Growth Hormone (GH)
Gene Polymorphism and Its Association with Meat Productivity in Two Rough Wool
Sheep Breeds Grown in Russia's Dry Zone
Ivan Fiodorovich
Gorlov1,2*, Nadezhda Vasilievna Shirokova1,3, Arkadiy
Kanurovich Natyrov4, Yuriy Anatolievich Kolosov3, Marina
Ivanovna Slozhenkina1,2, Anatoliy Yurievich Kolosov3,
Natalia Ivanovna Mosolova1,2, Elena Yurievna Anisimova1,
Ekaterina Vladimirovna Karpenko1,5 and Dmitriy Vladimirovich
Nikolaev1
1Volga Region Research Institute of
Manufacture and Processing of Meat-And-Milk Production, Rokossovskogo Street,
6, Volgograd, 400131, Russian Federation
2Volgograd State Technical University,
Lenina Avenue, 28, Volgograd, 400005, Russian Federation
3Laboratory of Molecular Diagnostics and
Biotechnology of Farm Animals, Don State Agrarian University, Persianovsky,
Rostov Province, 346493, Russian Federation
4Kalmyk State
University, 358000, Pushkina street, 11, Elista, The Republic of Kalmykia,
Russian Federation
5Volgograd State University, University
Avenue, 100, Volgograd, 400062, Russian Federation
*For correspondence:
niimmp@mail.ru
Received 26 August 2020; Accepted 10 October 2020; Published 10 December
2020
Abstract
The article considers the methods of efficient use of
the gene pool of rough wool sheep breeds in order to improve the level and
quality of meat production. The research material was Edilbay (n=100) and
Kalmyk fat-tailed sheep (n=100) breeds. The influence of the polymorphism of
the genotypes of the growth hormone gene on meat indices were established; and
the desired genotypes for further breeding work to increase their specific
weight in the population structure were determined. The AB/GH genotype associated with the best meat production in carriers
of this genotype has been identified as desirable. The results of the control
slaughter have indicated Kalmyk fat-tailed rams of the AB/GH genotype to be superior to their AA/GH and BB/GH genotypes
peers in terms of their pre-slaughter weight. The conducted research indicated the
feasibility of genetic markers to optimize breeding programs in flocks of rough
wool sheep in order to increase the meat production level. The obtained data
confirmed the need to investigate the DNA markers associated with the
productive qualities of sheep to get greater efficiency of breeding and enhance
profitability of the sheep industry. © 2021 Friends Science Publishers
Keywords: Growth
hormone gene polymorphism; MAS; PCR-RFLP; Sheep breeding; Slaughter traits; SNP
Introduction
Currently, the Russian Federation has great potential
both for increasing the sheep population and for the growth in production of
all types of products in sheep industry (Lescheva and Ivolga 2015). On its
territory there is large space of natural pastures that farmers can rationally
use to meet the need of sheep for feed without considerable material costs;
there are unique sheep breeds (Edilbay and Kalmyk fat-tailed) characterized by
a high genetic potential of production and good ability to adapt to various
climatic conditions; there is a substantial demand for organic meat products of
sheep production both in Russia and abroad. Over the past 20–25 years, the
consumption of mutton in the country has remained stable (1–1.2 kg/person per
year), and a positive shift in the industry can only be expected when a steady
consumer demand is formed (Fisinin 2017). In the early 2018, Russia imported
1.7 thousand tons, with the export of mutton amounting to 4.1 thousand tons
against 41 tons for the same period last year. Iran was the main consumer of
Russian lamb. This can be largely explained by the fact that in May, on the
margins of the Astana Economic Forum, the EAEU countries and Iran signed a free
trade agreement that fixed that the import duty on these products would not
exceed 5%. Thus, taking into account the export of mutton, there was recorded a
high positive dynamics of consumer demand among foreign trade partners. This
indicates good prospects for sheep industry.
According to
the Federal State Statistics Service, at the beginning of 2018, the sheep livestock
in farms of all categories amounted to 23 million head; 146.2 thousand of them
belonged to the Edilbay breed and 44.1 thousand to the Kalmyk fat-tailed breed.
Having large areas of natural rangelands, the regions of the Southern Federal
District are a traditional place of breeding sheep of unique breeds
characterized by a high genetic potential of productivity and adapted to
breeding in various climatic conditions. In the Southern Federal District, the
sheep population numbers 6.3 mil. head; 56.5 thousand of them are of Edilbay
breed, and 43.3 thousand of Kalmyk breed.
Each sheep
breed is characterized by a unique gene pool and results from long, focused and
hard work. Long-term intensive selection of breeds creates stable co-adaptive
gene complexes that determine specific characteristics of a particular breed
and the adaptive rate of populations (Rasali et al. 2006; Deniskova et al.
2018).
Sheep
breeding has historically been an integral part of the national economy,
especially in the North Caucasus and Southern Federal Districts, providing
industry’s needs for specific types of raw materials (wool, krimmer skin and
sheepskins) and the population’s needs for food (lamb and milk) (Zinovieva et al. 2015). In the recent past, the
economy of sheep farming in our country was based mainly on wool production.
Its share in the total cost of this industry production was 60–80%, and the
selling price of 1 kg of wool was equivalent to 20 kg of mutton in live weight.
At the same time, the ratio of prices for wool and lamb was 1:3 in the world
market.
Lamb is one
of the most valuable types of meat products and is in high demand in the world
market (Buschulte et al. 2005).
Resulting from the increasing economic importance of meat production, the rough
wool sheep breeds – Edilbay and Kalmyk fat-tailed with their all-purpose
production, especially meat production – have become important. The uniqueness
of rough wool sheep breeds lies in their abilities and traits being not
characteristic of animals in other production areas, i.e., high
resistance to infectious diseases, adaptability to rapid temperature changes
and ability to overcome long distances during drifts to pastures.
Developing
methods for more efficient use of the gene pool of rough wool sheep to enhance
the quality of meat production, reduce feed costs per unit of production,
genetic control and breeding management and find additional reserves that are
able to improve the economic performance of the industry are the most important
tasks at the present stage of sheep breeding development (Deniskova et al. 2016; Gorlov et al. 2018a; Bayram et al.
2019; Ekegbu et al. 2019). The growth
hormone (GH) gene is considered as a
marker of sheep meat production (Tahmoorespur
et al. 2011). The GH is of
great importance for regulating growth processes, cell proliferation and
differentiation in all mammalian species. Somatotropin has a powerful anabolic
and anti-catabolic effect, enhances protein synthesis, inhibits its breakdown
and helps reduce subcutaneous fat deposition, increase fat burning and increase
the ratio between muscle and adipose types of mass. The GH is a protein with a
molecular weight of 22,000; its polypeptide chain consists of 191 amino acid
residues (Gorlov et al. 2017).
The purpose
of our investigation was to study the GH
gene polymorphism that allows selection of rough wool sheep genetically
predisposed to high meat production. Furthermore, the obtained data confirmed the
need to study the DNA markers associated with the productive qualities of sheep
to get greater efficiency of breeding and enhance profitability of the sheep
industry.
Materials and Methods
Sample collection
and genomic DNA isolation
The research material was sheep of Edilbay (n=100) and
Kalmyk fat-tailed (n=100) breeds (males) of the herd in the Kirovsky breeding
plant, the Yashkulsky rayon in the Republic of Kalmykia. For molecular genetic
studies of sheep (n=200), tissue samples of 1 cm˛ were taken from the auricle.
The DNA was isolated using Nexttec columns (Nexttec GmbH, Germany) in
accordance with the manufacturer's recommendations.
PCR analysis and SNP genotyping
The data were analyzed by the method of PCR-RFLP. For
amplification of a fragment of the GH gene 934 bps long, there were used
oligonucleotide primers: 5'- GGAGGCAGGAAGGGATGAA-3' and 5'-CCAAGGGAGGGAGAGACAGA-3'
(Gorlov et al. 2017). The composition
of PCR mixture for amplification was following (in a final reaction volume of
25 µL): about 100–150 ng of isolated DNA (5 µL in average); 0.5 µL
of each oligonucleotide primers (forward and reverse); 1 µL mixture of
dNTPs; 5 µL of PCR buffer, and 0.3 µL of Taq DNA Polymerase
(Tersus Plus PCR kit, Evrogen, Russia).
The
amplification mode contained pre-denaturation at 95°C for 5 min. and then 33
cycles: 95°C for 45 s, 60°C for 45 s and 72°C for 45 s; final synthesis at 72°C
for 10 min. The PCR was done on a thermocycler Tertsik, Russia. The amplified
fragment was reduced by the endonuclease HaeIII.
For the restriction analysis of the obtained PCR products in a final volume of
10 μL, there were mixed 6 μL of a PCR product, 2.5 μL
of ddH2O, 0.5 μL of the Hae
III endonuclease restriction enzyme (SibEnzyme-M, Russia) and 1 μL
of buffer for the enzyme. The hydrolysis was carried out at a temperature of 65
0С for 1 h in a thermostat ТТ-2 Termit (NPO DNA-Tekhnologiya
LLC, Russia). The sizes of the fragments were determined in comparison with the
molecular weight marker M100 (Izogen, Russia, the length of the fragments 100
to 1000 bp) supplied with 1 mL of 6xDNA dye on 4% agarose gel (Helicon, Russia)
and stained with ethidium bromide (Helicon, Russia). The resulting restriction
fragments were visualized in ultraviolet light. The presence of 10 restriction
sites corresponded to allele A, the presence of 11 sites to allele B. The size
of the restriction fragments obtained was determined by electrophoresis in a 4%
agarose gel with ethidium bromide. The molecular genetic analysis established
the presence and frequency of alleles and genotypes.
The presence
and the frequency of alleles and genotypes were established similarly Gorlov et al. (2017). The allelic and genotypic
frequencies, the heterozygosity observed (Ho) and expected (He), and the
Hardy-Weinberg equilibrium tests were calculated by Pop Gene 3.1 software. The
research allowed us to solve the problem of assessing the state of the sheep
populations under study in terms of the statistical significance of differences
in the values of the heterozygosity observed and expected. The frequency of
heterozygotes is an important indicator, since each heterozygous individual carry
different alleles and thus indicates the presence of variability.
Slaughter traits
The control slaughter of rams aged 4 months helped
establish the meat qualities (in accordance with the requirements of the GOST
31777-2012 “Sheep and goats for slaughtering. Mutton, lambs and goats in
carcasses specifications”) with respect to parameters, i.e., the
pre-slaughter weight (kg), weight of fresh carcass (kg), weight of chilled
carcass (kg), slaughter weight (kg), and slaughter yield with fat-tail (%). All
animals studied (100 animals in each group) were the same year of birth with
minimal differences in age, kept in the same feeding conditions and daily
routine and served by the same employees.
Statistical analysis
The data on different obtained variables were
statistically analyzed by Statistica 10 package (StatSoft Inc.). The
significance of differences between the indices was determined by the criteria
of nonparametric statistics for linked populations (differences with P
< 0.05 were considered significant; NS = not significant at P <
0.95). Student’s t-test was applied for the statistical analysis (Johnson and
Bhattacharyya 2010).
Results
Molecular genetic studies of biological samples obtained
from sheep of Kalmyk fat-tailed breed (Fig. 1A) allowed establishing a GH polymorphism pattern caused by the
ratio between alleles A and B. The allele A and AA genotype had the
highest frequency in the sheep group under study. The frequencies of the AA, AB
and BB genotypes were set at a ratio of 52.0, 35.0 and 13.0%, respectively
(Fig. 2). The average value of the heterozygosity observed (Ho) in Kalmyk
fat-tailed breed was found 0.350 and expected (He) was higher – 0.574. The
research studies of the biomaterial obtained from Edilbay sheep (Fig. 1B)
found, that the GH gene polymorphism
represented by A and B alleles had a slightly different
structure. In the population studied, the highest frequency was also
characteristic of the allele A and
homozygous AA genotype (Fig. 2).
However, the picture of the
frequency of the AA, AB and BB genotypes identified in
Fig. 1: Electropherograms
of the PCR-RFLP result of the GH (HaeIII)
in 4% agarose gel (M is DNA marker – 100 bp): (A): Kalmyk fat-tailed sheep breed; (B): Edilbay fat-tailed sheep breed
Fig. 2: The frequency of alleles and genotypes of the GH gene in Kalmyk fat-tailed breed and
Edilbay breed
this group looked different than in Kalmyk fat-tailed
group, namely: 48.0, 35.0 and 17.0%, respectively. So, the proportion of AA homozygotes was noted to have
slightly decreased, and the BB
homozygotes proportion was registered to have increased. The proportion of
heterozygous AB genotype remained
virtually unchanged. The average value of the heterozygosity observed (Ho) in
Edilbay breed was found 0.350 and expected (He) was higher – 0.448.
The values of
the heterozygosity observed and expected in the studied populations of sheep of
Kalmyk fat tailed and Edilbay breeds are presented in Table 1. In our work, in
all cases, there was found no statistical significance of differences in the
values of the heterozygosity observed and expected. The distributions of
heterozygous genotypes observed reliably corresponded to those expected by
Hardy-Weinberg's equilibrium law; according to the χ˛ value obtained, the
populations under study were in equilibrium.
Further
studies on the relationship between the allelic GH gene variants and indicators of rough wool sheep meat production
showed that the best meat productivity belonged to the AB/GH rams that significantly exceeded their AA/GH analogs with respect to almost all traits analyzed (Table 2).
The analysis of the control slaughter data showed that Kalmyk fat-tailed sheep
of the AB/GH genotype surpassed their
AA/GH and BB/GH genotypes peers in terms of the pre-slaughter weight by 3.7 (P < 0.001) and 2.6 kg (P < 0.01). The slaughter weight and
slaughter yield of the AB/GH genotype
rams also exceeded these parameters in comparison with the AA/GH genotype rams by 2.2 kg (P
< 0.05) and 0.9%, respectively. A similar pattern was observed when
comparing the heterozygous AB/GH and
homozygous BB/GH genotypes of rams.
The slaughter weight and slaughter yield of the AB/GH genotype rams were more by 1.2 kg (P < 0.05) and 0.5%, respectively.
Table
1: Observed and expected heterozygosity of the growth
hormone gene in sheep populations
Breed |
Number of animals (n) |
Heterozygosity observed (HO) |
Heterozygosity expected (HE) |
χ˛ |
Kalmyk fat tailed breed |
100 |
0.350 |
0.574 |
3.04 |
Edilbay breed |
100 |
0.350 |
0.448 |
5.09 |
Table
2: Slaughter traits of rams of different GH genotypes (M ± SE)
Genotype |
Pre-slaughter live weight (kg) |
Carcass fresh weight (kg) |
Carcass cooled weight (kg) |
Slaughter weight (kg) |
Meat yield (per kg bones) |
Slaughter yield with fat-tail (%) |
Kalmyk fat-tailed breed (n=100) |
||||||
АА (n=52) |
38.0 ± 0.79a0 |
16.4 ± 0.81b0 |
16.0 ± 0.73bc |
19.3 ± 0.72c0 |
3.47 ± 0.03a*** |
50.7 |
АВ (n=35) |
41.7 ± 0.72*** |
18.9 ± 0.40*** |
18.4 ± 0.54*** |
21.5 ± 0.49*** |
3.56 ± 0.01*** |
51.6 |
ВВ (n=13) |
39.1 ± 0.45b0 |
16.9 ± 0.64bc |
16.3 ± 0.58bc |
20.1 ± 0.31c*** |
3.16 ± 0.04a*** |
51.1 |
Edilbay breed (n=100) |
||||||
АА(n=48) |
36.8 ± 0.46 |
14.9 ± 0.34 |
14.3 ± 0.38 |
18.3 ± 0.42 |
2.93 ± 0.04 |
48.9 |
АВ (n=35) |
38.4 ± 0.49C |
16.2 ± 0.36B |
15.6 ± 0.33C |
19.5 ± 0.28C |
3.03 ± 0.02c |
50.7 |
ВВ (n=17) |
37.4 ± 1.22NS |
15.3 ± 0.12NS |
14.9 ± 0.10NS |
18.6 ± 0.21NS |
3.02 ± 0.01c |
49.7 |
Note: a = P <
0.001, b = P < 0.01, c = P < 0.05, ns = not significant at
P<0.95 compared with data on the AB-genotype in Kalmyk fat-tailed breed
group; A = P < 0.001, B = P < 0.01, C = P < 0.05, NS = not significant at P < 0.95 compared with data on the AA-genotype in Edilbay breed
group; *** = P < 0.001, ** = P < 0.01, * = P < 0.05, 0 = not significant at P < 0.95 compared with data on the similar genotype in the
different breeds group
By all
parameters of the control slaughter, Edilbay rams of the AB/GH genotype exceeded their AA/GH
and BB/GH genotypes peers. So, in
Edilbay breed, the pre-slaughter live weight of the AB/GH genotype rams was higher than that of the AA/GH and BB/GH genotypes rams by 1.6 (P
< 0.05) and 1.0 kg (NS), and the slaughter yield by 1.8 and 1%,
respectively.
Discussion
In recent years, a number of reports have appeared that
noted the relationship between the different gene polymorphisms and the meat
production of sheep bred in Russia (Gorlov et
al. 2016; Trukhachev et al. 2016,
2017; Gorlov et al. 2018b). The
purpose of these studies was to enhance the level and quality of meat
production, as well as reduce the cost of feed per unit of production in
Russian sheep breeding. The GH gene
studies by Gorlov et al. (2017) on
Salsk sheep of Russian breeding established all three AA, AB and BB genotypes’ frequencies of 57, 36 and
7%, respectively. The frequency of the allele B was 0.25 and alleleA0.75.
In the studies conducted, the heterozygous genotype of Salsk sheep was
associated with the best average daily gains in the period from 2nd
to 9th months.
Similar
results were obtained by Kolosov et al.
(2015), who proved the positive effect of the heterozygous AB/GH genotype on the average daily gains and meat production of
Merino sheep breeds.
Most
researchers believe that the issue of increasing the level and quality of meat
production can be solved by genetic methods. In this regard, the GH gene looks
the most promising. So, in the studies conducted by Hajihosseinlo et al. (2013) on Makooei sheep, the best
indices of meat production were associated with the AB genotype.
During
previously conducted studies to identify the relationship between the
polymorphism and the growth and weight characteristics of sheep of various
breeds, similar results were obtained. According to the research by Palmer et al. (1998) on crossbred sheep from
crossing Dorset and Coopworth breeds, it was found that the AB genotype sheep gained more than the AA genotype sheep by 123 g per day or
18%. Thus, the sheep GH gene polymorphism
can be used as a marker for weight gain and higher meat production.
Conclusion
The results of the GH
gene polymorphism in sheep of two rough wool breeds grown in the arid zone of
Russia were obtained for the first time; and significant associations between
the GH gene genotypes and sheep meat
productivity were revealed. The influence of the polymorphism of the genotypes
of the growth hormone gene on meat indices were established; and the desired
genotypes for further breeding work to increase their specific weight in the
population structure were determined. The conducted research indicated the
feasibility of genetic markers to optimize breeding programs in flocks of rough
wool sheep in order to increase the meat production level.
Acknowledgements
The authors are grateful to the Russian
Science Foundation for the financial support in the implementation of this
research according to the scientific project # 19-76-10010, NIIMMP. The authors
are also grateful to Badma Esinovich Garyaev (Director General of Plemzavod
"Kirovskiy") for kindly agreeing to use of his animals in this study.
Author Contributions
Conceptualization:
IFG, YuAK, NVSh; Data curation: AYuK, NIM, AKN; Formal analysis: MIS, AKN;
Funding acquisition: EYuA, DVN; Investigation: NVSh, AYuK; Methodology: IFG,
YuAK; Resources: MIS, AKN; Supervision: IFG, YuAK; Writing-original draft:
EYuA.
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