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Department of Poultry Science, North Carolina State University, Raleigh, NC 27695
2To whom correspondence should be addressed. E-mail: pemozdzi{at}unity.ncsu.edu.
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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KEY WORDS: • lacZ • gene insertion • whey • milk • poultry
Because they do not feed their offspring milk, it is unlikely that chickens have made any evolutionary adaption to utilize milk in their diets or to hydrolyze the milk sugar lactose into glucose and galactose. Lactose has been evaluated in the literature as a poultry feed ingredient. Rutter et al. (1) found that the inclusion of 20% lactose in poultry diets results in growth impairment and diarrhea. Furthermore, they found lactose, but not galactose, in the serum of experimental chickens indicating the absence of the enzymes necessary to hydrolyze lactose into glucose and galactose (1). In addition, lactase activity has been previously shown to be at low levels in the enterocytes isolated from the small intestine of broiler chickens (2).
Lactose has been included in poultry diets in an attempt to increase calcium absorption, which may increase eggshell strength. Hurwitz et al. (3) found that including lactose in poultry diets has no effect on eggshell strength. However, Gleaves and Salim (4) found that including 1% lactose in a poultry diet increases eggshell strength, but concluded this is of no practical importance. Studies have also focused on employing lactose or whey (
80% lactose) in poultry feed to decrease endogenous Salmonella typhimurium. Deloach et al. (5) found that including 5% whey in a broiler diet is effective at lowering Salmonella typhimurium numbers in the broiler intestine. However, others have concluded that including lactose in broiler diets is not effective at reducing Salmonella typhimurium levels in processed broiler carcasses (6), and it was also concluded that including lactose in the drinking water of chickens during the 5–11-d before slaughter is not an effective Salmonella typhimirium control strategy (7).
Recently, lines of transgenic chickens carrying the E. coli lacZ gene and expressing bacterial ß-galactosidase have been generated at North Carolina State University (8). Bacterial ß-galactosidase has the ability to hydrolyze lactose, which cannot be utilized as a source of energy by birds, to glucose and galactose, which can participate in glycolysis to generate ATP. The objective of this study was to determine the ability of wild-type and transgenic chickens that carry the lacZ gene and express ß-galactosidase to hydrolyze lactose in the intestinal mucosa. No previous researchers have, to our knowledge, generated a transgenic chicken that has demonstrated altered nutrient utilization in the intestine.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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All experimental procedures involving animals were approved by the North Carolina State University Institutional Animal Care and Use Committee. One-d-old White Leghorn chicks were obtained from two different transgenic lines (P1 and P2) carrying the lacZ gene and expressing bacterial ß-galactosidase (8). The lines of transgenic chickens were created by injecting a replication-defective retrovirus (9) into the subgerminal cavity of freshly laid eggs. The retroviral vector carried a nuclear localized ß-galactosidase signal. A rooster from these initial injections that carried the lacZ gene in his semen was mated with hens until two male progeny were generated that carried the lacZ gene. The P1 line originated from one sire that carried the lacZ gene, and the P2 line originated from a second sire that also carried the lacZ gene. Heterozygous male and female birds from the second generation (G2) of each line were mated. Therefore, it was expected that some of the G3 birds from each line would be lacZ negative and would not express bacterial ß-galactosidase. Using sibling wild-type control birds from each line that did not carry the lacZ gene enabled the performance of the lactase assays in a blind fashion.
Immediately after hatching, the birds were placed in a brooder receiving a standard starter diet containing corn and soybean meal consisting of 88% dry matter, 3% fat and 18% crude protein. Water was consumed ad libitum. At 21-d of age, blood was collected from a wing vein and the chickens were killed by an overdose of Euthasol (0.25 mL/kg body weight; Delmarva Laboratories, Midlothian, VA). Immediately after death, the small intestine was removed from the body, placed on ice and separated at the ileal-cecal junction. The small intestine was opened longitudinally and the mucosa was scraped with a glass slide (10). The mucosa was weighed and homogenized with four parts (w/v) of cold PBS using a Tissumizer (Tekmar, Cincinnati, OH). All samples were frozen at -20°C until further analysis.
DNA isolation and PCR screening.
All DNA isolation and PCR screening procedures were as previously described by Mozdziak et al. (8). Genomic DNA was extracted from the chicken blood using a protocol modified from Petitte et al. (11). Briefly, blood was diluted 1:10 with PBS, mixed with lysis buffer (10 mmol/L of Tris HCl, pH 7.5, 5 mmol/L of MgCl2, 0.32 mol/L of sucrose and 1% Triton X-100), microfuged for 15 s and the supernatant placed in a fresh tube. The DNA containing solution was mixed with SDS and digested overnight with proteinase-K at 37°C with constant rotation. Subsequently, the protein was precipitated using saturated NaCl, and the DNA was precipitated using ethanol. All DNA was resuspended in Tris-EDTA buffer.
The presence of the lacZ gene in the offspring was determined using PCR. Briefly, Taq polymerase (Fisher Scientific, Pittsburgh, PA) was used to amplify a 588-bp fragment of lacZ using the forward primer 5'-TTCTGTATGAACGGTCTGGTC- 3, and the reverse primer 5'-ACTTACGCCAATGTCGTTATC- 3. The DNA was amplified using 35 cycles of 95°C for 30 s, 54°C for 1 min and 72°C for 1 min using a thermocycler (PTC-200; MJ Research, Waltham, MA). Subsequently, the amplification products were fractionated through a 1.5% agarose gel to reveal the presence of the 588-bp lacZ fragment.
Lactase assay.
The lactase assay was performed with the genetic origin of the samples unknown to the operator employing procedures modified from Oliver et al. (12). Briefly, the small intestinal mucosa homogenates were thawed at room temperature and centrifuged at 1800 x g for 10 min. The supernatant was removed from the pellet and diluted 1:2 with cold 9 g/L (w/v) NaCl. The diluted supernatant (100 µL) was added to a 13- x 100-mm borosilicate glass test tube with 100 µL of 0.056 mol/L lactose solution as a substrate. Blanks of all samples with the same composition were prepared, and immediately after mixing of the enzyme and substrate, were immersed in a boiling water bath for 2 min. The tubes, blanks excluded, were immersed in a constant-temperature water bath at 37°C. After 60 min of incubation, 0.80 mL of distilled water was added in each tube and immediately immersed in a boiling water bath for 2 min to stop the enzymatic reaction. The tubes were cooled and the glucose concentration was determined using a diagnostic glucose kit (Sigma Diagnostic, St. Louis, MO). Standard solutions were prepared in concentrations of 40, 80, 120, 160 and 200 mg/L of glucose. Briefly, 250 µL of each standard, blank and sample were transferred to 16 x 100-mm tubes. Combined enzyme-color reagent solution (2.5 mL) (Fisher Scientific, Chicago, IL) was added to each tube. The tubes were immersed in a constant-temperature water bath at 37°C for 30 min. The absorbance was read at 420 nm against reagent blanks. A biuret protein assay (13) was used on the homogenates of the intestinal mucosa, and the results of the biuret protein assay were used to normalize the enzyme activity per gram of protein used in the enzyme assay. Enzyme activity was expressed as µmol of glucose·min-1·g protein-1.
Whole mounts of the small intestines were fixed at 4°C with 2% formaldehyde and 0.2% glutaraldehyde in PBS, pH 7.4, for 30 min, rinsed in PBS and incubated in X-Gal solution [1 g/L X-Gal (5-bromo-4-chloro-3-indolyl-ß-D-galactoside) in PBS, pH 7.4, 5 mmol/L of potassium ferrocyanide, 5 mmol/L of potassium ferricyanide, 2 mmol/L of MgCl2 and 0.2% Triton X-100] overnight in the dark at 37°C. Subsequently, the intestines were washed with PBS and stored in 70% ethanol before microscopic evaluation. For cryosections, intestines were freshly collected, rinsed with PBS, fixed with 4% paraformaldehyde for 30 min at 4°C, rinsed with PBS, infiltrated with 20% sucrose in PBS and frozen in isopentane that was precooled in liquid nitrogen. Sections (10–15 µm) were cut on a cryostat, air dried and incubated in the same X-Gal formulation as used for the whole mounts except that Triton-X 100 was included at 0.1%. Subsequently, the sections were fixed in 2% paraformaldehyde, washed in PBS, dehydrated, cleared and mounted in permount. The X-Gal incubation was stopped after 3 h to preserve tissue architecture. Longer incubation times resulted in overstaining of the intestinal villi.
Statistical analysis.
Data were analyzed using the General Linear Models procedure of SAS (14) to perform a one-way ANOVA. The mean lactase activity between the groups of birds was separated using least significant differences (15). Means were considered significantly different at P < 0.05.
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RESULTS AND DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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The P1 line exhibited a higher lactase level than the P2 line. Although gene silencing has been an obstacle to generating transgenic chickens and quail (19), it does not appear that it is an issue in the P2 line because X-Gal-positive nuclei have been revealed in all tissues examined from each line (Borwornpinyo, S., Mozdziak, P. & Petitte, J. N., unpublished observations). Gene expression is a highly complex process and there may be slight differences in transcriptional regulation between lines. Although Mendelian inheritance has been observed in the P1 and P2 lines [50% germline transmission from the G1 to the G2 birds (8) and 75% germline transmission when the G2 birds are mated with each other; Borwornpinyo, S., Mozdziak, P. & Petitte, J. N., unpublished observations], it remains possible that the P1 line has two gene insertions on the same chromosome that would provide the observed germline transmission rate and explain the higher level of lactase activity. Secondly, the experiments were performed without knowing whether the birds were heterozygous or homozygous for lacZ, and it is possible that there were more heterozygous chickens analyzed from the P1 than the P2 line. Homozygous lacZ-positive chickens and those with multiple copies of the lacZ transgene may have a higher level of lactase activity through a gene dosage effect. Specifically, two copies of an inserted gene may result in a higher level of protein expression compared with one copy. Transgenic chickens expressing ß-lactamase have been successfully generated by other researchers (20), and ß-lactamase is higher in the egg white from homozygous G3 transgenic compared with heterozygous transgenic chickens (21). Overall, this study confirms that wild-type chickens have a low level of lactase activity and demonstrates that insertion of the lacZ gene accompanied by ß-galactosidase expression in the intestine results in an elevated lactase activity in the intestinal mucosa.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Manuscript received 4 June 2003. Initial review completed 8 July 2003. Revision accepted 30 July 2003.
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LITERATURE CITED |
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TOPABSTRACTMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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1. Rutter, W. J., Krocjevsly, P., Scott, H. M. & Hansen, R. G. (1953) The metabolism of lactose and galactose in the chick. Poult. Sci. 32:706-715.
2. Chotinsky, D., Toncheva, E. & Profirov, Y. (2001) Development of disaccharidase activity in the small intestine of broiler chickens. Br. Poult. Sci. 42:389-343.[Medline]
3. Hurwitz, S., Bar, A. & Bornstel, S. (1967) Effect of lactose on egg production and shell quality. Poult. Sci. 46:1024-1025.
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5. DeLoach, J. R., Oyofo, B. A., Corrier, D. E., Kubena, L. F., Ziprin, R. L. & Norman, J. O. (1990) Reduction of Salmonella typhimurium concentration in broiler chickens by milk or whey. Avian Dis. 34:389-392.[Medline]
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13. Gornall, A. G., Bardawill, C. J. & David, M. M. (1949) Determination of serum proteins by means of the biuret reaction. J. Biol. Chem. 177:751-766.
14. SAS Institute (1985) SAS User’s Guide: Statistics Version 5 Edition 1985 SAS Institute Cary, N.C.
15. Ott, L. (1993) An Introduction to Statistical Methods and Data Analysis 1993 Duxbury Press Belmont, Calif.
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17. Golovan, S. P., Meidinger, R. G., Ajakaiye, A., Cottrill, M., Wiederkehr, M. Z., Barney, D. J., Plante, C., Pollard, J. W., Fan, M. Z., Hayes, M. A., Laursen, J., Hjorth, J. P., Hacker, R. R., Phillips, J. P. & Forsberg, C. W. (2001) Pigs expressing salivary phytase produce low-phosphorus manure. Nat. Biotechnol. 19:741-745.[Medline]
18. Turner, N. D., Knapp, J. R., Byers, F. M. & Kopchick, J. J. (1998) Expression of mutant bovine growth hormone genes in mice perturbs age-related nutrient utilization patterns. J. Nutr. 128:520-524.
19. Mizuarai, S., Ono, K., Yamaguchi, K., Nishijima, M., Kamihira, M. & Iijima, S. (2001) Production of transgenic quails with high frequency of germ-line transmission using VSV-G pseudotyped retroviral vector. Biochem. Biophys. Res. Commun. 286:456-463.[Medline]
20. Harvey, A. J., Speksnijder, G., Baugh, L. R., Morris, J. A. & Ivarie, R. (2002) Consistent production of Transgenic chickens using replication-deficient retroviral vectors and high-throughput screening procedures. Poult. Sci. 81:202-212.
21. Harvey, A. J. & Ivarie, R. (2003) Validating the hen as a bioreactor for the production of exogenous proteins in egg white. Poult. Sci. 82:927-930.
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