Growth, pectoralis muscle performance, and testis of pelung cockerels (Gallus gallus gallus [Linnaeus, 1758]) supplemented with blood clam shell powder (Anadara granosa [Linnaeus, 1758])

Background and Aim: Pelung cockerels (Gallus gallus gallus [Linnaeus, 1758]) are different from other native cockerels in that they have a long and unique voice, in addition to their tall, large, and sturdy body with a relatively heavy body weight (BW). The sound quality of pelung cockerels is affected by the structure of the syrinx and their large and strong chest muscles. The performance of the chest muscles, and subsequently its voice, is influenced by the hormone testosterone. The shell of blood clams (Anadara granosa Linnaeus, 1758), a saltwater bivalve is known to contain a natural aromatase blocker (NAB) capable of blocking the aromatase enzyme from converting testosterone to estradiol. This generates consistently high levels of testosterone. This study aimed to determine the effect of blood clam shell powder (BCSP) as an NAB on the growth, pectoralis muscle performance, and testes of pelung cockerels. Materials and Methods: The study design was a completely randomized design, with 16 pelung cockerels aged 40–56 weeks divided into four treatment groups: T0 (control); T1 (BCSP [A. granosa] 0.9 mg/kg BW); T2 (zinc sulfate [ZnSO4] 0.9 mg/kg BW); and T3 (testosterone 3 mg/day). The animals were acclimatized for 7 days and then given dietary treatments for 56 days. The measurement of the comb, wattle, and chest circumference (CC) of pelung cockerels was performed on days 0, 14, 28, 42, and 56. At the end of the treatment, the pelung cockerels were sacrificed and the data of the pectoralis muscle weight (PMW), testis weight (TW), and area of the pectoralis muscle (APM) were measured. Samples of pectoralis muscle and testes were taken and fixed in 10% neutral buffer formalin for histology. The proliferating cell nuclear antigen (PCNA) was identified by immunohistochemical staining. To measure fascicle area (FA), myofiber area (MA), and enumerate, the fascicle myofibers (NM) histology preparations were stained with hematoxylin and eosin (H and E). Testicular preparations were stained with H and E to measure the diameter of the seminiferous tubules (DST) using ImageJ software. Results: The growth performance on day 56 showed significantly (p < 0.05) higher differences of CC in T1 compared to T2 and T0, in T1 and T3 compared to T0, and in T3 and T2 compared to T0. Pectoralis muscle results, that is, FA, NM, MA, and PCNA-positive cells, showed that cockerels on treatment T3 had significantly higher results than other treatments, T1 was significantly different from T2 and T0, and T2 was significantly different from T0. In addition, the TW and DST measurement of cockerels on treatment T3 were significantly reduced (p < 0.05) than the other treatment groups. Conclusion: The oral administration of BCSP in the role of a NAB at a dose of 0.9 mg/kg BW for 56 days improved the growth performance and pectoralis muscle, especially the CC, FA, NM, MA, and PCNA-positive cells parameters, but did not affect the PMW, APM, and testis of pelung cockerels. The administration of testosterone at 3 mg/day for 56 days contributed to the decrease in TW and DST, as well as atrophy of the seminiferous tubules of pelung cockerels.


Introduction
Pelung cockerels are native to Cianjur, a district in West Java, Indonesia [1][2][3]. The animal has been settled under the 2918/kpts/OT.140/6/2011 decree issued by the Ministry of Agriculture as native Indonesian freerange chickens and is obliged to be protected and conserved [4]. The chicken is reported to be threatened due to a decrease in population size [1]. The pelung cockerels have a long, distinct melodious voice and are in high demand for crossbreeding with other local chickens [5]. In addition, pelung is a relatively fastgrow chicken with a large posture and strong appearance [5]. Male pelung cockerels produce good quality meat as shown by their high muscle mass and increased strength [6,7]. The chest area or the pectoralis muscle has a low-fat content [8]. Improvement of pectoralis muscle is characterized by the increasing number of myofibers in one fascicle, myofiber area (MA), fascicle area (FA), and proliferation in the nucleus of muscle cells [9][10][11][12]. The increase in growth and the pectoralis muscle characteristics of pelung cockerels is triggered by the hormone testosterone [5,7,[13][14][15][16][17][18][19]. The hormone testosterone in male animals is produced by the testes [5]. A common practice to increase testosterone levels is through parenteral synthetic testosterone [18]. The continuous administration of parenteral synthetic testosterone can lead to decreased testis weight (TW), seminiferous tubule damage, and infertility [5,18,20]. Therefore, a natural agent capable of acting as a natural aromatase blocker (NAB) is required, and blood clam shells may be an option [21].
Blood clams (Anadara granosa) are classified as bivalves. The shells are generally discarded after the meat is harvested which creates environmental challenges [22,23]. According to Astuti et al. [24], the blood clam shells contain Zn, Mg, Fe, Ca, Na, and K, while the blood clam shell powder (BCSP) contains 30%-40% Ca, 1% P, and 3%-4% protein [25,26]. The Zn content in the BCSP acts as a NAB which is able to increase testosterone hormone levels, antioxidants, growth performance, and modulate the immune system [24,[27][28][29][30][31]. The BCSP also contains proteins which are required to boost chicken growth, pectoralis muscle area, MA, and stimulate satellite cell proliferation to regenerate myofiber for muscle growth [32][33][34][35][36]. The BCSP used as a NAB at a dose of 0.036 mg/40 g body weight (BW) has been proven to increase TW at the 5 th week and serum testosterone levels in male layer breeders at the 4 th week post-treatment [18]. Astuti et al. [24] showed that 0.18 mg/200 g BW of BCSP supplemented for 50 days was able to increase testosterone levels and showed aromatase enzyme blocking action in the brain and testes of rats as evidenced by reduced CYP19 aromatase expression in immunohistochemical (IHC) staining. However, Yuneldi et al. [5] showed that A. granosa shell powder as a NAB increased crowing frequency, BW, and testosterone levels. The application of the NAB from BCSP has been proven to increase testosterone levels. Aromatase blocker is known to affect growth, pectoralis muscle performance, and testicular organs in animals. However, the use of the NAB from BCSP in male pelung cockerels has not been investigated.
Therefore, this study aimed to evaluate the effect of BCSP as a NAB on growth, pectoralis muscle performance, and testicular organs in pelung cockerels.

Ethical approval
All the research procedures have been approved by the Ethics Committee of Integrated Testing and Research, Universitas Gadjah Mada (Approval no. 00020/04/LPPT/V/2020).

Study period and location
The study was conducted from September 2021 to March 2022 at the pelung cockerels farm in Bantul, Yogyakarta, Animal Development Structure Laboratory, Faculty of Biology and Physiology Laboratory, Faculty of Veterinary Medicine, Universitas Gadjah Mada, Indonesia.

Preparation of the A. granosa BCSP
Anadara granosa BCSP was processed according to the procedure outlined by Yuneldi et al. [5]. After discarding the meat from the shells by boiling, the shells were cleaned and sundried for 1-2 days. Subsequently, the shells were boiled in NaOH solution (1.0 N) at 50°C for 3 h, rinsed under running water, and dried at 120°C for 6-8 h before being ground into a powder. The prepared BCSP was analyzed for its mineral content by inductively coupled plasma analysis. The clam shell of A. granosa contains Zn=61.55 mg/kg, Mg=1666.09 mg/kg, Fe=600.54 mg/kg, Ca = 41.4 mg/dL, Na=9262.98 mg/kg, and K=369.29 mg/kg [24].

Experimental design and parameters
The study used 16 male pelung cockerels (sample size was small as the bird falls under the threatened species category) aged 40-56 weeks. The physiology performance of male pelung cockerels generally degrades from 40 to 56 weeks. To distinctively evaluate the effect of the NAB treatment, this specific age of the chicken was chosen in this study. The BW of the pelung cockerels was ±3 kg and they were randomly divided into four treatment groups: T0 (control); T1 (BCSP [A. granosa] at 0.9 mg/kg BW); T2 (zinc sulfate [ZnSO 4 ] at 0.9 mg/kg BW); and T3 (testosterone at 3 mg/day). Each treatment group contained four pelung cockerels (n = 4). The BCSP and ZnSO 4 were orally administered through gavage [5,18,24,37] and testosterone was administered subcutaneously [18,31]. The treatment was administered to the cockerels for 56 days. Drinking water and commercial breeder feed were provided ad libitum [38]. Before treatment, the pelung cockerels were allowed to acclimatize for 7 days. Measurements taken in growth performance were comb length and height, wattle area, and chest circumference (CC). The measurements for pectoralis muscle observation were pectoralis muscle weight (PMW), area of the pectoralis muscle (APM), FA, fascicle myofibers (NM), MA, and proliferating cell nuclear antigen (PCNA)-positive cells. The measurements for testes were TW and diameter of the seminiferous tubules (DST). Measurements of CC, comb length and height, and wattle area were collected fortnightly on days 0, 14, 28, 42, and 56.

Comb morphometry, wattle, and CC
Comb and wattle measurements were performed using a pair of calipers, while the CC was taken using a measuring tape [14,39,40].

Euthanasia and organ preparation
On the 56 th day, the cockerels were sacrificed using the halal method. The chest muscles and testicles were collected and washed with 0.9% NaCl [41,42]. The testes and left pectoralis muscle were weighed and right muscle was measured using ImageJ software [32]. Subsequently, they were processed for histological examination [18,32].

Hematoxylin and eosin staining (H & E)
For histological analysis, the pectoralis muscle and testes were fixed in 10% neutral buffer formalin (NBF) solution for 18-24 h and stained with H and E (Merck, Darmstadt, Germany) [24,32,43]. Furthermore, the slides were observed under a microscope (Leica DM 750, Germany) with magnifications of 10 × 10 and 40 × 10. The area of myofibers and fasciculus were measured, and the number of myofibers in one fascicle enumerated. Testicular histological slides were observed and the DST measured. These measurements were obtained using ImageJ software [32].

Immunohistochemical staining
The pectoralis muscle samples were fixed in 10% NBF solution for 18-24 h; they were, then cut into 5 µm thick sections and placed onto a poly-llysine coated slide. The slides were stained with the immunohistochemistry method using EnVision + system -HRP (DAB) with anti-PCNA primary antibody (PC10) ab29 (Abcam, USA) to detect cell proliferation. The secondary antibody used was polyclonal goat anti-mouse immunoglobulins (IgG) (Dako, Glostrup, Denmark) and color development was enhanced using diaminobenzidine (Dako). The preparations were mounted using Entellan (Merck) and covered with a 24 × 60 mm glass coverslip. Following indirect IHC staining, the slides were then observed for the muscle cell nuclei. Proliferating cell nuclear antigen-positive cells are indicated with brown nuclei in the pectoralis muscle cells (modification of 11,12). The percentage of PCNA-positive nuclei was calculated by counting the number of PCNA-positive nuclei divided by number of nuclei observed in the myofibers in one fascicle multiplied by 100% (modification of 11,12). Pectoralis muscle histological slides were examined, and the total myofiber in one fascicle was counted, and the area of fasciculus and myofiber was determined. The measurement of those parameters was obtained using ImageJ software [36,44].

Statistical analysis
All parameters were statistically analyzed with one-way analysis of variance using a statistical package for the social sciences software v.26.0 (IBM, NY, USA) with a 95% confidence level (α = 0.05). The analysis was confirmed with Duncan's test [45].

Comb and wattle
The comb length, height, and wattle area of the pelung cockerels at day 56 did not show significant differences (p > 0.05) between the treatments (Table-1).

Chest circumference
The CC at day 56 showed that T1 was significantly higher (p < 0.05) than those of T0 and T2 (Table-2). T1, T2, and T3 were significantly higher than T0 (Table-2 The weight and APM showed no significant difference between all the treatments (p > 0.05) ( Table-3). However, the FA, NM, MA, and PCNApositive cells showed that the T3 pelung cockerels had significantly higher values (p < 0.05) compared to the other treatments, T1 was significantly different (p < 0.05) from T2 and T0, and T2 was significantly different (p < 0.05) from T0 (Table-3 and Figure-1).

Testis weight (TW) and seminiferous tubule diameter
The statistical analysis result of DST (Figure-2) and TW showed that the T3 pelung cockerels were significantly reduced (p < 0.05) compared to the other groups (Table-4).

Comb and wattle 56 days post-treatment
The length and height of the comb, and the wattle area were not significantly different as chickens used in this study were adult pelung cockerels. The growth period of the comb and wattles was slow and some had reached full growth size. These results are in agreement with Astuti et al. [17], that demonstrated that the administration of Anadara nodifera shell powder as a NAB at doses of 3.3 and 6.6 g/day, ZnSO 4 0.45 mg/kg BW, and testosterone 0.1 mL/day for 35 days did not increase the size of the comb and wattle in adult Bangkok cockerels. This was also shown by Yuneldi et al. [18], where the administration of BCSP as a NAB at a dose of 0.036 mg/40 g BW and ZnSO 4 0.018 mg/40 g BW for 35 days could not increase the length and height of the day old chick (DOC) comb of male layers. The results showed that the administration of BSCP was able to increase the CC in pelung cockerels. This may be due to the Zn contained in the BCSP as a NAB, which contributes to the biological activity and metabolic action in the digestion of pelung cockerels. This is in agreement with Sandoval et al. [46] and Bartlett and Smith [47] that Zn can improve the growth and health performance of broilers. In addition, Kakhki et al. [48] reported that supplementation with Zn at a dose of 120 mg/kg affected the growth performance of broiler breast muscles. Besides acting as a NAB, Zn also acts as an antioxidant [49]. Reed Available at www.veterinaryworld.org/Vol.16/March-2023/7.pdf et al. [50] reported that Zn positively contributes to the digestive system in chickens. It is estimated that the mineral composition and physiological function of the BSCP are involved in the metabolic system which has an impact on increasing the pelung cockerels CC. Another possibility is the added protein content in the BCSP. The previous research have been reported that the protein content in chicken feed has a considerable influence on the growth of chickens and increases the efficiency of their digestive system [32,34,35].
This study showed that the treatments in groups T1, T2, and T3 were effective in improving the FA, NM, MA, and PCNA-positive cells of pelung cockerels compared to T0. Group T3 was significantly higher than the other groups. Group T1 was significantly higher than groups T2 and T0. T2 group was higher than T0. The Zn contained in BCSP can increase testosterone which further affected the FA, NM, MA, and PCNA-positive cells. These results are in accordance with Yuneldi et al. [5] and Astuti et al. [19,24], who showed that A. granosa shell powder at 0.9 mg/kg BW, 0.3 mg/30 g BW, and 0.18 mg/200 g BW was capable of increasing the testosterone levels in pelung cockerels, canaries, and rats. According to Li et al. [7], the elevation of testosterone can significantly increase the FA, NM, and MA, and according to Taylor et al. [51],  Mean with different superscripts within the same column are significantly different (p < 0.05). g-i Mean with different superscripts within the same row are significantly different (p < 0.05). No superscript indicates no significant difference. T0=Control, T1=Blood clam shell powder (A. granosa) 0.9 mg/kg BW, T2=ZnSO 4 0.9 mg/kg BW, T3=Testosterone 3 mg/day, SD=Standard deviation, T=Treatment, CC=Chest circumference Table- [52] demonstrated that Zn plays an important role in cellular proliferation and differentiation. The possible mechanism by which BCSP as a NAB can increase testosterone levels is that in the endoplasmic reticulum, Zn 2+ as a second messenger activates CK2, which adds a phosphate group (phosphorylation) to aromatase [53][54][55]. High intracellular phosphorylation can inhibit aromatase enzyme activity which inhibits the conversion of testosterone to estradiol [5,24,55,56]. This causes an elevation of the hormone testosterone [19,51,[55][56][57]. This is supported by Yuneldi et al. [18], who demonstrated that BCSP at 0.036 mg/40 g BW as a NAB increased testosterone levels in male layer chicks. High testosterone levels and balanced nutritional intake (adequate protein) can affect muscle mass and strength [58]. High testosterone from the mechanism of Zn as a NAB increases the number of active satellite cells in the skeletal muscle and transcription processes occur in the myonucleus [6].
There are two pathways of testosterone action in skeletal muscle. First, the androgen receptor located in the myonucleus binds to the hormone testosterone. This process increases the production of insulin-like growth factor 1, which stimulates increased protein synthesis and suppresses protein catabolism [59]. Second, the activated satellite cells express myogenic regulatory factors during skeletal muscle development, initiate proliferation and differentiation processes, and fuse to form new myotubes [6]. Furthermore, it undergoes myofiber maturation and increases the number of new myonuclei [59]. The above explanation demonstrates the process that occurs during muscle hyperplasia and hypertrophy. A similar concept has been affirmed by the work of Hughes et al. [60]. High testosterone accompanied by adequate nutrition can affect muscle mass and strength [58]. In addition, the myostatin gene or growth and differentiation factor 8 belong to the transforming growth factor-beta superfamily, serve as skeletal muscle growth regulation and differentiation factors [61,62].
Besides functioning as a NAB, Zn can also act as an antioxidant. According to Prasad and Kucuk [63] and Rouhalamini et al. [64], Zn can perform an antioxidant role that can reduce oxidative stress in chickens due to heat stress. Heat stress in chickens can cause muscle protein degeneration and a decrease in size and MA, which ultimately reduces muscle mass [31]. According to Chand et al. [65], Khan et al. [66], and Naz et al. [67], the function of Zn in chickens is to overcome heat stress. Zinc administration as an antioxidant can reduce oxidative stress and therefore improves meat quality [64] by increasing MA and FA [31]. Increased FA may be associated with increased NM and MA. According to Shah Mean with different superscripts within the same column are significantly different (p < 0.05). T0=Control, T1=Blood clam shell powder (BCSP) (A. granosa) 0.9 mg/kg BW, T2=ZnSO 4 0.9 mg/kg BW, T3=Testosterone 3 mg/day, SD=Standard deviation, T=Treatment, TW: Testis weight  et al. [68], an increase in FA is associated with an increase in the amount of myofibers and MA because the fasciculus consists of a collection of myofibers, and the increased fascicle cross-sectional area is associated with the increased diameter and cross-sectional area of myofibers in the pectoralis muscle of chickens [31]. Muscle growth can be observed from FA, NM, and MA measurements. Muscle growth after birth is noted by the increased size of myofibers or number of myofibers [69]. The increase of muscle mass during postnatal growth occurs due to muscle hypertrophy and hyperplasia [70][71][72]. Another possible factor affecting the parameters of FA, NM, and MA other than the minerals, is the protein content of the BCSP. According to Saragih et al. [36], the MA of broiler and pelung chickens that are fed a high protein feed on days 7 and 14 was increased compared to the broiler and pelung chickens fed a low protein feed. These studies results are consistent with those of Saragih et al. [32] and Paunesku et al. [33] in that providing sufficient protein in chicken feed is important for pectoralis muscle growth and protein is known to stimulate satellite cell proliferation in regenerating myofibers for improved muscle growth.
The results of FA, NM, MA, and cells that were positive for PCNA measurements of pelung cockerels treated with testosterone (T3) showed a significant increase compared to the other treatments. According to Li et al. [7], the administration of exogenous testosterone significantly increases the NM and MA of the pectoralis muscle of chickens and supported muscle growth by increasing the proliferation of chicken muscle cells. According to Josiak et al. [6], it was suggested that the active satellite cells proliferate and differentiate to form new myotubes. Furthermore, myofiber maturation occurs and increases the number of new myonuclei. This supports the process of muscle hyperplasia and hypertrophy [6]. Other studies have affirmed this concept [7,[73][74][75][76].

Testicular weight and DST 56 days post-treatment
The TW and DST of the pelung cockerels on treatments T0, T1, and T2 did not show any significant differences. The effect of BCSP on the DST has not been previously reported. According to Yuneldi et al. [18], using BCSP as a NAB at a dose of 0.036 mg/40 g BW and treatment with ZnSO 4 0.018 mg/40 g BW had no effect on the TW of DOC male layers. A similar result with A. nodifera shell powder as a NAB was obtained at a dose of 3.3 g/day and ZnSO 4 0.45 mg/kg for 35 days for Bangkok chickens [17]. According to Adelakun et al. [77], the ZnSO 4 treatment in rats for 56 days did not affect the DST compared to the controls. In addition, ZnSO 4 treatment to mice for 30 days did not affect the DST compared to the controls [78]. Thus, in accordance with the above-mentioned studies, BCSP and ZnSO 4 treatment did not affect the TW and DST.
The results of this study on the TW and DST showed that pelung cockerels treated with testosterone (T3) experienced a significant decrease compared to other treatments. According to Yuneldi et al. [18], the continuous administration of 3 mg/day of testosterone for 35 days in male layer chicken significantly reduces the TW. Similar results of continuous administration of testosterone at 4 mg/head/day to Bangkok chickens for 35 days led to a decrease in TW [17]. The application of exogenous testosterone suppresses the secretion of endogenous testosterone through a negative feedback mechanism in the hypothalamic-pituitary-anterior axis, luteinizing hormone (LH), and follicle-stimulating hormone (FSH) [79]. Disturbances in the secretion of endogenous testosterone, LH, and FSH hormones can lead to decreased TW or testicular atrophy in rats [80]. According to Amer and Selim [20], administering exogenous testosterone at a dose of 5 mg/kg for 6 days/week for 4 weeks caused seminiferous tubule atrophy and reduced the DST in rats. Testosterone propionate at a dose of 3 mg/100 g BW for 60 days also reduced the DST in rats [81], and the increased serum testosterone levels caused by the administration of exogenous testosterone inhibited the hypothalamic-pituitary-testicular axis in rats [20]. According to Yama et al. [82], the decrease in DST could be caused by the inhibition of LH secretion by the anterior pituitary, which functions to stimulate the growth and number of Leydig cells in mice. Furthermore, when LH is inhibited that the Leydig cells are inactivated and do not produce testosterone, resulting in a decreased testosterone level in the testes [83]. This lack of testosterone and FSH levels is predicted to cause seminiferous tubular atrophy in rats [20]. When the Leydig cells are not stimulated by the hypothalamus and pituitary gland to synthesize testosterone for a relatively extended period of time, testicular degeneration occurs and inhibition of FSH and LH secretion causes a reduction in the DST [83].

Conclusion
The oral administration of BCSP as a NAB at a dose of 0.9 mg/kg BW (T1) for 56 days improved the growth performance and pectoralis muscle, especially the CC, FA, NM, MA, and PCNA-positive cells, but did not affect the PMW, APM, and testes of the pelung cockerels. However, administering testosterone at 3 mg/day (T3) for 56 days caused the side effect of reduced TW and DST and induced atrophy of the seminiferous tubules of pelung cockerels.

Authors' Contributions
PA, CMA, HTSS, and RFY: Planned, designed, and contributed to the experiment. PA, CMA, HTSS, RFY, SS, ARA: Recorded and analyzed the samples and drafted and edited the manuscript. All authors have read, reviewed, and approved the final manuscript.