By selecting the best performing animals on phenotypic variants (milk,
fat, or protein yield) as reproducers, breeders have increased the frequency
of favorable alleles influencing the interesting traits. Moreover, in
dairy cattle, genetic progress is essentially based on the use of genetically
superior sires whose identification requires time (5-6 years) and money
(1000, 000 US$) (Parmentier et al., 1999). Furthermore, the exact
molecular natures of the target genes remain essentially unknown, particularly
for production traits that are often controlled by a pool of genes. At
the present time, the two major strategies developed to detect Quantitative
Trait Loci (QTL) are the candidate gene approach and the positional genetic
approach. The somatotropin axis contains the most promising candidates
in this respect, as it strongly regulates milk production. Current knowledge
in dairy biology indicates that genetically superior animals differ from
lesser animals mainly in their regulation of nutrient utilization and
that growth hormone exerts a key control in nutrient use, mammary development,
growth milk yield (Bauman, 1999) and also modulates intermediary metabolism
and other physiological processes e.g., aging and immune responsiveness.
Thus the Growth Hormone (GH) gene is a promising candidate gene worth
studying for its effects on milk-related traits. GH is a part of a multiple-gene
family that contains prolactin and the placental lactogens and has been
mapped by in situ hybridization to bovine chromosome 19 (Zwierzchowski
et al., 2002) bovine Growth Hormone (bGH) is a 22 kDa pituitary
hormone composed of 191 amino acid residues.
Selection for milk yield has been shown to be associated with increased
blood levels of GH (Bauman, 1999). GH is synthesized in the pituitary
gland and coded for by a single gene consisting of five introne and four
exon. Several base substitution polymorphisms have been detected both
in the promoter and the coding and none coding regions. One of the base
substitutions gives rise to an amino acid change at position 127 in the
peptide, where a Leusine is exchanged for Valine. Identification of such
mutations would permit selection at the DNA level without necessitating
measurement of GH levels.
Danish Jersey calves with Leu/Leu genotype had a higher PEAK than calves
with the Val/Val genotype, whereas the Leu/Val genotype had an intermediate
response. PEAK was the mean GH of blood sampled at 10, 15 and 20 min following
inducement with GHRH (Sorensen et al., 2002).
Grochowska et al. (2001) reported that differences between the
Leu/Leu and the Val/Val genotypes for carcass gain and weight of meat
in the carcass were significant (p≤0.05). Moreover, differences in
the size of the GH peak between the two homozygotes approached significance
(p≤0.10). Schlee et al. (1994) reported significant effects
for the LV genotype for carcass gain and meat value but reported no effects
for milk breeding values. Associations between Leu/Val polymorphism and
milk production traits of cows were found in first lactation (Dybus, 2002).
Cows with LL genotype had higher milk, fat and protein yield compared
to LV individuals (p≤0.01).
Objectives of this study were to detect genetic variants in bGH gene
as candidate gene in Holstein proven bulls and to test the effect of the
genetic polymorphisms on milk production traits and also to estimates
the average effect of allele substitution.
MATERIALS AND METHODS
Semen samples from 134 Holstein proven bulls were obtained from Animal
Breeding Center of Iran. Genomic DNA was extracted according to Zadworney
and Kuhnlein (1990).
DNA Amplification With PCR-RFLP
bGH genotypes were identified according to Sorensen et al.
(2002). The 282 bp fragment of intron 4 and exon 5 of the bGH gene was
amplified using fallowing primers: 5` GTG GGC TTG GGG AGA CAG AT 3` (position
1940) and 5` GTC GTC ACT GCG CAT GTT TG 3` (position 2202).
A single reaction (25 μL) contained the fallowing constituents:
10X PCR buffer (16 mM (NH4)2SO4, 67 mM
Tris HCl pH 8.8, 0.1% Tween-20), 2.5 mM MgCl2, 200 μM
dNTP, 5 pmoles of each primers, 1 unit Taq polymerase (Metabion) and 50-100
ng of genomic DNA. The amplification program consisted of an initial denaturation
at 94°C for 2 min, then 30 cycles of 94°C for 45 sec, 62°C
for 60 sec and 72°C for 60 sec and a final extension of 72°C was
maintained for 3 min.
A single digestion reaction consisted of 10 μL of PCR product, 4
units (0.4 μL) of AluI enzyme (Fermentas), 1.5 μL Tango buffer
and 3.15 μL nuclease-free water. The final reaction volume of 15
μL was incubated at 37°C for 12 h. The fragments were separated
on a 3% agarose gel by electrophoresis.
Calculation of gene frequency was based on direct gene count method
and standard error of frequency was calculated as:
||The sample size
||The frequency of L allele
|nLL and nLV
||No. of LL and LV types, respectively
The Χ2-test was used to determine Hardy-Weinberg equilibrium
Breeding values for milk related traits (milk, fat and protein yield;
and fat and protein percent) were estimated with the Best Linear Unbiased
Procedure (BLUP) based on an animal model with a relationship matrix.
The analysis was conducted using AI-REML procedures as programmed in MATVEC
software (Wang et al., 2002). The model included animal effect
as random effect and age of calving as covariate factor and fixed effect
The effect of bGH genotypes on the estimated breeding values for milk
related traits was analyzed using least square method of GLM procedure
of SAS (2002) software. As the breeding values are the best available
estimates of the additive genotype of the bulls, no environmental effects
were included in the model. The used model was as fallows:
||The breeding value for milk related traits
||The least square means of the traits
||The effect of the ith genotype (I = 1, 2, 3)
||The random residual effect
Type III sum of squares were used to evaluate the effect of bGH polymorphism.
Regression analyses were performed in which EBV for milk, fat, protein
yield; and fat and protein percent were the dependent variables and the
genotype was the independent variable. Average effect of allele substitution
was determined by coding genotypes as 0(VV), 1(LV) and 2(LL) to represent
the number of L alleles present for the bGH polymorphism. As described
by Falconer and Mackay (1996), the regression coefficient (α) estimates
the average effect of allele substitution, or the average effect of replacing
a V allele with an L allele.
RESULTS AND DISCUSSION
The PCR amplified a 282 bp fragment from intron 4 and exon 5 of the bGH
gene. The resulting digestion with AluI enzyme was two alleles. The leucine
(L) allele had fragment sizes of 150, 82 and 50 bp, whereas the valine
(V) allele had fragments of 150 and 132 bp (Fig. 1).
Gene frequencies of L and V alleles were 0.936 and 0.064, respectively.
Genotypes are distributed according to the Hardy-Weinberg equilibrium.
The L allele frequency obtained in our study is agreement with those reported
by the other researchers, for example, Danish Holstein, 0.93; Danish Red,
0.85; Danish Jersey, 0.51 (Sorensen et al., 2002), Holstein
cows, 0.863 (Lee et al., 1996), Polish Friesian bull, 0.64 (Grochowska
et al., 2001), Holstein bulls, 0.91 (Yao et al., 1996),
Brown Swiss, 1.00; Guernsey, 0.92; Ayshire, 0.72; Jersey, 0.56 (Lucy et
al., 1993), Aberdeen Angus, 0.77, Simmental, 0.82 (Luciana et al.,
2003). Holstein, Brown Swiss and Guernsey breeds had higher frequencies
of the L allele of bGH whereas highest frequencies for the V allele were
found in Jersey and Airshire cows. Variants of bGH at position 127 are
the result of a point mutation (from CTG to GTG) in the nucleotide sequence.
This change converts the leucine codon to the valine codon (Lucy et
||A 3% agarose gel displaying an AluI restriction digest
on an amplified portion of bGH. Lane 4 is 50 bp ladder (Fermentase)
||Least square means of breeding values for milk yield and fat
and protein yield and percent in Iranian Holstein bulls with two bGH genotypes
|Values are means ± SE
The effect of the genotypes of AluI polymorphism on the breeding
values for milk related traits was examined using least square methods.
Least square means of the two bGH genotypes are shown in Table
Differences in the milk and fat yield between the two genotypes approached
significance (p≤0.10). Bulls with LL genotype had higher milk, fat
yield compared to LV genotypes. Present study showed that the L allele
was associated with protein yield (p≤0.021). No associations (p>0.1)
were found between the genotypes and fat and protein percent. Amino acid
position 127 is located in the third α-helix of the bGH molecule
and is close to residues that are involved in receptor binding. Therefore,
changes in the bGH molecule at this location may change interactions of
bGH with its receptor and affect growth or milk production.
These results for the GH RFLP are consistent with the results of (Lee
et al., 1996; Lucy et al., 1993) who reported that in American
Holstein-Friesian cows, the V allele is associated with low milk production.
Differences were found between the LL and the VV genotypes for carcass
gain and weight of meat in the carcass (Grochowska et al., 2001).
Shariflou et al. (2000) reported that the L allele of GH favoured higher
production of milk, fat and protein yield and no significant differences were
found between genotypes with percentages of fat and protein.
||Average additive gene substitution effects of alleles of AluI-GH
polymorphisms on milk, fat and protein yield; and fat and protein percent
Schlee et al. (1994) also reported that in AI bulls of three cattle
breeds in Germany, the LL genotype is associated with higher concentrations
of circulating growth hormone in different physiological conditions. Yao et
al. (1996) reported that there were no significant differences between genotypes
LL and LV in Canadian Holstein bulls for milk yield, fat percent and protein
The average effects of replacing a V allele with an L allele are shown
in Table 2. The average effects of the gene substitution
for AluI were amounted to 177 kg for milk yield, 3.8 kg for fat yield
and 5.4 kg for protein yield and -0.04 and 0.03 for fat and protein percent,
The dominant L allele at the GH locus favours higher production of milk
protein. It can be concluded that this locus is a QTL or is in disequilibrium
linkage with one or more tightly linked QTL. Either way, it could be a
useful DNA marker for milk production traits in dairy cattle breeding.
This research was financially supported by University of Tehran, Iran.
Data and pedigree records and semen of Iranian Holstein bulls was provided
through the Animal Breeding Center of Iran.