Genetic engineering of livestock.

Genetic engineering of livestock is expected to have a major effect on the agricultural industry. However, accurate assessment of the consequences of transgene expression is impossible without multigenerational studies. A systematic study of the beneficial and adverse consequences of long-term elevations in the plasma levels of bovine growth hormone (bGH) was conducted on two lines of transgenic pigs. Two successive generations of pigs expressing the bGH gene showed significant improvements in both daily weight gain and feed efficiency and exhibited changes in carcass composition that included a marked reduction in subcutaneous fat. However, long-term elevation of bGH was generally detrimental to health: the pigs had a high incidence of gastric ulcers, arthritis, cardiomegaly, dermatitis, and renal disease. The ability to produce pigs exhibiting only the beneficial, growth-promoting effects of growth hormone by a transgenic approach may require better control of transgene expression, a different genetic background, or a modified husbandry regimen.

Induction of lactogenesis in transgenic virgin pigs: evidence for gene and integration site-specific hormonal regulation.

Five month-old transgenic female pigs from three lines carrying the mouse whey acidic protein (WAP) gene and nontransgenic female littermates were implanted with slow-release estrogen and progesterone pellets. Histological analysis of biopsies taken at the time of implantation and 4 weeks later revealed that mammary alveolar development had occurred upon hormonal stimulation in vivo. beta-Casein and beta-lactoglobulin mRNA was found in all induced animals, and WAP mRNA was detected in two of the three transgenic pigs. Differential hormonal regulation between the transgenes in the three lines and also between endogenous milk protein genes was observed in induced mammary tissue cultured in vitro. In the presence of insulin, hydrocortisone, and PRL, beta-casein and WAP mRNA levels increased in all transgenic pigs. In contrast, beta-lactoglobulin mRNA had reached or exceeded lactational levels in response to the in vivo induction, and no further increase was observed in vitro. This suggests that the regulation of the beta-lactoglobulin gene is distinct from that of beta-casein and WAP. Differences were also observed during pregnancy; whereas beta-lactoglobulin gene expression was induced in early pregnancy, a time when PRL levels are low, WAP mRNA levels increased sharply around parturition. Finally, the observation that hormonal regulation of WAP transgenes greatly differed between the three lines suggests that chromatin surrounding the integration site can modify the response of transcription elements.

Follicular expression of a human beta-cell leukaemia/lymphoma-2 (Bcl-2) transgene does not decrease atresia or increase ovulation rate in swine.

Transgenic (TG) gilts carrying a human Bcl-2 cDNA transgene driven by mouse inhibin-alpha subunit promoter were produced and evaluated to determine if ectopic expression of Bcl-2 in the ovaries would decrease the frequency of atresia in antral follicles and increase ovulation rate. Immunohistochemical analysis showed that the Bcl-2 transgene protein was expressed in granulosa and theca cells, in 86% of healthy and 54% of atretic follicles analysed in TG prepubertal and Day 50 pregnant gilts combined (n = 24). In contrast, Bcl-2 transgene protein was expressed in only 1.4% of healthy and 0% of atretic follicles in non-TG littermates (n = 13). Real-time reverse transcription-polymerase chain reaction analysis confirmed that human Bcl-2 was expressed in follicles of TG gilts. The atresia rate for the TG and non-TG groups did not differ (P > 0.05) for prepubertal (45 v. 59%) and Day 50 pregnant gilts (53 v. 52%) respectively. The mean +/- s.e.m. ovulation rate did not differ (P > 0.5) between TG (15.9 +/- 0.8, n = 12) and non-TG (16.4 +/- 0.6, n = 7) Day 50 pregnant gilts. The molecular basis of the failure of ectopic Bcl-2 expression to increase the ratio of healthy to atretic follicles is unknown, but it is possible that the activity of the mitochondrial-dependent cell death pathway was not neutralized by ectopic expression of human Bcl-2 or that other cell death pathways compensated for the decreased mitochondrial-dependent cell death.

Expression of human or bovine growth hormone gene with a mouse metallothionein-1 promoter in transgenic swine alters the secretion of porcine growth hormone and insulin-like growth factor-I.

Endocrine profiles were examined in swine that had integrated and expressed a fusion gene consisting of mouse metallothionein-1 (MT) promoter fused to either a human (h) or bovine (b) GH structural gene. Eleven of 18 pigs that had integrated MT-hGH and eight of nine pigs that had integrated MT-bGH expressed the genes. The level of expression varied widely among pigs (14-4551 micrograms/l for MT-hGH and 23-1578 micrograms/l for MT-bGH). The level of expression varied over time within each pig with no general pattern. Concentrations of porcine GH (pGH) were lower in MT-hGH pigs that expressed the gene than in non-expressors or in littermate controls. Insulin-like growth factor-I (IGF-I) concentrations increased with age in all pigs and were raised threefold in pigs expressing either the MT-hGH or MT-bGH genes. Measurement of the foreign GH in samples taken at 15-min intervals failed to reveal any short-term fluctuations in concentration. Administration of hGH releasing factor (GRF) to pigs expressing MT-bGH resulted in attenuated release of pGH compared with that of contemporary controls. Concentrations of bGH did not change after GRF injection. Human and bovine GH expressed in transgenic pigs appear to be biologically active in that they induce IGF-I and suppress endogenous pGH secretion. The failure to find short-term fluctuations and the lack of response to GRF injections are consistent with a non-pituitary and non-GRF regulatable site of production.

Dual-energy X-ray absorptiometry measurements of the body composition of pigs of 90- to 130-kilograms body weight.

We used the pig as an in vivo model for evaluating the effects of extreme body depth on the measurement of body composition by dual-energy X-ray absorptiometry (DXA). One group of 17 pigs weighed an average of 90 kg and had a maximum body depth of approximately 30 cm; another group of 54 pigs weighed 123 kg on average and had a maximum body depth of about 35 centimeters. In the larger pigs, DXA tended to measure a higher percentage of total body fat relative to the chemical analysis than in the smaller pigs. In both groups, but more predominantly in the larger pigs, there were areas in the bone of extreme thickness and/or density that were excluded from the analysis, which caused underestimation of bone mineral content and total tissue mass.

Progress on gene transfer in farm animals.

Transgenic pigs and sheep have been produced by the microinjection of single-cell zygotes and two-cell ova with linear molecules of mouse metallothionein I (MT) promoter/regulator fused to either the human growth hormone (hGH) or bovine growth hormone (bGH) structural genes. The foreign genes integrated into the chromosomes of 3 of 111 lambs or fetuses and 31 of 341 pigs or fetuses examined. Immunoreactive hGH or bGH was present in the plasma of two transgenic lambs and 19 transgenic pigs. The hGH concentration in plasma varied greatly among pigs and was unrelated to the number of gene copies that had integrated. Rate of growth was not enhanced in any of the transgenic pigs in comparison to their littermate controls. However, bGH and hGH exerted definite biological effects in transgenic pigs as evidenced by significantly depressed backfat measurements, elevated levels of insulin-like growth factor (IGF-I), stimulation of mammary development (by hGH) and reduction in porcine growth hormone (pGH) to nondetectable levels in plasma. Five of six founder transgenic pigs transmitted the MT-hGH gene construct to one or more progeny. Three progeny of a boar that expressed hGH also expressed the foreign gene.

Culturing the epiblast cells of the pig blastocyst.

Pig epiblast cells that had been separated from other early embryonic cells were cultured in vitro. A three-step dissection protocol was used to isolate the epiblast from trophectoderm and primitive endoderm before culturing. Blastocysts collected at 7 to 8 days postestrus were immunodissected to obtain the inner cell mass (ICM) and destroy trophectodermal cells. The ICM was cultured for 2 to 3 days on STO feeder cells. The epiblast was then physically dissected free of associated primitive endoderm. Epiblast-derived cells, grown on STO feeders, produced colonies of small cells resembling mouse embryonic stem cells. This primary cell morphology changed as the colonies grew and evolved into three distinct colony types (endodermlike, neural rosette, or complex). Cell cultures derived from these three colony types spontaneously differentiated into numerous specialized cell types in STO co-culture. These included fibroblasts, endodermlike cells, neuronlike cells, pigmented cells, adipogenic cells, contracting muscle cells, dome-forming epithelium, ciliated epithelium, tubule-forming epithelium, and a round amoeboid cell type resembling a plasmacyte after Wright staining. The neuronlike cells, contracting muscle cells, and tubule-forming epithelium had normal karyotypes and displayed finite or undefined life spans upon long-term STO co-culture. The dome-forming epithelium had an indefinite life span in STO co-culture and also retained a normal karyotype. These results demonstrate the in vitro pluripotency of pig epiblast cells and indicate the epiblast can be a source for deriving various specialized cell cultures or cell lines.

Continuous cultures of macrophages derived from the 8-day epiblast of the pig.

Secondary macrophage cell cultures were generated from the primary culture of epiblasts of 8-d-old pig blastocysts. The epiblast-derived macrophagelike (EDM) cells have a morphology and ameboid behavior that is typical of tissue histocytes. The cells reacted positively with monoclonal antibodies specific for pig granulocyte-macrophage lineage cells, and were not reactive with monoclonal antibodies specific for pig B and T lymphocytes. Marked phagocytic behavior and the formation of phagosomes were demonstrated following incubation with FITC-labeled bacteria. The EDM cells stained positively for nonspecific acid esterase that was not inhibited by sodium fluoride. DiI-acetylated-LDL was rapidly taken up by the cells. Transmission electron microscopy of the EDM cells showed phagolysosomes, numerous cytoplasmic vacuoles, large, lobed nuclei, and numerous pseudopods or filopodia at the cell surface. Strong reactivity of the cells with anti-CD14 monoclonal antibody was observed. Further, cytotoxic activity was produced from the EDM cells after exposure to lipopolysaccharide in a concentration and time-dependent manner. The cultures could be maintained and expanded for several months on STO co-culture. Their derivation from the epiblast of the pig demonstrates the possibility of obtaining hemopoietic cell cultures from the preimplantation blastocysts of all mammals.

A continuous culture of pluripotent fetal hepatocytes derived from the 8-day epiblast of the pig.

Continuous cultures of pluripotent parenchymal hepatocytes were derived from the epiblasts of 8-day-old pig blastocysts. The cells were polygonal and had phase-contrast dark, granular cytoplasm with prominent nuclei and nucleoli. These feeder-dependent cell cultures differentiated into large, multicellular, secretory, duct-like structures or formed small canaliculi between individual cells. Alternatively, the cells accumulated droplets that stained intensely with Oil Red O, a lipid-specific stain. Alpha-fetoprotein (AFP), albumin, and beta-fibrinogen mRNAs were expressed as the cells differentiated in culture. Serum-free medium that was conditioned by the cells contained transferrin, AFP, and albumin. The growth and viability of the cells were inhibited by transforming growth factor beta 1 (TGF beta 1) at concentrations > or = 1 ng/ml. The cell cultures grew slowly with doubling times of 2 to 3 d. One of the cultures, pig inner cell mass-19 (PICM-19), was passaged continuously for over 2 yr [> 100 population doublings (PD)] and appears to be an infinitely self-renewing cell population. The stem cell characteristics of the epiblast-derived fetal hepatocytes indicate that the cells may be unique for investigations of liver differentiation and organogenesis.

Colony isolation and secondary culture of fetal porcine hepatocytes on STO feeder cells.

The secondary culture of non-transformed parenchymal hepatocytes has not been possible. STO feeder cell-dependent secondary cultures of fetal pig hepatocytes were established by colony isolation from primary cultures of 26-d fetal livers. The liver cells had the typical polygonal morphology of parenchymal hepatocytes. They also spontaneously differentiated to form small biliary canaliculi between individual cells or progressed further to large multicellular duct-like structures or cells undergoing gross lipid accumulation and secretion. The secondary hepatocyte cultures expressed alpha-fetoprotein (AFP), albumin, and beta-fibrinogen mRNA, and conditioned medium from the cells contained elevated levels of transferrin and albumin. STO feeder cell co-culture may be useful for the sustainable culture of hepatocytes from other species.

Effect of bovine growth hormone gene expression, sex and age on plasma gonadotropins, estrone and testosterone in prepuberal pigs.

Chronic supraphysiological blood levels of growth hormone (GH) may retard sexual maturation in swine. Pigs used in this study included four founder transgenic pigs (two gilts and two boars) expressing a mouse transferrin (TF) promoter fused to a bovine (b) GH structural gene, 13 second- or third- generation transgenic pigs (seven gilts and six boars) expressing a mouse metallothionein (MT) promoter fused to a bGH structural gene and 16 control littermates (eight gilts and eight boars). Blood plasma levels of LH, FSH, estrone and testosterone were measured to determine whether expression of bGH genes altered secretion of hormones between 80 and 180 days of age. Presence of a bGH gene was detected by hybridization of DNA in dot blots of tail biopsies. Expression of a bGH gene was detected by radioimmunoassay of plasma bGH. In four TFbGH founder transgenic pigs bGH ranged from 164 to 1948 ng/ml; in one MTbGH transgenic boar of line 3104 bGH was 1211 ng/ml; and in 12 pigs of line 3706 bGH ranged from 25 to 190 ng/ml. Expression of bGH in transgenic pigs lowered (P = .0192) plasma LH with no significant differences between sexes, had no significant effect on plasma FSH and lowered plasma estrone (P = .0001) and testosterone (P = .0269) in boars (but not gilts). Plasma estrone and testosterone were higher (P = .0001) in boars than in gilts. Plasma FSH was higher (P = .0001) in gilts than boars and decreased (P = .0001) with advancing age in gilts but not in boars.(ABSTRACT TRUNCATED AT 250 WORDS)

Duration of thawing on post thaw acrosome morphology and motility of boar spermatozoa frozen in 5-ml maxi-straws.

Ejaculates from six boars were frozen in 5-ml maxi-straws and stored in liquid nitrogen. Maxi-straws were thawed by submersion in a 52 degrees C circulating waterbath for 28, 34, 40, 46, 52, or 58 sec. The post thaw percentages of motile sperm and acrosomes with a normal apical ridge (NAR) at the initial microscopic examination were significantly higher (P < 0.05) for maxi-straws submerged for 40 sec than for any other duration of submersion. The mean core temperature of maxi-straws after 40 sec of submersion was 4.8 degrees C. Based on regression equations for post thaw motility and NAR acrosomes, a 37 sec thawing time would be expected to provide the maximal post thaw survival for boar sperm frozen in maxi-straws.

Effects of transferred ova per recipient and dual use of donors as recipients on production of transgenic swine.

The purpose of the study was to determine whether the number of transferred ova per recipient influenced the efficiency of producing transgenic pigs and whether donor gilts were as effective as unmated gilts as recipients of microinjected ova. Eight genes were microinjected into 4,232 ova that were transferred into 169 recipients over a 5-yr period. Although the farrowing rate and litter size was highest for recipients receiving 31 to 41 ova per recipient, the percentage of transferred ova developing into piglets was highest for recipients receiving 13 to 20 ova (P = 0.021 for all recipients and P = 0.011 for pregnant recipients). Based on these data we conclude transferring more than 20 ova per recipient may incur some loss due to uterine crowding. Gilts used as recipients of microinjected ova immediately after their own ova were flushed from their oviducts had the same farrowing rate, litter size, and ovum development efficiency as unmated gilts that were only used as recipients. However, donor-recipients that ovulated 21 or more ova had smaller litters (P = 0.009) and were less efficient in producing pigs (P = 0.024) and transgenic pigs (P = 0.054) from transferred ova than donor-recipients that ovulated 20 or fewer ova. Dual use of donors as recipients was an effective method of reducing the number of recipients in a transgenic pig project by nearly one-half.

Mating by vasectomized boars failed to improve fertility in gilts inseminated with frozen semen.

One-hundred sixty-four gilts were artificially inseminated (AI) with frozen-thawed boar semen and, of these, 78 were immediately bred by a vasectomized boar after AI. The farrowing rate and litter size were 37.2 and 7.2 for mated gilts and 38.4 and 7.5 for control gilts, respectively. Mating by a vasectomized boar did not improve fertility or litter size.

Effect of incubation conditions, inhibitors, 2-mercaptoethanol and testosterone on RNA synthesis in ram spermatozoa.

In vitro studies on RNA synthesis using washed ram spermatozoa were carried out by measuring the incorporation of (3)H-uridine into RNA. Penicillin-G (100 mug/ml medium) was added to prevent contamination by microorganisms. Spermatozoa were quickly separated from seminal plasma by washing twice in Tris-HCl buffer (at pH 7.2) and centrifuged at 1,000 g for 5 min. Washed spermatozoa were then diluted to 1 10 , 1 20 or 1 40 (v/v) by the same buffer system (containing 400 mg% glucose) and were incubated in air at 37 degrees C for 1, 2 and 4 h. Results indicated that the rate of RNA synthesis was maximal at 1 40 semenbuffer dilution (5-8 x 10(7) spermatozoa/ml) and increased linearly up to 4 h of incubation. The rate of RNA synthesis at 1 40 dilution also increased linearly as the dose of exogenous glucose substrate was increased up to 400 mg%. Denaturation of the ram spermatozoa by 1% HgCl(2) caused almost complete inhibition of RNA synthesis that amounted to 97% of the control samples. Incubation of spermatozoa with 50, 100 or 200 mug/ml chloramphenicol also inhibited uridine incorporation by 86 to 94%, while equivalent doses of cycloheximide did not. On the other hand, the incorporation of (3)H-uridine into the RNA of ram spermatozoa was significantly enhanced by graded doses of 2-mercaptoethanol (0.2, 0.4 and 0.8 muM) and of testosterone (15 and 30 mug/ml). The results of this study indicate RNA synthesis, mainly of mitochondrial origin, by mature ram sperm. The data also suggest a role for intracellular cyclic adenosine monophosphate in the regulation of sperm RNA synthesis.

Effect of cyclic AMP on fructose utilization, progressive motility and protein synthesis by ram spermatozoa.

Ejaculated washed ram spermatozoa showed consistent increases in the intracellular concentration of cyclic 3', 5'-adenosine monophosphate (cAMP) after incubation for 15 minutes with the phosphodiesterase (PDE)-inhibitors, theophylline and caffeine. In vitro addition of cAMP or PDE-inhibitors to ram semen also stimulated and maintained sperm motility and enhanced the rate of fructose utilization. The same doses of cAMP or theophylline significantly stimulated the rate of protein synthesis by the washed spermatozoa, while the PDE-stimulator, imidazole, inhibited protein synthesis significantly. The stimulatory effect of cAMP on sperm protein synthesis was not affected by cycloheximide, but was abolished by the mitochondrial inhibitor, chloramphenicol. The present results indicate a positive correlation between the intracellular concentration of cAMP and the rates of progressive motility, fructose utilization, and protein synthesis by ram spermatozoa. The results suggest that the effect of cAMP is associated with the synthesis of mitochndrial proteins which may be involved with the observed enhancement of sperm motility and metabolism. The data also indicate that cAMP map act either as a first or a second messenger in mature spermatoza.

Effect of cyclic AMP, imidazole and triiodothyronine on the rate of RNA synthesis by ejaculated ram spermatozoa.

The effects of cyclic AMP (cAMP) and of triiodothyronine (T(3)) on the enhancement of sperm motility and metabolism are well documented, and the present study was undertaken to investigate the mechanisms underlying these effects in terms of their influence on sperm RNA synthesis in vitro. Washed ram sperm were diluted to 1 40 (v/v) with incubation buffer that contained 100 mug/ml penicillin-G and 400 mg% glucose, followed by incubation at 37 degrees C for a period of 4 h. Washed ram spermatozoa incubated with graded doses of cAMP (10(-8), 10(-6) and 10(-4) M) showed significant enhancements of the rate of (3)H-uridine incorporation into RNA, with maximal effect occurring at 10(-8) M. The presence of 3.75, 7.50 or 15.00 muM T(3) also stimulated the rate of RNA synthesis by the washed ram sperm, with maximal effect occurring at 7.50 muM. On the contrary, imidazole (a compound known to stimulate phosphodiesterase activity and consequently to decrease the intracellular cAMP levels in many tissues) was found to cause consistent inhibition of spermatozoal RNA synthesis. The inhibition was 47, 90 and 92% of control for 10, 50 and 100 mM imidazole, respectively. The results obtained indicate that cAMP may act either as a first or a second messenger in the mature sperm. The data also indicate that T(3) (possibly mediated by cAMP) may act on the ram sperm by the induction of enzymes, which are required for the well-known effects of this hormone in enhancing the sperm metabolic activity.

Effect of uterine ligation and cervical plugs on retention of frozen-thawed boar sperm.

Two experiments were conducted to determine whether the prevention of retrograde sperm expulsion from the uterus would enhance ovum fertilization by frozen-thawed boar sperm and whether insertion of a cervical plug would reduce the loss of frozen-thawed boar sperm from the uterus. In Exp. 1, 25 gilts were surgically inseminated and one uterine horn was ligated to prevent expulsion of sperm. Nine gilts were killed 4 h after insemination, and the sperm were recovered from the uterine horns and uterotubal sections. Significantly more sperm were recovered from the ligated than from the unligated uterine horns. Fertilized ova were recovered from nine of 16 gilts killed 48 to 90 h after insemination. More ova (P less than .005) were fertilized on the ligated than on the unligated sides of the reproductive tracts (87 vs 64%). In Exp. 2, 24 gilts were artificially inseminated with frozen-thawed boar sperm and then randomly selected to receive the intracervical insertion of a cotton tampon, a plastic plug or no treatment (control). Sperm were recovered from the uterine horns and uterotubal sections 4 h after insemination. The cervical plugs failed to significantly increase the retention of sperm in the uterotubal sections and uterine horns.

Effect of processing of semen on capacitation time of fresh and frozen-thawed boar spermatozoa.

Ovulation time of gilts was controlled by oral treatment with altrenogest (17-allyl-hydroxyestra-4,9,11-trien-3-one) followed by gonadotropic hormone injections. Gilts were laparotomized 42 to 44 h after injection of human chorionic gonadotropin. Spermatozoa were surgically placed into ligated oviducts of gilts after one of the following semen treatments: frozen and thawed in Beltsville thawing solution (F-BTS) or in seminal plasma (F-SP); unfrozen, processed like the frozen semen, but not cooled, frozen or thawed, and extended in Beltsville thawing solution (U-BTS) or in seminal plasma (U-SP). Ova were recovered 5 or 24 h after insemination and their stage of development was determined. At 5 h, F-BTS and U-BTS spermatozoa had fertilized significantly more ova (45 and 69%, respectively) than the F-SP and U-SP spermatozoa (5 and 34%, respectively). Ova fertilized by F-BTS and U-BTS spermatozoa had reached a more advanced stage of development than ova fertilized by F-SP and U-SP spermatozoa at both recovery times. Unfrozen spermatozoa (U-BTS or U-SP) fertilized more ova by 5 h after tubal deposition than F-BTS or F-SP spermatozoa (P less than .005 for each comparison). By 24 h, the percentage of fertilized ova was similar for all treatments. The results indicate that semen processing reduced capacitation time of boar spermatozoa to an extent similar to passage through the uterus. The capacitation time of frozen-thawed spermatozoa appeared to be similar to spermatozoa that were processed, but not cooled, frozen or thawed.

Status of research with transgenic farm animals.

Microinjection is the predominant method used to transfer genes into farm animals. Current research is devoted to improvement of productivity traits, enhancement of animal health, and production of biomedically useful human health products. Initial transgenic research primarily involved genes coding for growth hormone (GH), growth hormone-releasing factor (GRF), and insulin-like growth factor I (IGF-I). More recent investigations have attempted to stimulate muscle development, to use bacterial enzymes so animals can synthesize certain essential amino acids, to induce expression of specific immunoglobulin or disease-resistance genes, and to direct expression of human proteins to the mammary gland, specific organs, or specific cells for production of useful human health products. The main limitations to progress are the lack of useful cloned genes for productivity traits and disease resistance and the insufficient knowledge of mechanisms involved in regulation of transgenes.