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ANIMAL GROWTH, PHYSIOLOGY, AND REPRODUCTION |
* Department of Animal and Food Sciences, Texas Tech University, Lubbock, TX 79409;
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Center for Cancer Research and Therapeutic Development, Clark Atlanta University, Atlanta, GA 30314;
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Department of Poultry Science and Veterinary Pathobiology, Texas A&M, College Station, TX 77843;
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Poultry Science Department, University of Arkansas, Fayetteville, AR 72701; and
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# Oklahoma State University, Stillwater, OK 74078
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Abstract |
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 Top Abstract INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Key Words: chicken gonadotropin-releasing hormone-II • immunization • lamprey gonadotropin-releasing hormone-III • pig
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INTRODUCTION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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; Yu et al., 2002; Okada et al., 2003). In addition, other areas of investigation stress the need to reconsider the traditional conjecture that a single GnRH molecule controls reproduction (Padmanabhan et al., 1997; Padmanabhan and McNeilly, 2001). Active immunization against mamGnRH in pigs decreases the concentration of LH but has no effect on plasma and pituitary concentrations of FSH (Awoniyi et al., 1988; Wagner and Claus, 2004). The question of the presence of 2 or more forms of GnRH is important to the understanding of the control of reproductive function. It is possible that the coordinated action of more than 1 form of GnRH may be crucial in some aspects of reproduction, such as the regulation of FSH, puberty, and testis development. The effect of active immunization against GnRH isoforms in pigs has not been evaluated.
Therefore, the objective of this experiment was to determine the effects of active immunization against cGnRH-II and lGnRH-III on gonadotropin secretion and testicular function of pigs.
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MATERIALS AND METHODS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The experimental procedures were approved by the Texas Tech University Animal Care and Use Committee. Twelve crossbred sows that farrowed within 4 d were randomly chosen to provide the piglets used in the present experiment. The day after parturition (d 1), piglets were weighed, their tails were docked, and their ears were notched. On d 3, 4 males from each of the 12 litters were randomly assigned to 1 of 4 treatments. At 13 wk of age (WOA), the pigs on treatment 1 were actively immunized against cGnRH-II, whereas the pigs on treatment 2 were actively immunized against lGnRH-III. Control animals were actively immunized against the carrier protein, which was BSA (boars; treatment 3), and pigs on treatment 4 were castrated and actively immunized against the carrier protein (barrows). Eight experimental pigs (2 per treatment) were assigned to each of 6 pens designed to provide at least 0.8 m2 per animal. Throughout the study, all animals were maintained on ad libitum food and water intake.
Immunizations
Synthetic cGnRH-II and lGnRH-III peptides, with the common pGlu-His-Trp-Ser sequence at the N-terminal omitted and a C-terminal Cys residue added, were used in the conjugation process (Table 1). The side-chain sulfhydryl group of Cys was used to conjugate the peptides to maleimide (m-maleimidobenzoyl-N-hydroxy-succinimide ester)-activated bovine serum albumin (Grieger and Reeves, 1990). Mass spectrometry (matrix assisted laser desorption ionization-time of flight; MALDI-TOF) was used to assess the incorporation of each of the GnRH peptide fragments to the carrier protein, as previously described (Lewis et al., 1993; Nakanishi et al., 1994). Briefly, radiation from a nitrogen laser was used to desorb ions from the target, and data were collected and accumulated from 128 to 256 laser pulses. The matrix solution used for sample preparation was a saturated sinapinic acid solution in a solvent system consisting of a 1:1 mixture of acetonitrile and water containing 0.1% (vol/vol) trifluoroacetic acid.
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Blood Sampling
Blood samples were collected via venipuncture weekly, beginning at 11 WOA, for 15 consecutive weeks. A 5-mL Vacutainer tube equipped with a 20-ga, 3.8-cm-long needle, and containing EDTA to prevent clotting, was used to withdraw 5 mL of blood from the jugular vein. Samples were placed on ice and centrifuged (1,800 x g for 15 min) within 4 h. Plasma was separated and stored at –20°C until antibody titers and concentrations of testosterone, LH, and FSH were determined by RIA.
Isolation and Treatment of Leydig Cells
One week after the last blood sample (27 WOA), the pigs (n = 48) were exsanguinated at the Texas Tech University Meat Science Laboratory. Testes were removed from the intact (noncastrated) pigs and weighed. The left testis was immersed in Bouin’s fixative, and the right testis was hemisected; one-half was frozen and stored at –80°C, whereas the other half was used to isolate Leydig cells. Leydig cells from testes (n = 3/ treatment) were isolated as previously described (Khan et al., 1992).
Briefly, testis pieces were minced in minimal essential media (MEM; Gibco-BRL, Grand Island, NY) and treated with 0.5 mg/mL of collagenase (Type I, Sigma Chemical, St. Louis, MO). Interstitial cells and seminiferous tubules were separated by unit gravity sedimentation, and the supernatant containing interstitial cells was centrifuged (1,000 x g for 10 min) and decanted. The interstitial cell pellet was resuspended in fresh MEM and then purified by centrifugation in a Percoll gradient (50%) for 1 h at 1,000 x g. The purity of Leydig cells was assessed by 3-beta-hydroxysteroid dehydrogenase staining, as described previously (Geiger et al., 1999).
Leydig cell function was assessed in vitro to determine the effects of immunization on functional differentiation. Isolated Leydig cells were preincubated at 37°C in MEM for 1 h, followed by washing and resuspension in MEM. Cells were counted and tested for viability with trypan blue, using a hemocytometer and a light microscope. Percentage viability was then calculated and used to adjust the number of cells used in each tube. Leydig cells (100,000 viable cells/tube), were placed in sterile glass tubes, resuspended in 300 µL of MEM, and treated with LH (0, 1, or 10 ng/mL) for 24 h. The conditioned media were collected and analyzed for testosterone content by RIA.
RIA
Testosterone.
Concentrations of testosterone in plasma from weekly samples (n = 3 samples·treatment–1·wk–1) and testosterone production from Leydig cells were quantified using a solid-phase RIA (ICN testosterone kit, Pharmaceuticals Inc., Costa Mesa, CA) as previously described (França et al., 2000; Vizcarra et al., 2004). The inter- and intraassay CV were 10.1 and 6.6%, respectively. The average sensitivity was 0.03 ng/mL.
Luteinizing Hormone.
Concentrations of LH in plasma samples (n = 12 samples·treatment–1·wk–1) were quantified using a procedure similar to that previously described (Guthrie and Bolt, 1983). Briefly, reagents from the National Hormone and Peptide Program were used in a homologous RIA. Sodium phosphate buffer, 125I (1 mCi), and chloramine-T (20 µg) were added to a vial that contained 2.5 µg of porcine LH (AFP-11043B). After 90 s of incubation, the reaction was terminated with 50 µg of sodium metabisulfite. The mixture was transferred to an anion-exchange column (BioRad Laboratories, Hercules, CA) that was used to separate [125I]LH from free 125I. Antiserum against porcine LH (AFP-15103194) was diluted in sodium phosphate buffer (1:600,000, vol/vol). Two hundred microliters of the dilution was added to culture tubes containing standards (AFP-11043B) or unknowns. Iodinated LH (100 µL) was added to each tube, and the tubes were incubated for 24 h at room temperature (RT) followed by the addition of 200 µL of sheep anti-rabbit gamma globulin (1:40 dilution). After incubation for 24 h at RT, 2.0 mL of PBS (4°C) was added to each tube. The tubes were centrifuged for 30 min (1,900 x g), the supernatant was aspirated, and radioactivity in the pellet was quantified with a gamma counter. The addition of 5 and 10 ng of LH to 1 mL of serum resulted in 108 and 95% recovery (n = 7). When different volumes of serum (50, 100, and 150 µL) were assayed, the resulting concentrations were parallel to the LH standard curve. Inter- and intraassay CV were 6.2 and 8.2%, respectively (n = 7). The average sensitivity was 0.08 ng/mL.
Follicle Stimulating Hormone.
Concentrations of FSH in plasma samples (n = 12 samples·treatment–1·wk–1) were quantified by using procedures similar to those previously described (Guthrie and Bolt, 1983). Briefly, reagents from the National Hormone and Peptide Program were used in a homologous RIA. Sodium phosphate buffer, 125I (2 mCi), and chloramine-T (20 µg) were added to a vial that contained 2.5 µg of porcine FSH (AFP-10640B). After 60 s of incubation, the reaction was terminated with 50 µg of sodium metabisulfite. The mixture was transferred to a BioGel P-60 column (BioRad Laboratories) that was used to separate [125I]FSH from free 125I. Antiserum against porcine FSH (AFP-2062096) was diluted in sodium phosphate buffer (1:80,000). Two hundred microliters of the dilution was added to culture tubes containing standards (AFP-10640B) or unknowns. Iodinated FSH (100 µL) was added to each tube, and the tubes were incubated for 24 h at RT followed by addition of 200 µL of sheep anti-rabbit gamma globulin (1:40 dilution). After incubation for 24 h at RT, 2.0 mL of PBS (4°C) was added to each tube. The tubes were centrifuged for 30 min (1,900 x g), the supernatant was aspirated, and radioactivity in the pellet was quantified with a gamma counter. The addition of 5 ng of FSH to 1 mL of plasma resulted in 90% recovery (n = 7). When different volumes of plasma (50, 100, and 150 µL) were assayed, the resulting concentrations were parallel to the FSH standard curve. Inter- and intraassay CV were 2.4 and 12.2%, respectively (n = 7). The average sensitivity was 0.04 ng/mL.
Antibody Titers.
Antibody titers against cGnRH-II, lGnRH-III, and mamGnRH were quantified in weekly plasma samples (n = 12 samples·treatment–1·wk–1) with a procedure similar to that described by Esbenshade and Britt (1985). Briefly, iodination of full-length mamGnRH and cGnRH-II was performed by Bachem Bioscience Inc. (Torrance, CA), using the chloramine-T technique. Iodination of full-length lGnRH-III was performed by Bachem Bioscience Inc., using the Bolton-Hunter technique. Plasma was diluted (1:100) in PBS-EDTA and incubated at 4°C overnight with 125I-cGnRH-II, 125I-lGnRH-III, or 125I-mamGnRH. After incubation, 100 µL of Bovine IgG/PBS solution (1 mL of IgG + 3 mL of PBS-BSA) and 500 µL of cold 24% polyethylene glycol were added. The tubes were vortexed and then incubated at 4°C for 10 min. After incubation, the tubes were centrifuged (1,900 x g for 15 min at 4°C), the supernatant was aspirated, and the pellet was counted in a gamma counter.
Antibody Specificity. To evaluate the specificity of the antibodies to the GnRH isoforms, dose response curves were constructed. The ability of cGnRH-II and lGnRH-III (full sequence; Bachem Bioscience Inc.) to inhibit binding of 125I-cGnRH-II or 125I-lGnRH-III to antibodies in plasma of 1 selected animal per treatment (wk 8) was evaluated in duplicate. Concentrations of cGnRH-II and lGnRH-III (0 to 10,000 ng/mL) were incubated with antibodies (1:100 dilution), and 125I-cGnRH-II or 125I-lGnRH-III, at 4°C overnight, as described above. The percentage of radioactive peptide displaced by the nonlabeled peptide was measured. Dose response curves to the different GnRH isoforms were constructed and evaluated.
Statistical Analysis
Antibody titers were analyzed using repeated measurements over time (PROC MIXED, SAS Inst. Inc., Cary, NC). If a significant treatment x week interaction existed, the SLICE option of SAS was used to test for significant difference between treatments each week. Immunization treatments were in the main plot and day of bleeding in the subplot. Animals within pen (experimental unit) were used to test treatment effects, and the residual error was used to test day and the interaction of day x treatment. Similarly, LH, FSH, and testosterone concentrations in weekly samples were analyzed using repeated measurements over time. If a significant treatment x week interaction existed, polynomial response curves over time were characterized, and significant differences were computed using dummy variables (Steel and Torrie, 1980).
Analysis of variance for a 3 x 3 factorial arrangement of treatments was used to evaluate the effect of immunization treatment (cGnRH-II, lGnRH-III, and boars) and LH challenges (0, 1, and 10 ng/mL) on in vitro testosterone secretion from Leydig cells. Dose response curves to the different GnRH isoforms were constructed by plotting the logit of the ratio of bound:free against the log of the concentration of the antigen (Goldsmith, 1975). Regression and correlation variables were obtained from those plots.
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RESULTS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Mass spectrometry (MALDI-TOF) was used to measure the masses of BSA, BSA conjugated to cGnRH-II, and BSA conjugated to lGnRH-III (Figure 1). There was a 14.4% incorporation of cGnRH-II into the carrier protein (BSA) and a 15.9% incorporation of lGnRH-III into BSA. These data allowed proper adjustment in the dose of each conjugate to provide treated animals with a similar amount of conjugated peptide per immunization.
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Body weights were obtained weekly starting 5 wk before primary immunization. Treatments had no effect on BW; however, there was a time effect (P < 0.001). On average, pigs gained 413 ± 14 g/d over the course of the experiment.
Antibody Titers
Antibody titers were detectable in cGnRH-II and lGnRH-III immunized animals 1 wk after the first booster immunization. Titers continued to increase after booster immunizations were given (Figure 2A and 2B). Antibodies from animals immunized against lGnRH-III did not bind 125I-cGnRH-II (Figure 2A). Similarly, antibodies from animals immunized against cGnRH-II did not bind 125I-lGnRH-III (Figure 2B). Antibody titers against cGnRH-II and lGnRH-III were not detected in control animals. None of the animals produced antibodies that recognized mamGnRH (Figure 2C). However, when a plasma sample (1:100 dilution) from a cow previously immunized against mamGnRH was used (OSU #302; Vizcarra and Wettemann, 1995), 22% binding to 125I-mamGnRH occurred (see inset; Figure 2C).
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Testosterone and Gonadotropin Concentrations
A treatment x week interaction (P < 0.01) for testosterone concentrations was best described by linear regression (Figure 3). Concentrations of testosterone 7 wk after primary immunization (linear intercept) were influenced by treatment. At 20 WOA (wk 7 of treatment), concentrations of testosterone were greater (P < 0.05) in boars (8.7 ± 3.4 ng/mL) than in all other treatments (barrows, 0.5 ± 0.5; cGnRH-II, 2.5 ± 1.7; lGnRH-III, 2.0 ± 1.0 ng/mL). The slope of control barrows was not different from zero, and no differences in the slopes were observed between treatments.
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; P < 0.03). At the initiation of the experiment, FSH concentrations were greater (P < 0.05) in barrows (2.9 ± 0.7 ng/mL) than in all other treatments (boars, 0.2 ± 0.1; cGnRH-II, 0.4 ± 0.2; lGnRH-III, 0.2 ± 0.1 ng/mL). Concentrations of FSH in plasma decreased more rapidly (P < 0.001) during the experimental period in barrows compared with boars, cGnRH-II, and lGnRH-III animals.
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). However, there was no treatment x week interaction on concentrations of LH. Concentrations of LH were greater (P < 0.05) during the experimental period in barrows (1.0 ± 0.1 ng/mL) than in all other treatments (boars, 0.3 ± 0.1; cGnRH-II, 0.3 ± 0.1; lGnRH-III, 0.3 ± 0.1 ng/mL). Leydig Cells
Leydig cells from animals immunized against cGnRH-II, and incubated with 0, 1, or 10 ng/mL of LH, released consistently less testosterone (P < 0. 01) in the media compared with control boars (Figure 5). Leydig cells, with and without LH stimulation, from pigs immunized against lGnRH-III released less testosterone (P < 0.01) in the media compared with cGnRH-II immunized animals and control boars (Figure 5).
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DISCUSSION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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), pigs (Esbenshade and Britt, 1985), and cattle (Prendiville et al., 1995; Vizcarra and Wettemann, 1995). Because GnRH is a small peptide, it must be conjugated to a foreign protein. Proteins that have been used for immunization against GnRH include keyhole-limpet hemocyanin (Adams and Adams, 1986), BSA (Fraser et al., 1974), ovalbumin (Goubau et al., 1989), and human serum albumin (Schanbacher, 1982). Nonetheless, BSA has been used as the carrier protein of choice in pigs immunized against mamGnRH (Esbenshade and Britt, 1985; Traywick and Esbenshade, 1988).
We have previously used MALDI-TOF to evaluate the mass of BSA and BSA conjugated peptides (Vizcarra et al., 2006). Animals in each treatment received the same amount of conjugated peptide after proper adjustment of the dose. We chose DEAE + FIA because it resulted in a satisfactory antibody production with few granulomas at the site of injection in cattle (Vizcarra and Wettemann, 1995).
Treatments did not affect BW, indicating that treatments did not affect growth rate. Average daily gain ranges from 222 to 907 g in swine (Davis et al., 2004; Omogbenigun et al., 2004). The ADG observed in the present experiment was within the normal range reported.
When immunizing, it is common practice to use synthetic peptides larger than 6 AA, typically around 15 AA in length, to generate more specific antibodies (Lerner, 1982). However, immunization against peptides smaller than 6 AA is feasible. For instance, production of monoclonal and polyclonal antibodies directed against thyrotropin-releasing hormone (TRH; pGlu-His-Pro) and against a cGnRH-II fragment have been reported (Klootwijk et al., 1995; Clerens et al., 2003).
In the present experiment, we omitted the common pGlu-His-Trp-Ser sequence in both GnRH isoforms to minimize possible cross-reactivity of the resulting antibodies. Therefore, only 6 AA were used in the conjugate to immunize experimental animals. Despite the limited number of AA, immunized animals had specific antibody titer production with minimal cross reactivity between isoforms. These data suggest that pigs can be immunized against each isoform individually without affecting the function of the other isoforms. None of the animals produced antibodies that recognized the mammalian form of GnRH (mamGnRH). Chicken GnRH-II and lGnRH-III have 3 and 4 AA substitutions compared with mamGnRH [(His5 Trp7 Tyr8) and (His5 Asp6 Trp7 Lys8); cGnRH-II and lGnRH-III, respectively; Millar et al., 2004]. We hypothesize that the omission of the first 4 common AA allowed each immunogen to elicit isoform-specific antibodies. No antibodies were detected in castrated or intact control animals (barrows and boars), which was to be expected because they were immunized against BSA. To the best of our knowledge this is the first report on the use of GnRH isoforms for immunization purposes in mammalian species.
Even though cGnRH-II has been detected in mammalian brains, the function of this peptide in mammals is unknown (Rissman et al., 1995; Lescheid et al., 1997). In the monkey, cGnRH-II is synthesized in a distinct population of hypothalamic neurons in the mediobasal hypothalamus in males and females, suggesting a different neuroendocrine pathway compared with mamGnRH (Latimer et al., 2000, 2001). Nevertheless, most of the evidence suggests that the actions of cGnRH-II are similar to those of mamGnRH (Densmore and Urbanski, 2003; Okada et al., 2003).
Sower et al. (1993) first reported the isolation of the molecular form of lamprey GnRH from the sea lamprey (Petromyzon marinus). Subsequently, the presence of lGnRH-III (or a closely related peptide) was also reported in brain extracts from humans and cows (Yahalom et al., 1999), sheep (Yu et al., 2000), and rats (Hiney et al., 2002; Yu et al., 2002). A selective lGnRH-III-induced FSH release have been reported in the literature (Yu et al., 1997, Dees et al., 1999; McCann et al., 2001); however, others suggest that lGnRH-III is not involved in the selective release of FSH (Kovacs et al., 2002; Amstalden et al., 2004).
In pigs, the presence or absence of cGnRH-II and lGnRH-III has not been reported. However, there are indications that lGnRH-III might be involved in the mechanism controlling gonadotropin secretion. Infusion of lGnRH-III in barrows stimulated FSH secretion within 2 h posttreatment but did not elicit an increase on LH secretion (Kauffold et al., 2005). In contrast to the response in pigs, lGnRH-III did not release FSH selectively in cattle (Amstalden et al., 2004). The contradiction between different research groups is not known; however, a plausible explanation relates to the fact that 2 GnRH receptors (GnRH-R) have been isolated in mammals (GnRH-I and GnRH-II receptor; Millar et al., 2004). Recently, the GnRH-II receptor has been identified and sequenced in pigs (Neill et al., 2004). Interestingly, this same receptor type is not present in cattle (Neill et al., 2004; Millar, 2005). It is possible that differences in the response to lGnRH-III-doses in pigs and cows are due to the 2 GnRH and GnRH-R systems. Because different receptor types provide different signaling pathways (Millar, 2005), the potential for differential FSH and LH secretion is apparent.
Concentrations of testosterone were maximal in control boars and minimal in control barrows. Pigs immunized against cGnRH-II and lGnRH-III had concentrations of testosterone that were less than control boars. Consequently, barrows had increased concentration of LH and FSH compared with boars, cGnRH-II, and lGnRH-III animals. We hypothesize that the absence of testicular negative feedback signal(s) governing LH and FSH secretion allowed the increased gonadotropin secretion observed in control barrows. Concentrations of FSH and LH in serum of cGnRH-II and lGnRH-III animals were similar to control boars. Whether the decreased gonadotropin secretion observed in GnRH immunized animals was due to the immunization protocol, negative steroid feedback, or a combination of both cannot be established.
Immunizations against cGnRH-II and lGnRH-III decreased the ability of Leydig cells to release testosterone in response to LH challenges. The in vivo and in vitro decrease of testosterone concentrations in cGnRH-II and lGnRH-III immunized animals compared with control boars suggests that the immunization protocol had an effect at the level of the gonads to decrease testosterone synthesis and secretion. The increased in vitro LH-induced testosterone secretion (from 0 to 1 ng/mL) in cells isolated from control animals was approximately 70%, whereas the response in cells isolated from cGnRH-II pigs was approximately 54%. No response to LH-induced testosterone secretion was observed in cells isolated from lGnRH-III animals. The physiological relevance of the Leydig cell challenge was apparent when the differential testicular response between immunized groups observed in the in vitro approach was not evident in the in vivo data (cGnRH-II vs. lGnRH-III). Normal testosterone concentrations in individual boars vary throughout the day and between animals (Wettemann and Desjardins, 1979; Juniewicz and Johnson, 1983; Lubritz et al., 1991). We speculate that variation of daily endogenous testosterone concentrations in immunized animals provided a source of variation that decreased the power of the statistical analysis. Nevertheless, the controlled environment used in the in vitro approach allows for a clear distinction of the Leydig cells responsiveness to LH challenges in immunized pigs.
Secretion of LH and FSH is episodic in boars (Liptrap et al., 1986). In the present experiment, no differences on gonadotropin secretion among control boars, cGnRH-II, and lGnRH-III were observed with weekly samples. We theorize that alterations in maximum amounts of gonadotropins may have occurred, and this could have influenced testicular function. Additionally, a possible autocrine-paracrine regulation of steroido-genesis at the testicular level by GnRH and GnRH-R cannot be excluded. In fact, testicular tissue and seminiferous tubules express GnRH mRNA, whereas mRNA for GnRH-R is found in Leydig cells (Clayton et al., 1980; Bahk et al., 1995; Botte et al., 1998). The short half-life (Eskay et al., 1977), and the reduced concentration of hypothalamic GnRH in systemic circulation (Nett et al., 1974) suggests the presence of other GnRH-like peptides as ligands for the gonadal GnRH-R. The existence of such peptides has been demonstrated in Sertoli cells by competitive binding studies and immuno-chemistry assays (Paull et al., 1981; Sharpe and Fraser, 1983). Exogenous GnRH and GnRH analogs infused in hypophysectomized male and female rats caused a direct inhibition of Leydig cell steroidogenesis and ovarian luteal function by decreasing the number of LH receptors (Bambino et al., 1980; Jones and Hsueh, 1980). Therefore, in the present experiment, immunization treatments may have had a direct steroidogenic effect in immunized, intact pigs.
Taken together, our data suggest that pigs can be immunized against cGnRH-II and lGnRH-III with minimal cross-reactivity when only 6 AA are used for conjugation. Immunized animals had concentrations of LH and FSH that were less than control barrows and similar to control boars. Immunizing against cGnRH-II and lGnRH-III caused a decrease in plasma testosterone and affected testicular function. Even though the presence of cGnRH-II and lGnRH-III in the brain of pigs has not been reported to date, our data strongly suggest that cGnRH-II and lGnRH-III, or close related molecules, may be involved in the regulation of testicular function in the pig.
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Footnotes |
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2 Corresponding author: jorge.vizcarra{at}ttu.edu
Received for publication April 12, 2006. Accepted for publication June 20, 2006.
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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