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Poult Sci 2007. 86:1754-1759
© 2007 Poultry Science Association
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PHYSIOLOGY, ENDOCRINOLOGY, AND REPRODUCTION

Effect of Caponization and Testosterone Implantation on Hepatic Lipids and Lipogenic Enzymes in Male Chickens

K. L. Chen*, W. T. Chi*, C. Chu{dagger}, R. S. Chen{ddagger} and P. W. S. Chiou§,1

* Department of Animal Science, {dagger} Department of Applied Microbiology, and {ddagger} Department of Molecular and Biological Chemistry, National Chiayi University, Chiayi, Taiwan, China; and § Cheng-Jen College of Nursing, Health Sciences and Management, Chiayi, Taiwan, China

1 Corresponding author: wschiou{at}dragon.nchu.edu.tw


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
This study was conducted to determine the role and effects of testosterone in lipogenesis by measuring and analyzing the lipid composition and lipogenic enzyme activity of livers from capons treated with various doses of exogenous testosterone implant. Healthy and uniform male Single Comb White Leghorn chickens were caponized at 12 wk of age. Sixteen-week-old capons were randomly selected for a 10-wk experiment. Fifteen intact males and 15 capons were used for trial 1. In trial 2, 10 sham-operated males and 40 capons were used. The capons were randomly divided into 4 independent treatments with sialistic implants of cholesterol (1.62 mm i.d., 3.6 mm o.d., 9.24 ± 0.36 mg; CHOL), low testosterone (1 mm i.d., 3 mm o.d., 5.88 ± 0.23 mg), medium testosterone (1.62 mm i.d., 3.16 mm o.d., 9.81 ± 0.17 mg), or high testosterone (2 mm i.d., 4 mm o.d., 16.7 ± 0.24 mg). In trial 1, the results showed that caponization increased total hepatic lipid and triacylglycerol contents and decreased the nonesterified fatty acid content (P < 0.05) compared with the intact male. Meanwhile, caponization increased nicotinamide adenine dinucleotide phosphate -malic dehydrogenase (MDH) activity and MDH mRNA content (P = 0.09) simultaneously. In trial 2, comparing treatments with the various implantation doses of testosterone, the liver triacylglycerol content of capons the medium-dose implantation was decreased as compared with those receiving CHOL (P < 0.05). The total lipid and phospholipid contents of liver were decreased in capons receiving the high-dose implantation (P < 0.05), whereas the relative weight and nonesterified fatty acid content were increased (P < 0.05) and reached the same level as those in the sham treatment (P > 0.05). With an increased implantation dose, MDH activity of capons receiving the medium dose or higher was not different from those receiving the CHOL and sham treatments (P > 0.05). The increase in MDH activity at the transcriptional and translational levels suggests that caponization may positively regulate hepatic lipogenesis. In contract, implantation of testosterone up to the threshold concentration depressed hepatic lipogensis and lipid accumulation.

Key Words: cockerel • caponization • testosterone implantation • hepatic lipid composition • hepatic lipogenic enzyme


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
Capons are male chickens whose testes have been surgically removed. Because of the resultant androgen deficiency, secondary male sexual characteristics, including the comb, wattle, fighting behavior, and vocalization, degenerate and regress to an immature stage. In addition, capons also accumulate lipids in the body, which enhances flavor, texture, and meat juiciness when compared with intact cockerels (Chen et al., 2000b, 2005, 2006).

Caponization reduces the plasma testosterone concentration and not only stimulates fat accumulation in adipose tissue (Cason et al., 1988; Chen et al., 2000a,b, 2005, 2006) and subcutaneous and intramuscular tissues, but also increases blood lipids as compared with intact cockerels (Chen et al., 2005, 2006). Chen et al. (2005) reported that caponization changed the lipoprotein profiles, which resulted in an increased lipid storage capacity. This increase in blood lipid concentration may be associated with the up-regulation of hepatic lipogenesis. The liver is the main organ of lipogenesis in poultry; however, the impact of caponization on the mechanism of lipogenesis and on related hepatic lipogenic enzymes remains unclear. The purpose of this study was to determine changes in the liver lipid profile and lipogenic enzyme activity of male chickens after caponization. Implantation of different doses of testosterone was used to determine the role of testosterone in regulating hepatic lipid composition and related lipogenic enzymes.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 REFERENCES
 
Animal Management and Experimental Design
Healthy and uniform male Single Comb White Leghorn chickens were caponized at 12 wk of age and housed in individual 40 x 30 x 38 cm (length, width, height) cages. After a 4-wk adaptation period, 15 male and 15 caponized (capons, prominent degenerated comb) chickens were randomly selected for a 10-wk experiment in trial 1. In trial 2, 10 sham-operated chickens and 40 capons were used. The capons were randomly divided into 4 treatment groups implanted with either cholesterol (1.62 mm i.d., 3.16 mm o.d., 9.24 ± 0.36 mg; CHOL) or low (1 mm i.d., 3 mm, o.d., 5.88 ± 0.23 mg), medium (1.62 mm i.d., 3.16 mm, o.d., 9.81 ± 0.17 mg), or high (2 mm i.d., 4 mm, o.d., 16.7 ± 0.24 mg) doses of testosterone for the 10-wk experiment (to 26 wk of age). Feed (Table 1Go) and water were provided ad libitum throughout the experiment. The testectomy procedure was performed according to Chen et al. (2000a, 2005). The testosterone implantation procedure was performed according to the method of Fennell et al. (1990) with modifications. A 1-cm implantation tube (Tygon clear tubing R-3603, Saint-Gobain Performance Plastics Corp., Akron, OH) was used in this trial with different inner diameter sizes to control the testosterone dose. The tubes were implanted subcutaneously at the back of the chicken’s neck at 16, 20, and 24 wk of age.


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  		Table 1. Basal diet composition
 
Measurements and Analysis
At the end of the experiment, birds were euthanized, the BW was recorded, and the liver was weighed after being cleaned with cool saline. Two grams of liver was sampled and stored in liquid nitrogen for RNA extraction. The rest of the liver was stored at –40°C to measure lipid composition and enzyme activity.

Hepatic Lipid Composition.
Five grams of liver was homogenized with 10 vol of chloroform-methanol solution (2:1 vol/vol) for 1 min. The mixture was mixed with 10 mL of saline and then centrifuged at 1,734 x g for 10 min. The bottom-layer supernatant was collected for analysis of hepatic lipid composition. Triacylglycerol (TG) and CHOL contents were measured using human testing kits (Triglyceride LiquiColor Mono, R&D Systems Inc., Wiesbaden, Germany) and Infinity cholesterol reagent kits (Sigma-Aldrich Corp., St. Louis, MO) separately. Phospholipids (PL) and nonesterified fatty acids (NEFA) were determined according to the method of Dryer et al. (1957) and Chromy et al. (1977), respectively.

Hepatic Lipogenic Enzymes.
Approximately 5 g of liver was placed into 15-mL tubes containing buffer solution (0.25 M sucrose, 1 mM EDTA, pH 7.4), and the solution was homogenized at 4°C for 2 min (12,000 rpm). The supernatant was taken after centrifugation at 10,000 x g, 4°C, for 10 min. The supernatant was centrifuged again at 105,000 x g, 4°C, for 60 min to precipitate cell microsomes. The cytoplasm in the supernatant was collected for hepatic enzyme and protein concentration analysis. Activities of adenosine triphosphate-citrate cleavage enzyme (CCE; EC 4.1.3.8 [EC] ), nicotinamide adenine dinucleotide phosphate-malic dehydrogenase (MDH; EC1.1.40), fatty acid synthetase (FAS), acetyl-coenzyme A carboxylase (ACC; EC 6.4.1.2 [EC] ), and glucose-6-phosphate dehydrogenase (G-6-PDH; EC 1.1.1.49 [EC] ) were determined by the methods of Takeda et al. (1969), Ochoa (1955), Kumar et al. (1970), Numa (1969), and Lhr and Wallex (1974), respectively, with modifications. Protein content of the homogenized liver was measured according to the method of Lowry et al. (1951) with modifications and was used for calculating enzyme activity.

Gene Expression Analyses.
Approximately 1 g of liver was sampled for RNA extraction. Total RNA was extracted and purified by the REzol C&T reagent according to the manufacturer’s protocol (GRP1A, Genesis Biotech Inc., Boca Raton, FL). Ribonucleic acid integrity was assessed via agarose gel electrophoresis, and RNA concentration and purity were determined spectrophotometrically by the measurements of A260 and A280. Ribonucleic acid was used to synthesize of cDNA by reverse transcription (RT). The RT reaction mixture (20 µL) consisted of 1 µg of total RNA, 200 U of Moloneymurine leukemia virus reverse transcriptase (Promega, Madison, WI), 2.5 mmol/L of deoxy nucleotide 5'-triphosphate, and 0.5 µg of random hexamer primers (Promega). Nested PCR was performed according to the primer sets of first and second PCR (Table 2Go). Twenty-five microliters of PCR reaction mixture consisted of 2.5 µL of 10x buffer (pH 8.4, 50 mmol/L of KCl), 1.0 µL of the RT reaction mixture, 1.0 U of YEA (Yeastern Biotech Co., Taiwan, China) DNA polymerase, 2.5 mmol/L of deoxy nucleotide 5'-triphosphate, and 5 pmol each of the gene-specific primers. Thermal cycling parameters were as follows: 1 cycle at 94°C for 2 min, followed by 25 cycles at 94°C for 30 s, 56°C for 30 s, and 72°C for 1 min, with a final extension at 72°C for 5 min. The PCR products were separated by gel electrophoresis and quantified. The relative level of MDH gene expression was determined as the ratio of integrated peak area for each PCR product of individual MDH genes relative to that of the coamplified ß-actin internal standard.


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  		Table 2. Primers used for nested PCR
 
Statistical Analysis
A complete randomized design was applied to examine treatment effects. Analyses of variance were calculated using the GLM procedure (SAS Institute, 1985). Duncan’s new multiple-range test was used to compare mean differences between treatments according to Steel and Torrie (1997).


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
REFERENCES
 
Trial 1
Hepatic Lipid Composition.
The effect of caponization on the hepatic composition of male chickens in trial 1 is shown in Table 3Go. Caponization increased total hepatic lipid and TG contents and decreased the NEFA content (P < 0.05). Chen et al. (2005) observed that the increase in abdominal fat weight in capons was mainly due to the increase in TG content, based on a similar experiment design. In trial 1, a simultaneous increase was obtained in TG and liver lipid contents. The liver is the main organ of lipogenesis and generates the lipids of very low density lipoproteins (VLDL) for whole-body use. Chen et al. (2005) reported that caponization changes the lipoprotein composition and increases lipid retention. Previously reported increases in VLDL-TG contents of capons were consistent with the higher liver TG content observed in trial 1.


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  		Table 3. Effect of caponization on the hepatic composition of male chickens (trial 1)
 
Under normal conditions in poultry, blood NEFA are mainly hydrolyzed by lipoprotein lipase from portomicrons and VLDL. When the available energy is sufficient to meet the body’s requirements, NEFA binds to albumin and is then transported into the liver (Pownall, 2001; Rodoman et al., 2001). In intact male chickens, compared with capons, androgen reduces lipid accumulation and energy retention; therefore, more NEFA can be transported into the liver in intact male chickens than in capons.

Hepatic Lipogenesis Enzymes and MDH mRNA.
The effects of caponization on hepatic lipogenic enzymes and MDH gene expression in male chickens are shown in Table 4Go and Figure 1Go. Although there were no changes in the activity of CCE, G-6-PDH, ACC, and FAS (P > 0.05), caponization up-regulated the activity of hepatic MDH (P < 0.05) and the MDH mRNA content (P = 0.09). In poultry, MDH catalyzes the oxidative decarboxylation of malate and simultaneously generates reduced nicotinamide adenine denucleotide phosphate, which can be utilized in de novo synthesis of fatty acids. Legrand et al. (1987) previously reported that the activity of hepatic malic enzyme was positively correlated with the rate of fatty acid synthesis. In trial 1, only MDH activity was significantly increased in capons (P < 0.05). Chen et al. (2006) reported that caponization had no influence on G-6-PDH and CCE activities, and that it increased MDH activity only in the Taiwanese country chicken. Grunder et al. (1987) reported that liver MDH and CCE activities were higher in female chickens than in males. Tanaka et al. (1986) also showed that blood estrogen was positively related to liver FAS, MDH, and CCE activities and to blood lipids. This implies that the mechanisms of body lipid accumulation in capons differ from those of female chickens.


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  		Table 4. Caponization effect on the hepatic lipogenic enzymes and malic dehydrogenase gene expression of male chickens (trial 1)
 

Figure 1
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  		Figure 1. Electrophoretic patterns of PCR products amplifying hepatic malic dehydrogenase (MDH) mRNA from the male chicken and capon. M = marker.

 
Trial 2
Hepatic Lipid Composition.
The effects of testosterone implantation on hepatic lipid composition in capons are shown in Table 5Go. With the implantation of different testosterone doses, liver TG content of the medium-dose implantation was decreased, as compared with that of CHOL (P < 0.05). Liver total lipid and PL contents were decreased by the high-dose implantation (P < 0.05), whereas the relative liver weight and NEFA content were increased (P > 0.05) to the level of the sham-treated group. Liver weight and relative liver weight also did not differ between the CHOL and sham groups in both trial 1 and trial 2 (P > 0.05), implying that androgen does not affect the liver weight. However, the relative liver weight of capons in the high-dose implantation group was heavier than that of the CHOL group (P < 0.05) because of the much lighter BW of the high-dose implantation group. Liver total lipid and TG contents of the CHOL implantation group did not differ from those of the sham group in trial 2 (P > 0.05). This result was inconsistent with trial 1, in which liver total lipid and TG contents of capons were higher than those in intact male chickens (P < 0.05). This decrease in lipid production can be attributed to the stress from CHOL implantation every 4 wk in this trial, as compared with the capons in trial 1.


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  		Table 5. Effect of testosterone implantation on the hepatic lipid composition of capons (trial 2)
 
Liver total lipid, TG, and PL contents of the capons were reduced with the increase in testosterone implantation dose. With high-dose implantation, total lipid and PL contents were reduced, as compared with those of the CHOL group (P < 0.05). Triacylglycerol content was reduced by the medium-dose implantation and reached levels similar to those of the sham group (P > 0.05). These results demonstrate that androgens inhibit liver lipogenesis, especially in the production of TG and PL. Testosterone implantation dosage was negatively correlated with hepatic lipids. It was clear that androgen affected hepatic lipogenesis directly and thereby significantly reduced abdominal fat weight (Chen et al., 2006). The CHOL group had a significantly decreased ratio of NEFA in the liver compared with the sham group in the current trial 2 as well as in trial 1. However, capons implanted with the high-dose testosterone obtained a higher ratio of NEFA in the liver than did the CHOL group (P < 0.05) and reached the level of the sham group (P < 0.05). This result indicates that NEFA will bind to albumin, be transported into the liver, and be utilized de novo by the body.

Hepatic Lipogenesis Enzyme and MDH mRNA.
The effects of testosterone implantation on hepatic lipogenic enzyme activity and MDH mRNA content of the capons are shown in Table 6Go. In comparison with the sham group, none of the implantations in capons significantly changed the activity of lipogenic enzymes, including CCE, G-6-PDH, ACC, and FAS. With the testosterone implantation dose increased to medium dose or higher, MDH activity was not different from that of the CHOL group (P > 0.05) but was similar to that of the sham group (P > 0.05).


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  		Table 6. Effect of testosterone implantation on hepatic lipogenic enzyme activity in capons (trial 2)
 
Obviously, caponization decreased the androgen concentration and increased lipogenesis and lipid accumulation; however, these changes were not related to the hepatic lipogenic enzymes, including CCE, G-6-PDH, ACC, and FAS. With the medium- or high-dose testosterone implantation, MDH activity and mRNA content of the capons were not reduced, as compared with those of the CHOL group (P > 0.05). These nonsignificant changes might result from the low testosterone concentration in the capon. We observed that the blood testosterone concentration differed between capons implanted with high-dose testosterone (444 pg/mL) and intact male chickens (817 pg/mL; Chen et al., 2005). Therefore, the implantation dose used in this study may not have elevated plasma testosterone to levels similar to those of birds in the sham group.


   ACKNOWLEDGMENTS
 
The authors wish to thank the National Science Council of Taiwan for financially supporting this project (project no. NSC92-2313-B005-041).

Received for publication January 11, 2007. Accepted for publication March 21, 2007.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
Cason, J. A., D. L. Fletcher, and W. H. Burke. 1988. Effects of caponization on broiler growth. Poult. Sci. 67:979–981.[ISI][Medline]

Chen, K. L., W. T. Chi, and P. W. S. Chiou. 2005. Caponization and testosterone implantation effects on blood lipid and lipoprotein profile in male chickens. Poult. Sci. 84:547–552.[Abstract/Free Full Text]

Chen, K. L., T. Y. Hsieh, and P. W. S. Chiou. 2006. Caponization effects on growth performance and lipid metabolism in Taiwan country chicken cockerels. Asian-australas. J. Anim. Sci. 19:438–443.

Chen, K. L., C. P. Wu, and R. G. R. Chou. 2000a. Effect of castration age on growth performance and postmortem change in muscles of Taiwan country chicken. J. Agric. Assoc. Chin. 1:54–63.

Chen, K. L., C. P. Wu, and Y. M. Hong. 2000b. Meat quality and carcass traits of capon in comparison with intact male and female Taiwan country chickens. J. Chin. Soc. Anim. Sci. 29:77–88.

Chromy, V., J. Gergel, and J. Voznicek. 1977. Assay of free fatty acids by extraction-photometric procedures. Clin. Chem. Acta 80:327–332.[ISI][Medline]

Dryer, R. L., A. R. Tammes, and J. I. Routh. 1957. The determination of phosphorus and phosphatase with N-phenyl-(-phenylenediamine. J. Biol. Chem. 225:177–183.[Free Full Text]

Fennell, M. J., A. L. Johnson, and C. G. Scanes. 1990. Influence of androgens on plasma concentration of growth hormone in growing castrated and intact chickens. Gen. Comp. Endocrinol. 77:466–475.[ISI][Medline]

Grunder, A. A., J. R. Chambers, and A. Fortin. 1987. Plasma very low density lipoproteins, abdominal fat lipase, and fatness during rearing in two strains of broiler chickens. Poult. Sci. 66:471–479.[ISI][Medline]

Kumar, S., T. A. Dorsey, R. A. Muesing, and J. W. Porter. 1970. Comparative studies of the pigeon liver fatty acid synthetase complex and its subunit. J. Biol. Chem. 245:4732–4744.[Abstract/Free Full Text]

Legrand, P., J. Mallard, M. A. Bernard-Griffiths, M. Douaire, and P. Lemarchal. 1987. Hepatic lipogenesis in genetically lean and fat chickens. In vitro studies. Comp. Biochem. Physiol. 87B:789–792.[Medline]

Lhr, G. W., and H. D. Wallex. 1974. Methods of Enzymatic Analysis. Acad. Press, New York, NY.

Lowry, O. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265–275.[Free Full Text]

Numa, S. 1969. Acetyl-CoA carboxylase from chicken and rat liver: EC 6.4.1.2 acetyl-CoA:carbon-dioxide ligase (ADP). Pages 9–17 in Methods in Enzymology. Vol. 14: Lipids. J. M. Lowenstein, ed. Acad. Press, New York, NY.

Ochoa, S. 1955. Malic enzyme. Pages 739–753 in Methods in Enzymology. Vol. 1. S. P. Colowick and N. O. Kaplan, ed. Acad. Press, New York, NY.

Pownall, H. J. 2001. Cellular transport of nonesterified fatty acids. J. Mol. Neurosci. 16:109–115.[ISI][Medline]

Rodoman, G. V., T. I. Shalaeva, and G. E. Dobretsov. 2001. Blood fatty acid transporters in acute pancreatitis. Vopr. Med. Khim. 47:633–641.[ISI][Medline]

SAS Institute. 1985. SAS/STAT. Guide for Personal Computers (Version 6.08). SAS Inst. Inc., Cary, NC.

Steel, R. G. D., and J. H. Torrie. 1997. Principles and Procedures of Statistics. 3rd ed. McGraw-Hill, New York, NY.

Takeda, Y., F. Suzuki, and H. Inoue. 1969. ATP citrate lyase (citrate-cleavage enzyme). Pages 153–160 in Methods in Enzymology. Vol. 13:Citric Acid Cycle. J. M. Lowenstein, ed. Acad. Press, New York. NY.

Tanaka, K., J. C. Hsu, S. Ohtani, and C. M. Collado. 1986. Changes in the activities of hepatic lipogenic related enzymes and in the concentrations of various plasma and liver lipid fractions in hens before and after the onset of laying. Jpn. Poult. Sci. 23:203–210.





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