Reproductive performance of a transgenic boar carrying the bovine growth hormone gene (bgh)

Transcription

1 J. Appl. Genet. 43(3), 2002, pp Case report Reproductive performance of a transgenic boar carrying the bovine growth hormone gene (bgh) Barbara REJDUCH, 1 Ewa S OTA, 1 Pawe³ SYSA, 2 Marian RÓ YCKI 3 1 Department of Immuno- and Cytogenetics, National Research Institute of Animal Production, Balice, Poland 2 Department of Morphological Sciences, Warsaw Agricultural University, Warszawa, Poland 3 Department of Pig Breeding, National Research Institute of Animal Production, Balice, Poland Abstract. The aim of this study was to obtain information on the possible influence of the bovine growth hormone gene (bgh) on gametogenesis and reproductive parameters of a 2-year-old Polish Landrace transgenic boar. The bgh gene construct had been introduced into the zygote of the boar with the use of the microinjection technique. On the basis of the available documentation we established that the fertility of the investigated transgenic (bgh) boar was low in comparison with other animals of the Polish Landrace breed, with a poor libido, ineffective matings and, on average, 3 live piglets less per litter. Samples of testis tissue from the boar were obtained after castration. In total, we observed 100 spermatocytes and all of them had normally paired bivalents. It is possible that the boar s lower fertility was caused by some, as yet unknown factor. Key words: bgh transgenic boar, fertility, spermatogenesis, synaptonemal complexes. Gene transfer between mammalian organisms started in the early 1980 s after the first successful transfer of genes, which yielded the first transgenic mice. The constructs encoding growth regulators, such as the growth hormone (GH) or the growth hormone releasing factor (GHRF), were transferred using the microinjection technique in order to produce super mice (PALMITER et al. 1982). Received: March 13, Accepted: June 13, Correspondence: B. REJDUCH, Department of Immuno- and Cytogenetics, National Research Institute of Animal Production, ul. Krakowska 1, Balice, Poland, brejduch@izoo.krakow.pl

2 338 B. Rejduch et al. A large number of original research reports have focused on the influence of the growth hormone (GH) on reproductive traits in various mammalian species (PALMITER et al. 1982). In the 1990 s some experiments were carried out to study the influence of the bovine growth hormone gene (bgh) transferred into the genome of the pig on the growth and development of the animal (SOLOMON et al. 1994, RÓ YCKI et al. 1999). Gene transfer in animals is required to evaluate the potential for improving production efficiency, carcass quality and disease resistance of livestock. It has been established that administration of exogenous porcine somatotropin (pst) to pigs at different stages of growth and development, results in alteration of body composition (CAMPBELL et al. 1988, SMITH, KASSON 1990). The studies of PURSEL and REXROAD (1993) showed that the bovine growth hormone gene (bgh) transferred to the pig genome results in stimulation of muscular development. The trangenic pigs showed muscular hypertrophy at approximately 3 months of age and had longer hams. Furthermore, SOLOMON et al. (1994) showed that carcass tissue from bgh transgenic pigs had much more favourable levels of fatty acids than control pigs. For these reasons it was important to obtain information about the possible influence of the bgh gene transferred into boar on his gametogenesis and in consequence on his reproductive parameters and fertility. Case report We analysed the reproductive performances of a 2-year-old Polish Landrace transgenic boar, which weighed above 300 kg (average weight for two year old Polish Landrace boars ranged from 200 to 250 kg) and carried the bgh gene. The boar was kept at Experimental Station in Grodziec Œl¹ski. The bgh gene construct had been introduced into the zygote with the use of the microinjection technique. The presence of the bgh gene construct on the investigated boar s genome was screened by PCR and Slot Blot method (RÓ YCKI et al. 1999, SMOR G et al. 1999). Chromosomal integration of the transgene is not known. On the basis of the available documentation we established that the fertility of the investigated transgenic (bgh) boar was low in comparison with other animals of the Polish Landrace breed. This mature boar showed a very poor libido and it was rather difficult to obtain his semen for artificial insemination. The boar s semen was used for insemination of seven sows and one of them was mated twice (her first mating was effective and the second was ineffective). The ratio of effective: ineffective matting was 6:2. The transgenic boar s progeny totalled 47 piglets, but only 38 were born alive. Average number was established as 6.7 live born piglets per litter, while the average number of live born piglets for Polish Landrace boars in that Experimental Station was 9.5. Unfortunately, we could not obtain any information about parameters of his semen.

3 Reproductive performance of a bgh - transgenic boar 339 Samples of testis tissue from the boar were obtained after castration. The preparations of meiotic chromosomes for observations under an electron microscope were made according to COUNCE and MEYERS (1973) method with minor modifications. The synaptonemal complexes (SC) were observed using a transmission electron microscope JEOL JEM100C. In total, 100 spermatocytes in different substages of pachytene were analysed. Among them, we found 11 spermatocytes in the Z/P stage (zygotene/pachytene), 20 in P1 (early pachytene), 44 in P2 (mid pachytene) and 25 in P3 (late pachytene). Only normally paired bivalents were found (Figure 1). Chromosome changes in pig karyotype may be the cause of reproductive failure due to disturbances of chromosome segregation during meiosis. Over the last decades, investigation of chromosome rearrangements and their impact on fertility in domestic animals was possible due to development of new research methods. The electron microscope examination of synaptonemal complexes (SC) at meiotic pachyten I in boars is a method for investigation of pairing bivalents (VILLAGOMEZ 1993, ŒWITOÑSKI, STRANZINGER 1998). Abnormal progress of the whole process of chromosome pairing may lead to production of unbal- Figure 1. Synaptonemal complexes (SC) of a transgenic (bgh) boar: all bivalents completely paired (early pachytene). ME JEOL JEM100C microscope,

4 340 B. Rejduch et al. anced gametes causing decreased fertility. It can result in the arrest of male gametogenesis and consequently in total sterility of the animal (ŒWITOÑSKI, STRANZINGER 1998). In this study, analysis of synaptonemal complexes in the transgenic boar carrier of bgh revealed exclusively normal pairing bivalents. However, the investigated transgenic boar showed a low fertility in comparison with other Polish Landrace boars. It is possible that the lower fertility was caused by some, as yet unclear reason. Today, one of the interesting questions is how a newly introduced genetic information is transmitted to future generations. Transmission rate of the transferred gene is one of the most awaited parameters in transgenic technology. Often an important gene construct is eliminated from the genome of the animal. In some cases the introduced gene can be integrated into the host genome but become silent for an unknown reason and later become awake in a relatively distant generation (SUTHERLAND et al. 2000). However, some authors were able to successfully produce the next generation of transgenic pigs (RÓ YCKI et al. 1999, JURA et al. 2000). The analysis of spermatogenesis is a very important tool for identifying the background of transgene elimination. Acknowledgements. This work was supported by State Committee for Scientific Research, Project No. 6PO6D REFERENCES CAMPBELL R.G., STEELE N., CAPERNA T.J., MCMURTY J.P., SOLOMON M.B., MITCHEL A.D. (1988). Interrelationships between energy intake and endogenous porcine growth hormone administration on the performance, body composition and protein and energy metabolism of growing pigs weighing 25 to 55 kilograms live weight. J. Anim. Sci. 66: COUNCE S.J., MEYER G.F. (1973). Differentiation of the synaptonemal complex and the kinetochore in Locusta spermatocytes studied by whole mount electron microscopy. Chromosoma 44: JURA J., SMOR G Z., SKRZYSZOWSKA M., KOPCHICK J.J., KELDER B., PRIETO P.A., KARETA W., PASIEKA J. (2000). Efektywnoœæ transgenezy u królika, kozy i œwini z zastosowaniem genu modyfikuj¹cego sk³ad mleka. [Effectiveness of transgenesis in the rabbit, goat and pig with the use of a gene modifying milk composition]. Rocz. Nauk. Zoot. 5: PALMITER R.D., BRINSTER R.L., HAMMER R.E., TRUMBAUER M.E., ROSENFELD M.G., BIRNBERG N.C., EVANS R.M. (1982). Dramatic growth of mice that develop from eggs micro-injected with metallothioein-growth hormone fusion genes. Nature (Lond.) 244: PURSEL V.G., REXROAD C.E. Jr. (1993). Status of research with trangenic farm animals. J. Anim. Sci. 71(3): RÓ YCKI M., SMOR G Z., KOPCHICK J.J., CHEN Wen Y., JURA J., PASIEKA J., ORZECHOWSKA B., GAJDA B., SKRZYSZOWSKA M. (1999). Performance of transgen-

5 Reproductive performance of a bgh - transgenic boar 341 ic pigs with the use of two different growth hormone gene constructs. J. Appl. Genet. 40(1): SMITH V.G., KASSON C.W. Growth performance and carcass characteristic of pigs administrated recombinant porcine somatotropin during 30 to110 kilogram live weight. J. Anim. Sci. 68: SOLOMON M.B., PURSEL V.G., PAROCZAY E.W., BOLT D.J. (1994). Lipid composition of carcass tissue from trangenic pigs expressing a bovine growth hormone gene. J. Anim. Sci. 72: SMOR G Z., GAJDA B., JURA J., SKRZYSZOWSKA M., PASIEKA J. (1999). Factors affecting the production of zygotes in superovulated pigs: seven-year retrospective studies. Ann. Anim. Sci. 26(4): SUTHERLAND H.G., KEARNS M., MORGAN H.D., HEADLEY A.P., MORRIS C., MARTIN D.I., WHITELAW E. (2000) Reactivation of heritably silenced gene expression in mice. Mamm. Genome. 11(5): ŒWITOÑSKI M., STRANZINGER G. (1998). Studies of synaptonemal complexes in farm mammals a review. J. Hered. 89: VILLAGOMEZ D.A.F. (1993). Zygotene-pachytene substaging and synaptonemal complex karyotyping of boar spermatocytes. Hereditas 118: