ištyrus 11 bazių porų delecijos mutaciją MSTN1 miostatino geno
laukinis alelis L buvo rastas 0,89 dažniu, o mutavęs alelis MSTN1 - 0,11
dažniu. 85 proc. tirtų gyvulių tur÷jo homozigotinį laukinį L/Lgenotipą, 15
proc. heterozigotinį L/MSTN1 genotipą ir buvo dvigubo raumeningumo
alelio nešiotojai. Gyvulių, turinčių du pakitusius miostatino alelius su genotipu MSTN1/MSTN1 tirtoje grup÷je nenustat÷me. Nustatyta, kad 100 proc.
Lietuvos juodmargiai, Limuzinai, Simentaliai ir Šarole galvijai tur÷jo laukinį, t.y nepakitusio miostatino geno genotipą L/L, o mišrūnai Lietuvos juodmargių su Belgijos m÷lynaisiais visi tur÷jo po vieną pakitusio miostatino
geno kopiją ir tur÷jo genotipą L/ MSTN1.
4. Ištyrus taškinę miostatino geno mutaciją MSTN2, nustatytas vienodas
visų gyvulių genotipas L/L, t.y. turintis laukinio tipo miostatino geno alelį.
Šioje galvijų grup÷je mutuotas alelis nerastas.
Leptino geno B alelis didina pieno kiekį per laktaciją.
6. Ištyrus genetinių veiksnių įtaką galvijų priesvoriui skirtingais pen÷jimosi laikotarpiais nustatyta, kad leptino genas statistiškai reikšmingai įtakojo priesvorį dviem pirmais pen÷jimosi ketvirčiais. Leptino genas įtakojo
0,1–4,4 proc. šių rodiklių įvairov÷s. Leptino geno A alelis didina galvijų
priesvorį. Augimo hormono geno AA genotipo galvijai tur÷jo didžiausią
priesvorį, tačiau skirtumai nebuvo statistiškai reikšmingi.
GYVENIMO APRAŠYMAS (CURRICULUM VITAE)
NATALIJA KRASNOPIOROVA (g. 1969-07-28, Kaune)
1976–1986 m.
mok÷si 19-oje vidurin÷je mokykloje.
1997–2002 m.
LVA Gyvulininkyst÷s technologijos
fakulteto student÷.
7. Augimo hormono geno BB genotipo galvijai tur÷jo didžiausią spermos tūrį, koncentraciją, jų buvo geriausias spermos judrumas ir eksterjero
vertinimas.
2002–2004 m.
8. Galvijų augimo hormono GH, leptino LEP, miostatino MSTN1 genai
rasti polimorfiški ir tai sudaro galimybes juos panaudoti kaip genetinius
biožymenis selekcijai. Miostatino MSTN2 genas nustatytas tirtoje galvijų
grup÷je monomorfiškas.
Gyvūnų genetikos katedros magistrant÷. 2004 m.
apgyn÷ magistrantūros baigiamąjį darbą tema
„Žem÷s ūkio gyvulių citogenetiniai tyrimai“.
2005–2010 m.
Gyvūnų veisimo ir genetikos katedros
doktorant÷.
1987–2004 m.
K. Janušausko gyvūnų genetikos laboratorijos
prie Gyvūnų veisimo ir genetikos katedros
laborant÷, o nuo 2004 m. Gyvūnų veisimo ir
genetikos katedros asistent÷.
PASIŪLYMAI
Rasti galvijų augimo hormono ir leptino genų polimorfizmai bei jų įtaka
tokioms galvijų ūkin÷ms savyb÷ms kaip pieno kiekis, sud÷tis bei pen÷jimosi
rodikliai sudaro prielaidas juos naudoti kaip biožymenis selekcijos procese,
taip ją paspartinant molekulinių technologijų pagalba, padidinant gyvulių
veislinę vertę bei pagerinant jų ūkines savybes bei produkcijos kokybę.
Maketavo R. Trainien÷,
Leidybos organizavimo specialist÷
Už teksto turinį ir redagavimą atsakingas autorius
Spausdino LSMU VA Spaudos ir leidybos skyrius
Tilž÷s g. 18, LT-47181 Kaunas
Tiražas 35. 2,00 sp. l. Užs. Nr. 33 d. 2010
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