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.

Growth and tissue accretion rates of swine expressing an insulin-like growth factor I transgene.

The goal of this research was to determine whether directing expression of an insulin-like growth factor I (IGF-I) transgene specifically to striated muscle would alter the growth characteristics in swine. Transgenic pigs were produced with a fusion gene composed of avian skeletal alpha-actin regulatory sequences and a cDNA encoding human IGF-I. Six founder transgenic pigs were mated to nontransgenic pigs to produce 11 litters of G1 transgenic and sibling control progeny. Birth weight, weaning weight, and proportion of pig survival did not differ between transgenic and control pigs. The ADG of pigs as they grew incrementally from 20 to 60 kg, 60 to 90 kg, and 90 to 120 kg, respectively, did not significantly differ between transgenic and control pigs. Efficiency of feed utilization (gain:feed) was also similar for transgenic and control pigs. Plasma IGF-I and porcine growth hormone (pGH) concentrations were determined at 60, 90, and 120 kg body weight. Plasma IGF-I concentrations were 19% higher in transgenic gilts than control gilts and 11.1% higher in transgenic boars than control boars (P=0.0005). Plasma IGF-I concentrations for boars were also higher than for gilts (P=0.0001). At 60, 90, and 120 kg body weight each pig was scanned by dual energy X-ray absorptiometry (DXA) to derive comparative estimates of carcass fat, lean, bone content of the live animal. Control pigs had more fat and less lean tissue than transgenic pigs at each of the scanning periods and the difference became more pronounced as the pigs grew heavier (P<0.005 at each weight). Transgenic pigs also had a slightly lower percentage of bone than control pigs (P<0.05 at each weight). While daily rates of lean tissue accretion did not differ for transgenic and control pigs, daily rates of fat accretion were lower in transgenic pigs than in control pigs (P<0.05). Based on these results we conclude that expression of IGF-I in the skeletal muscles gradually altered body composition as pigs became older but did not have a major affect on growth performance.

Cell donor influences success of producing cattle by somatic cell nuclear transfer.

To assess sources of variation in nuclear transfer efficiency, bovine fetal fibroblasts (BFF), harvested from six Jersey fetuses, were cultured under various conditions. After transfection, frozen-thawed lung or muscle BFF donor cells were initially cultured in DMEM in 5% CO(2) and air and some were transferred to MEM, with 5% or 20% O(2) or 0.5% or 10% serum and G418 for 2-3 wk. Selected clonal transfected fibroblasts were fused to enucleated oocytes. Fused couplets (n = 4007), activated with ionomycin and 6-dimethylaminopurine, yielded 927 blastocysts, and 650 were transferred to 330 recipients. Fusion rate was influenced by oxygen tension in a fetus-dependent manner (P < 0.001). Blastocyst development was influenced in a number of ways. Hip fibroblast generated more blastocysts when cultured in MEM (P < 0.001). The influence of serum concentration was fetus dependent (P < 0.001) and exposing fibroblast to low oxygen was detrimental to blastocyst development (P < 0.001). Cells from two of the six fetuses produced embryos that maintained pregnancies to term, resulting in eight viable calves. Pregnancy rates 56 days after transfer for the two productive donor fetuses, was at least double that of other recipients and may provide a fitness indicator of BFF cell sources for nuclear transfer. We conclude that a significant component in determining somatic cell nuclear transfer success is the source of the nuclear donor cells.

Prediction of pork carcass composition based on cross-sectional region analysis of dual energy X-ray absorptiometry (DXA) scans.

Dual energy X-ray absorptiometry (DXA) was used to measure pork carcass composition by performing a total scan of the right half of 262 pork carcasses (42.7±5.2 kg). The DXA scans were analyzed for percentage fat in the entire half-carcass as well as the shoulder, ham, loin, and side regions. In addition, a total of 14 cross-sections (57.6 mm wide) were analyzed: six in the shoulder/thoracic region, three in the loin region, and five in the ham region. Relative to the DXA measurement of total fat content, the coefficient of determination (R(2)) for a single cross-sectional slice ranged from 0.908 to 0.976. Relative to chemical analysis, a single slice from the ham region predicted the percentage of fat or lean in the half-carcass with an R(2) of 0.81 and a standard error of the estimate of 2.04. Prediction equations were used to analyze a separate group of 65 half-carcasses. These results indicate that carcass fat and lean percentages can be measured by performing a single-pass cross-sectional scan that would be compatible with on-line processing.

Inability of heterologous growth hormone (GH) to regulate GH binding protein in GH-transgenic swine.

Transgenic pigs expressing bovine, ovine, or human growth hormone (GH) structural genes fused to mouse metallothionein-I (mMT-bGH), ovine MT (oMT-oGH), or mouse transferrin (mTf-hGH) promoters were used to study the effects of GH on the regulation of serum GH-binding protein (GHBP). In the 14 transgenic pigs studied, circulating concentrations of heterologous GH ranged from 15 to 2,750 ng/mL. Using chromatographic methods, specific binding of GH was detected in serum from normal pigs but was undetectable in serum from all the transgenic pigs used, probably as a result of the high serum concentrations of heterologous GH present in these animals. Thus, to avoid interference of binding by high GH concentrations, serum samples were subjected to immunoblotting using a specific anti-GHBP antibody. A specific 54-kDa band was detected in normal pig serum as well as in sera from mMT-bGH, oMT-oGH, and mTf-hGH pigs. Additionally, sera from transgenic mMT-bGH pigs and their sibling controls were subjected to immunoprecipitation with an anti-GHBP antibody followed by immunoblotting with the same antibody. With this technique, we detected two specific bands of 53 and 45 kDa that could represent different degrees of glycosylation of GHBP. As determined by densitometric analysis the amount of GHBP in transgenic pig sera was similar to that detected in sera of the respective control animals. The amount of circulating GHBP remained unchanged even in oMT-oGH and mTf-hGH pigs that were exposed from birth to circulating concentrations of GH as high as 2,750 ng/mL. Thus, we conclude that heterologous GH do not act as modulators ofthe serum GHBP in pigs.

Total body and regional measurements of bone mineral content and bone mineral density in pigs by dual energy X-ray absorptiometry.

Dual energy X-ray absorptiometry (DXA) was used to make total body and regional measurements of bone mineral content, bone mineral density, and bone area during the growth of pigs from 3 to 138 kg. In all, 1,053 total body scans were performed on 587 live pigs. Regional measurements consisted of the front legs, trunk, and back legs. In addition, bone mineral density readings were recorded for the head, pelvis, spine, and ribs. From about 5 to 75 kg, a greater percentage of the total body bone mineral content (BMC) was located in the trunk region. However, the percentage of BMC in the front and back legs continued to increase linearly whereas the percentage of BMC in the trunk region peaked at about 25 kg and then decreased logarithmically. Allometric analysis revealed that up to about 30 kg the BMC increased more rapidly in the trunk region compared to the front or back leg regions (P > 0.05), but after 30 kg the increase in BMC was more rapid in the leg regions (P < 0.05). Overall, the rate of increase in BMC in the back legs was slightly more than in the front legs (P > 0.05). Positive allometric growth of BMC was observed when compared with the increase in bone area for the same region. By far, the highest measured level of bone mineral density (BMD) was in the head region (P < 0.05), followed in order by the front legs, spine, back legs, pelvis, and ribs. Over the entire range of growth from 3 to 138 kg, the highest relative growth coefficient for the increase in BMD occurred in the pelvic and back leg regions and the lowest was in the ribs (P < 0.05). For pigs < 30 kg, the highest growth coefficient for BMD relative to BW was in the spine (P > 0.05). The growth coefficients for BMD in the back legs and total body increased in pigs > 30 kg and those of the front legs and trunk regions decreased.

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.

Birth of piglets after transfer of embryos cryopreserved by cytoskeletal stabilization and vitrification.

Pig embryos suffer severe sensitivity to hypothermic conditions, which limits their ability to withstand conventional cryopreservation. Research has focused on high lipid content of pig embryos and its role in hypothermic sensitivity, while little research has been conducted on structural damage. Documenting cytoskeletal disruption provides information on embryonic sensitivity and cellular response to cryopreservation. The objectives of this study were to document microfilament (MF) alterations during swine embryo vitrification, to utilize an MF inhibitor during cryopreservation to stabilize MF, and to determine the developmental competence of cytoskeletal-stabilized and vitrified pig embryos. Vitrified morulae/early blastocysts displayed MF disruptions and lacked developmental competence after cryopreservation; hatched blastocysts displayed variable MF disruption and developmental competence. Cytochalasin-b did not improve morula/early blastocyst viability after vitrification; however, it significantly (P < 0.05) improved survival and development of expanded and hatched blastocysts. After embryo transfer, we achieved pregnancy rates of almost 60%, and litter sizes improved from 5 to 7.25 piglets per litter. This study shows that the pig embryo cytoskeleton can be affected by vitrification and that MF depolymerization prior to vitrification improves blastocyst developmental competence after cryopreservation. After transfer, vitrified embryos can produce live, healthy piglets that grow normally and when mature are of excellent fecundity.

Correlation between histochemically assessed fiber type distribution and isomyosin and myosin heavy chain content in porcine skeletal muscles.

Highly sensitive enzyme assays developed to differentiate skeletal muscle fibers allow the recognition of three main fiber types: slow-twitch oxidative (SO), fast-twitch oxidative glycolytic (FOG), and fast-twitch glycolytic (FG). Myosin, the predominant contractile protein in mammalian skeletal muscle, can be separated based on the electrophoretic mobility under nondissociating conditions into SM2, SM1, IM, FM3, and FM2 isoforms, or under dissociating conditions into myosin heavy chain (MHC) I, IIb, IIx/d, and IIa. The purpose of the present study was to determine whether the histochemical method of differentiation of fiber types is consistent with the electrophoretically identified isomyosin and MHC isoforms. These comparisons were made using serratus ventralis (SV), gluteus medius (GM), and longissimus muscles (LM) from 13 pigs. Two calculation methods for the histochemical assessed fiber type distribution were adopted. The first method incorporated the number of fibers counted for each fiber type and calculated a percentage of the total fiber number (fiber number percentage: FNP). The second method expressed the cross-sectional area of each fiber type as a percentage of the total fiber area measured per muscle (fiber area percentage: FAP). Independent of the calculation methods, correlation analyses revealed in all muscles a strong relation between SO fibers, the slow isomyosin (SM1 and SM2), and MHCI, as well as between the FG fibers, the fast isomyosin (FM3 and FM2), and MHCIIx/b content (P<.05). There were no correlations between FOG fiber population assessed by histochemical analysis and intermediate isoform (IM) or MHCIIa content. The present results did not provide conclusive evidence as to which of the calculation methods (FNP or FAP) was more closely related to myosin composition of skeletal muscles. Despite some incompatibility between the methods, the present study shows that histochemical as well as electrophoretic analyses yielded important information about the composition of porcine skeletal muscle. The combination of the two methods may be essential to accurately characterize porcine skeletal muscles.

Codon optimization, genetic insulation, and an rtTA reporter improve performance of the tetracycline switch.

The objective of this work was to further develop a tetracycline repressor (TetR) protein system that allows control of transgene expression. First, to circumvent the need for a binary approach, a single plasmid design was constructed and tested in tissue culture. To indirectly assay integrations that express the synthetic transcription factor (rtTA), a bicistronic gene was built which included an internal ribosome entry site (IRES) and a green fluorescent protein coding region (GFP) on the same expression cassette as the coding region of rtTA (pTetGREEN). This construct did not produce fluorescent colonies when stably integrated and provided minimal expression of GFP in the face of adequate expression of rtTA. The coding region for TetR was then altered by introducing 156 silent point mutations to simulate mammalian genes. Replacement of wild-type TetR gene (tetR) in pTetGREEN with 'mammalianized' tetR provided GFP expression. Adjustment of codon usage in the tetR region of rtTA nearly doubled the expression level of functional rtTA. To increase the number of rtTA expressing lines, the chicken egg-white lysozyme matrix attachment region (MAR) was introduced into the single plasmid design just upstream of the tetracycline operators (tetO). Inclusion of the MAR doubled the number of colonies that expressed rtTA (44% vs 88%). With the modifications described here, the number of lines that express rtTA and provide induction from a single plasmid design can be increased by the inclusion of a MAR and the level of rtTA expression can be further increased by adjusting the base composition of the TetR coding region. The MAR also insulates the inducible gene from the promoter driving rtTA.

Composition analysis of pork carcasses by dual-energy x-ray absorptiometry.

Dual-energy x-ray absorptiometry (DXA) was used as a noninvasive method to measure the composition of pig carcasses. A total of 181 half-carcasses (10 to 51 kg, from pigs slaughtered at approximately 30, 60, 90, and 120 kg) were scanned using a Lunar (Madison, WI) DPX-L densitometer. The DXA measurements of fat, lean, bone mineral, and total tissue mass were compared with chemical analysis for fat, water, protein, total ash, and scale weight. The mean value for total tissue mass by DXA was slightly less than the mean carcass weight (32.3 kg vs 33.6 kg, P > .05, R2 = .998). Although highly correlated (R2 = .81), the DXA measurement of the percentage of fat in the half-carcass was less (P < .001) than the chemical measurement (19.5 vs 24.9%). The DXA measurement of lean tissue mass (total mass less fat and bone mineral) was correlated with carcass protein (R2 = .97) and water (R2 = .99) content. The correlation (R2) between DXA bone mineral content and carcass ash content was only .68; however, DXA bone mineral content was more highly correlated with carcass weight (R2 = .93) than was carcass ash content (R2 = .70). When we used the DXA R value (ratio of the attenuation coefficients for fat and lean) to predict percentage of fat in the carcass, the mean value for predicted carcass fat was 25.9% (P > .05). Similarly, carcass protein and water content were predicted from DXA lean. Using DXA region of interest analysis, estimates of the fat content of the shoulder and ham regions were close to chemical values; however, DXA underestimated the fat content of the loin and side regions by 20 and 28%, respectively. When prediction equations were used to evaluate DXA measurements of the half-carcasses of 28 gilts and 37 boars slaughtered at approximately 120 kg, the half-carcasses of gilts contained more fat (33.9 vs 27.8%, P < .001), less protein (14.1 vs 16.1%, P < .001), and less water (45.9 vs 52.1%, P < .001) than those of boars. These results indicate that DXA could be a valuable research tool for measuring the composition of pig carcasses. On the basis of the results of this study, prediction equations were revised for the DXA estimation of fat, protein, and water content of the half-carcass: Fat (%) = 450 - (315 x DXA R value), Protein (g) = -145 + (.23 x DXA lean), and Water (g) = 150 + (.73 x DXA lean). Furthermore, it seems that separate prediction equations are needed for regional analysis.

Transfer of an ovine metallothionein-ovine growth hormone fusion gene into swine.

An ovine metallothionein-1a (oMT1a)-ovine growth hormone (oGH) fusion gene was microinjected into 400 pig zygotes, the zygotes were transferred into recipient females, and 15 founder transgenic pigs were born. Of 12 transgenic pigs assayed, five expressed high levels of oGH (> 900 ng/mL plasma), one expressed low levels of oGH (10 to 30 ng/mL), and six did not express oGH. Dietary supplementation with 2,000 ppm of zinc for 6 d induced a 20-fold increase in plasma oGH in the transgenic pig with low expression but did not induce expression in the six transgenic pigs with no constitutive oGH expression. The average daily gain of five transgenic pigs with elevated oGH was similar to that of non-transgenic littermates during a 9-wk feeding trial (P = .52). The liver, kidney, adrenal, and thyroid weights were all significantly heavier for the oGH-expressing transgenic pigs than for non-transgenic littermates. Total carcass fat, longissimus muscle fat, subcutaneous backfat thickness, and loin eye area were lower and carcass protein and water content and beta R fiber area of longissimus muscle were higher in the transgenic pigs with elevated oGH than in their littermate controls (P < .05 for each). The data indicate that even though the oMT1a promoter was more inducible by zinc than was previously reported for the mouse MT promoter in swine, the former provided a higher level of oGH expression than the mouse MT promoter.

Identification of pig primordial germ cells by immunocytochemistry and lectin binding.

Monoclonal antibodies anti-SSEA-1 and EMA-1, and the lectins DBA and LTA, bound to the surface of large, round cells randomly distributed in the 26-day pig genital ridge. Other antibodies, SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81, did not react with any cells in the pig genital ridge. SSEA-1-positive cells displayed pseudopods and appeared to migrate from the dorsal mesentery of the hindgut (18-day) to the primordium of the gonad (day 23) and entered the genital ridge by 26 days. The number of SSEA-1-positive cells associated with the dorsal mesentery and genital ridge markedly increased from the 18-day to the 26-day pig embryo. It was concluded that the SSEA-1-positive cells were primordial germ cells (PGCs). Using these markers and alkaline phosphatase histochemistry, pig PGCs derived from the 26-day genital ridge showed no proliferation when grown in STO co-culture in the presence of human LIF, bFGF and SCF.

Porcine follicle-stimulating hormone treatment of gilts during an altrenogest-synchronized follicular phase: effects on follicle growth, hormone secretion, ovulation, and fertilization.

Porcine FSH (SUPER OV), containing .03% LH activity, and equine chorionic gonadotropin (eCG) were administered during an altrenogest-synchronized follicular phase to determine their effects on follicle development, estrus, ovulation, and fertilization. Treatments were made by i.m. injection starting on d 1 (24 h after the last feeding of altrenogest): 1) saline, once, n = 14; 2) eCG (1,200 to 1,500 IU) once, n = 32; 3) FSH 14 (n = 2) or 21 (n = 6) NIH-FSH-S1 units/100 kg BW, divided among six injections at 12-h intervals (FSH14/21); 4) FSH, 28 NIH-FSH-S1 units/100 kg BW, divided among six injections at 12-h intervals, n = 12; and 5) FSH, 28 NIH-FSH-S1 units/100 kg BW and 100 IU hCG, two or six injections at 12-h intervals (FSH28+hCG), n = 13. Gilts were injected with 750 IU of hCG on d 5 to ensure ovulation. Twenty-eight eCG- and FSH-injected gilts (n = 6, 8, and 11 on treatments 3, 4, and 5, respectively) were bred and laparotomized on d 7 to recover ova and record ovulation rate. The mean number of ovulations and large (6- to 10-mm) follicles, respectively, on d 7 were as follows: saline (17, .7), eCG (43, .9), FSH14/21 (15, .6), FSH28 (12, 16), and FSH28+hCG (32, 21). Plasma FSH concentrations were at least threefold higher (P < .05) in gilts treated with FSH than in gilts not treated with FSH. The percentage in estrus was higher (P < .05) for saline- and eCG-treated gilts (100 and 87%, respectively) than for FSH-treated gilts (53%). Proportion of FSH28+hCG-treated gilts with fertilized ova (27%) was lower than for other groups (79 to 100%). In summary, the 3-d high dose FSH treatment (FSH28 and FSH28+hCG) during an altrenogest-synchronized follicular phase increased the number of potentially ovulatory follicles, but this potential benefit was not realized because many follicles failed to ovulate. The co-injection of low doses of hCG (FSH28+hCG) increased the ovulation rate and estradiol secretion but reduced ova recovery and fertilization rate compared with eCG and the other FSH treatments.

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.

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.

Ultrastructure, enzymatic, and transport properties of the PICM-19 bipotent liver cell line.

The pig epiblast-derived PICM-19 cell line was previously shown to spontaneously differentiate into liver-like cells and structures and to secrete serum proteins. A study was undertaken to further define the liver-like characteristics of the PICM-19 cell line. PICM-19 cells displayed in vitro ultrastructure, enzymatic, and transport characteristics similar to those of parenchymal hepatocytes and bile duct epithelium. The PICM-19 cells contained large oval nuclei, numerous oval to elongate mitochondria with flat cristae, extensive rough and smooth endoplasmic reticulum, Golgi complexes, lipid vacuoles, and glycogen granules. Biliary canaliculi with intraluminal projecting microvilli were delimited by the junctional apparatuses between adjacent PICM-19 cells. The PICM-19 cells rapidly transported fluorescein into their biliary canaliculi from the extracellular environment. PICM-19 cells that had differentiated into multicellular ductal structures had high gamma-glutamyltranspeptidase (GGT) activity at their apical surfaces as shown by histochemical staining. PICM-19 total GGT activity was at least 19 times higher than that found in porcine hepatocytes. Metyrapone induced cytochrome P-450 content of PICM-19 cells was at least one-fourth of that found in porcine hepatocytes. PICM-19 P-450 activity induced by 7-ethoxycoumarin was nearly equivalent to that of primary cultures of pig hepatocytes. The data support the proposal that differentiated PICM-19 cells resembled hepatocytes, or bile duct epithelium cells, and, therefore, the PICM-19 cell line behaved like early embryonic liver progenitor cells.

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.

Lipid composition of carcass tissue from transgenic pigs expressing a bovine growth hormone gene.

Fatty acid profiles and cholesterol content of whole-carcass ground tissue were compared from 26 transgenic (T) pigs expressing a bovine growth hormone gene (bGH) to 26 sibling control (C) pigs. All pigs were fed a common diet and were slaughtered at five different live weights: 14, 28, 48, 68, and 92 kg. The left side of each intact carcass was ground and tissue samples were analyzed for lipid composition and cholesterol content. At 14-kg body weight, carcasses from bGH-T pigs contained 38% less fat, 44% less saturated fatty acids (SFA), 48% less monounsaturated fatty acids (MUFA), and 38% less polyunsaturated fatty acids (PUFA) than C pigs. At 28 kg, bGH-T pigs had 38% less total carcass fat, 42% less SFA, 46% less MUFA, and 24% less PUFA than C pigs. At 48-kg body weight, bGH-T pigs contained 48% less carcass fat, 55% less SFA, 59% less MUFA, and 22% less PUFA than C pigs. At 68 kg, bGH-T pigs had 78% less carcass fat, 78% less SFA, 79% less MUFA, and 53% less PUFA than C pigs. At 92 kg, carcasses from bGH-T pigs contained 85% less carcass fat, 85% less SFA, 91% less MUFA, and 66% less PUFA than those from C pigs. Cholesterol content was not different between bGH-T pigs and C pigs at any of the carcass weights. The trend was for cholesterol content to decrease from the 14- to 92-kg weight group. These results suggest a dilution effect of carcass fat and fatty acids in carcass tissue from bGH-T pigs with increasing live weight.