УДК 636.4:636.018:[573.6.086.83+577.21]


L.K. Ernst1, N.A. Volkova2, N.A. Zinovieva2

The comparative study of effect of integrated gene of human growth hormone-releasing factor on the aggregate of phenotypic signs in transgenic animals in the early and late generations at the similar conditions of keeping and growing permit to establish, that such individuals have heightened content of growth hormone in the blood and exceed of the intact analogs on live weight and its average daily gain. It was shown, that some stabilization of new correlations takes place in the next generations.

Keywords: pigs, recombinant DNA, human growth hormone releasing factor.


Creation of transgenic farm animals with modified economically valuable traits is of a great practical interest (1). Targeted change of mammalians’ genome by transgenic methods provides the breeding effect on individual characteristics in a much shorter time than when using traditional techniques, and it results in new genetic variants that didn’t exist in animal species before (integration of a transgene) or were not available for selection (resistance to infection). In particular, an important role belongs to genes encoding a cascade of growth hormone proteins. Currently, these genes have been modified in rabbits, pigs and sheep (2-6), along with 12 generations of pigs transgenic for human somatoliberin gene, which have been obtained and studied in the All-Russia Research and Development Institute for Livestock Husbandry (VIZH) (7-12 .)
It is assumed that extra synthesis of somatotropic proteins improves growth rate of animals. However, the effects of directed diversity at transgenesis sometimes are seen very simplified without understanding complex interactions that determine phenotypic changes of transgenic genotype. The effects of integration of foreign genes on biochemical, physiological and morphological properties still remain poorly studied, as well as the degree and direction of correlations between them. Even less is known about the dynamics of phenotypic parameters over generations, which allows estimating the breeding value of a transgenic animal. At the same time it is expected that the organism will tend to maintain a balance (stabilizing selection).
The purpose of this study was to assess the degree of influence of introduced growth hormone genes on a complex of phenotypic traits in different generations of transgenic animals kept under similar farming conditions.
Technique. The study was conducted in the vivarium of VIZH on transgenic pigs transgenic for somatoliberin gene (growth hormone releasing factor) controlled by mouse metallic-thionein promoter (7-12). Transgenic nature of animals was confirmed by PCR analysis, which data were used to form experimental and control groups. DNA isolation from samples of ear tissue and PCR were performed as described (13).
The dynamics of growth and development of both transgenic and intact pigs was studied under control feeding (from 30 to 120 kg with subsequent slaughter) in two independent experiments on animals of different generations: I experiment – the 2nd and 8th, II experiment – the 3rd and 9th generations. A control group in both experiments was formed from analogs (by live weight, breed, growing conditions, etc.). Live weight and its daily gain were estimated along with meat quality properties at different ontogeny periods (morphological composition of a carcass and its quality - length, percentage of meat and fat, fat thickness), and weight of the liver, stomach, intestine, kidney, uterus, ovarian, thyroid and pituitary were compared in transgenic and intact animals.
The content of growth hormone in the blood of transgenic pigs was determined using ELISA-based diagnostic system (“Diagnostic Systems Laboratories Inc.”, USA).  The samples of serum for analysis were stored at -20 °C.
Histological samples were prepared according to conventional techniques (14). The samples of organs and tissues were fixed with 10% formalin solution. Slices for histological samples were cut on a rotary microtome (“TermoShandon”, UK). To assess the expression level of nucleic acids (NA) in cells of internal organs, histological samples obtained by Feulgen’s technique (DNA) were stained with a mixture of gallocyanin and chrome alum (DNA + RNA) (14). Relative content of nucleic acids in cells of investigated organs were determined according to I.Ya. Shikhov et al. (15). Relative content of NA was found under the formula: NA = [100 x (1/B)] x S, where B - average brightness of the nuclei, 100 x (1/B) - relative density of the nuclei, S - area of the nuclei. Relative content of RNA in the cytoplasm of cells was calculated as difference between the content of total nucleic acids (staining with gallocyanin - chrome alum mixture) and the content of DNA in nuclei (Feulgen stain). Localization of growth hormone in the pituitary cells of pigs were detected immunohistochemically using Novostatin Super ABC Kit (“Novocastra”, USA) followed by detection with AEC substance (3-amino-9-ethylcarbazole) (“Sigma”, USA). Relative content of growth hormone in the pituitary cells was determined using the same formula as for NC. Histological samples were photographed using the microscope “Opton” (Germany) with a digital camera and analyzed in the computer program Image Scope (“Systems for Microscopy and Analysis”, Moscow).
Statistical calculations of results was performed by standard methods (16) in Microsoft Excel.
Results. The nature of inheritance of genetic structure in different generations of pigs was not significantly different; it was dependant on type of pairing and animal’s origin. Thus, if one of the parents was transgenic, the proportion of transgenic animals in the progeny averaged from 28,4 to 40,0%, and if both parents were transgenic, this value reached 50,0-62,5%. At the same time, there were observed individual effects of some animals on the efficiency of transgene inheritance: during the testing crosses between transgenic boars and non-transgenic sows, a high degree of inheritance of recombinant DNA was established in progeny of the boar № 831 (51,0%) and, conversely, a low degree - in progeny of № 046 (10,0%).

1. Growth hormone gene expressionоn in pigs transgenic for human somatoliberin gene (experiment)  and in intact analogs (control) in different generations (Х±х, vivarium of the All-Russia Research and Development Institute for Livestock Husbandry)


ELISA, ng/ml (Cv, %)

IHC, conv. units (Cv, %)






6,20±1,11 (54)

3,31±0,44 (56)

106,7±5,1 (45)

98,3±2,9 (28)


7,15±1,34 (56)

3,11±0,37 (51)

91,4±4,2 (44)

82,8±3,7 (42)


4,60±1,10 (72)

3,31±0,44 (56)

100,5±4,7 (44)

98,3±2,9 (28)


6,16±1,12 (54)

3,11±0,37 (51)

99,3±4,2 (33)

82,8±3,7 (42)

Note. ELISA – enzyme-linked immunoenzyme assay, IHC – immunohistochemical analysis, Cv — coefficient of variation



Fig. 1. Expression of growth hormone in hypophysis of a pig transgenic for human somatoliberin gene (A) and non-transgenic pig (B) (the darker areas indicated accumulated hormone). Immunohistohemical staining with AEC chromogen (3-amino-9-ethylkarbazol); magnification х400 (vivarium of the All-Russia Research and Development Institute for Livestock Husbandry).

The own level of growth hormone is one of key indicators reflecting the expression of human somatoliberin transgene in the organism of transgenic pigs. ELISA has shown that transgenic animals had 1,5-2,0 times higher content of growth hormone in the blood than their intact analogs (Table 1). In earlier generations (2nd and 3rd) this parameter was higher than in later generations (8th and 9th). These results of ELISA were confirmed by immunohistochemical studies: transgenic animals had 20% higher relative content of growth hormone in the pituitary cells than the control group (Table 1, Fig. 1).


Fig. 2. The dynamics of growth in different generation of  transgenic pigs carrying human somatoliberin gene and non-transgenic analogs (control) in two independent fattening experiments:  А — I experiment, 2nd and 8th generations (resp., 1 and 2), B – II experiment, 3rd and 9th generations (resp., 4 and 5); 3 and 6 – control (vivarium of the All-Russia Research and Development Institute for Livestock Husbandry).

А Б – A B
abscissa – Age, days
ordinate – Live weight, kg

Effects of the integrated recombinant DNA on animals’ productive properties (particularly, growth rate) were assessed. Transgenic animals were found to exceed the intact group by live weight and its daily gain in almost all studied periods of ontogenesis (Fig. 2). The greatest difference between these groups was, respectively, 11,9 and 35,7%. At the same, animals of earlier generations manifested higher growth rate than individuals of later generations. Thus, two independent experiments on control fattening have shown that the 2nd and 3rd generations of pigs exceeded the animals of 8th and 9th generations by live weight and its daily gain by 1,8-12,4 and 3,0-14, 5%, respectively.
Morphological composition of carcasses and their quality differed in transgenic and control pigs as well. Transgenic animals demonstrated changes in length of carcasses, percentage of meat and fat, fat thickness (Table 2). Fat thickness reduced in the line of transgenic generations – early generations exceeded the control by 16,9% for this parameter, while later generations - by 3,3-6,1% .

2. Morphological composition and quality parameters of carcasses of pigs transgenic for human somatoliberin gene compared to non-transgenic analogs (control) in different generations  (vivarium of the All-Russia Research and Development Institute for Livestock Husbandry).


Length of half-carcass, cm (Х±х)

Carcass output, %

Carcass composition, %

Fat thickness, cm (Х±х)




I  experiment






















II  experiment






















Note. Average fat thickness was determined in four points:  on withers, between 6th and 7th thoracic vertebrae, on lumbar and sacrum.

Transgenic animals are also superior to intact animals by weight of the liver, stomach, intestine, kidney, uterus, ovaries, thyroid and pituitary glands, and this advantage amounted to 65,2% for weight of the gastrointestinal tract and reproductive organs
Histological investigations of internal organs and tissues of transgenic pigs revealed no deviations of inflammatory or pathological nature. At the same time, morphometric distinctions from control were found in most of the samples. Thus, almost all transgenic generations showed the increased thickness of hepatic cords (up to +22,7%, p <= 0,01), glandular layer of the stomach (up to +11,2%, p £0,01), and height of duodenal villi (up to +24,3%, p <=0,05) that indicates more intense digestive function. Transgenic animals of earlier generations showed the increased diameter of pancreas acini and thyroid follicles (by 7,3%, p <= 0,05 and 6,5-19,1%, respectively), while individuals of later generations, in turn, yielded the intact analogs for these parameters by 6,4-8,8 (р <= 0,01) and 2,6-21,8 % (р<= 0,001), respectively. Significant changes were found in the structure of cardiovascular and excretory systems, as well as in muscle, but these differences varied in a number of generations (Table 3).
All studied generations of transgenic pigs had the greater size of cells owing to larger volume cytoplasm. Cells of transgenic animals were found to contain higher content of RNA compared to control (up to +34,6%, p <= 0,001), which indicates intense protein synthesis.

3. Morphometric histological parameters of internal organs and tissues of pigs transgenic for human somatoliberin gene compared to non-transgenic analogs (control) in different generations  (vivarium of the All-Russia Research and Development Institute for Livestock Husbandry).


Thickness, um

Diameter of thyroid follicles, um

Hepatic cords

Muscle fibers


I  experiment
















II  experiment
















The abovementioned facts suggest that increased levels of growth hormone cannot be fully manifested in transgenic pigs since auxiliary factors, processes and interaction mechanisms remain unchanged. For example, there could be a mismatch of the growth hormone content and the number of receptors for it. The obtained results agree with the theory of I.I. Schmalhausen who considered an organism as a whole system subject to individual and historical development. According to this theory, all body organs form a unified system whose parts are independent and interrelated to each other, so changes in one part of the organism cause corresponding changes in all other parts. At the same time, modifications induced in transgenic animals change the existing system of correlations, which undoubtedly affects individual development.
Thus, human somatoliberin gene integrated into the genome of pigs causes multiple effects on a number of traits. Some changes were manifested as a clear trend over all generations (eg, weight of the gastro-intestinal tract and reproductive organs, and reduction of fat thickness with increasing number of generations) while the lack of stably inherited reliable differences of growth parameters at early age. The latter fact could be the result of a long-term selection for rapid growth significantly reducing natural reserves for this trait compared to laboratory animals. Findings of this research reveal the ambiguous influence of the integrated somatotropin gene and its subsequent expression in the organism, which effect is manifested in transgenic individuals as changes of different traits at both cellular and at the organism level.


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8. Ernst L.K., Zinovyeva N.A., Volkova N.A., Parkhomenko E.G., Shatailo V.N. and Brem G., Influence of Integration of the Gene for Growth Hormone Releasing Factor on Some Economically Important Traits of Pigs, Zootekhniya, 2007, no. 5, pp. 2-5.
9. Volkova N.A., Volkova L.A., Klenovitsky P.M., Gusev I.V., Bagirov V.A., Ernst L.K., Zinovyeva N.A. and Brem G., Cytogenetic Profiles of Pigs Transgenic on Human Somatoliberin Gene Construct, Dokl. RASKhN, 2007, no. 5, pp. 37-38.
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1Russian Academy of Agricultural Sciences, Moscow 117218,
e-mail: ernstrashn@mail.ru;
2All-Russia Research and Development Institute for Livestock Husbandry, RAAS,
Moscow province, Podolsk region, Dubrovitsy 142132, Russia,
e-mail: natavolkova@inbox.ru

Поступила в редакцию
23 августа 2011 года