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. 2024 Jun 26;103(10):104030. doi: 10.1016/j.psj.2024.104030

A comparative study between curcumin and curcumin nanoparticles on reproductive performance and antioxidant system of aged roosters

Hamid Reza Behboodi ⁎,1, Firooz Samadi *, Ahmad Riasi †, Mojtaba Najafi *, Mahdi Ansari *, Mehdi Ebadi ‡
PMCID: PMC11364113  PMID: 39127009

Abstract

Increased lipid peroxidation and decreased antioxidant status can result in reduced reproductive activity and fertility in aged male broiler breeders. This study was conducted to evaluate the effects of curcumin supplements (natural or nanoparticles) on the sperm characteristics, antioxidant system, fertility, and hatchability of aged roosters (54–64 wk), and to estimate the relative bioavailability value (RBV) of nano-curcumin on the measured parameters including the hypo-osmotic swelling test (HOST), motility, viability, sperm count, volume, the concentration of testosterone, glutathione peroxidase (GPx), and superoxide dismutase (SOD), diameter of the spermatogenic tube (DST), epithelium thickness (EpiTh), spermatogonia count (SPcount), fertility, hatchability, and relative weight of testis (RW-testis). A total of 30 roosters were individually caged and randomly assigned to 5 treatments comprising control (without curcumin as the basal diet), basal diet + 15 mg/kg curcumin (CUR15), basal diet + 30 mg/kg curcumin (CUR30), basal diet + 15 mg/kg nano-curcumin (Nano15), and basal diet + 30 mg/kg nano-curcumin (Nano30) for 10 wk. The slope ratio method was used to estimate the bioavailability of nano-curcumin by regressing each response on supplemental curcumin intake. Increasing dietary curcumin (P < 0.001) elicited a linear response to all studied traits. The RBV for volume, viability, motility, HOST, RW-testis, and GPx were estimated as 135 (CI: 115–156%), 143 (CI: 114–173%), 159 (CI: 122–196%), 132 (CI: 107–157%), 195 (CI: 126–264%), 176 (CI: 103–249%), and 178% (28–328%), respectively. Our findings revealed that curcumin nanoparticles enhance the reproductive efficiency of aged breeder roosters. In addition, the curcumin nanoparticles RBV exceeded that of natural curcumin, suggesting that lower concentrations of curcumin nanoparticles could have a significant effect on reproductive characteristics.

Key words: curcumin nanoparticle, fertility, reproductive performance, aged rooster

INTRODUCTION

Fertility and hatchability have a great impact on economic outcome in poultry industry. The reproductive rooster begins to ejaculate at the 25th week, the maximum fertility rate is reached around 40 weeks. Decline of fertility after45-50th week of age is one of the problems that exists in broiler breeder flocks (Yarmohammadi Barbarestani et al., 2023). The usual male to female proportion in broiler breeder flocks is of 1 rooster to each 10 hens, revealing the role and importance of roosters in broiler breeding. Curcumin, also known as diferuloylmethane (C21H20O6), is a hydrophobic polyphenolic phytocompound present in the rhizomes of the turmeric plant (Curcuma. spp) belonging to the family of Zingiberaceae. It is the primary component of turmeric powder, commonly gathered as a culinary spice and traditional drug in various Asian countries. Turmeric, extracted from the rhizome of the plant C. longa, has been acknowledged for its medicinal advantages for many years (Sureshbabu et al., 2023). However, its low bioavailability is a major limitation to realizing its full potential (Moniruzzaman and Min, 2020). Various techniques, including the production of nanomedical formulations, have been used to enhance the bioavailability of curcumin. One such formulation is the nanocurcumin particles, which have been shown to improve the oxidative stability of broiler chicken breast meat (Partovi et al., 2020). Oxidative stress, which occurs when the production of reactive oxygen species (ROS) in the body exceeds the antioxidant capacity, has been linked to various adverse effects on meat, including discoloration, off-flavors, and loss of nutritive value (Archile-Contreras and Purslow, 2011). The supplementation of nanocurcumin to a broiler chicken diet decreased the extent of oxidative damage in the meat, resulting in improved meat quality and stability (Partovi et al., 2020). Likewise, diminishing the potency of the antioxidant system with aging results in sperm abnormalities and low fertility in aged roosters (Lagares et al., 2017). Akhlaghi et al. (2014a) demonstrated that improving the antioxidant system of old Cobb 500 breeder males significantly mitigated the effects of oxidative stress on spermatozoa along with a higher fertility rate without adversely affecting their hatching traits. Curcumin has shown many pharmacological activities in both preclinical and clinical studies. Many technologies have been developed and applied to improve the solubility and bioavailability of curcumin, especially the nanotechnology-based delivery systems (Sun et al., 2012). However, there has been evidence that specific nanoparticles have potential reproductive toxicity in practice. However, the clinical application of curcumin in animal production has long been hampered by its poor solubility in aqueous solvents, its low oral bioavailability, chemical instability, and the rapid degradation in practice (Karthikeyan et al., 2020). Therefore, many technologies have been developed to overcome this limitation, including liposomes, phospholipid complexes, micro-emulsions, polymeric micelles, and nanoparticles (Bisht and Maitra, 2009; Liu et al., 2016). Wu et al. (2023) showed that dietary curcumin supplementation (200 mg/kg of diet) in breeder roosters challenged with H2O2 decreased the abnormal sperm rates in the semen, which led to an improvement in seminiferous tubules and an increase in testis scores and serum testosterone levels. The study further showed that curcumin has potent antioxidant properties, as it reduces the oxidative damage caused by H2O2. This was shown by an increase in the capacities of antioxidant enzymes, such as glutathione peroxidase (GPx) and superoxide dismutase (SOD). Zhou et al. (2020) found that curcumin could positively impact asthenospermia, a condition in which sperm has impaired motility or movement ability. Curcumin effectively reduces the production of reactive oxygen species (ROS), which are molecules linked to various disorders or conditions, including infertility, by activating the nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway and suppressing the expression of ROS-generating enzymes, ultimately enhancing sperm quality (Lin et al., 2019). Curcumin enhanced both the reproductive performance of aged roosters and the laying hen performance, positively impacting egg production and quality (Mirbod et al., 2017).

Overall, it is well known that curcumin has antioxidant properties. It can help to protect sperm from oxidative stress, an overproduction of ROS, resulting in the damage of sperm DNA and decreased motility. The present study is the first to explore the relative bioavailability value of 2 sources of curcumin, natural and nanoparticles, specifically for reproductive performance and antioxidant status in aged roosters, which is a critical area of research given the significant decline in reproductive performance and antioxidant capacity that occurs with age in these birds.

MATERIALS AND METHODS

Bird Management

Thirty 50-wk-old Ross 308 breeder roosters were placed in individual cages (40 × 30 × 50 cm) and randomly assigned to 5 treatment groups (6 replicates of 6 birds each) in a completely randomized design. During a 10-wk experimental period, roosters were weighed weekly. Meanwhile, the photoperiod remained constant at 14 hours of light and 10 h of darkness; the room temperature was maintained at a comfortable range of 21 to 23°C. This study was approved by the Animal Care Committee of the Gorgan University of Agricultural Sciences and Natural Resources, Gorgan, Golestan, Iran (Protocol No; 1399/165943/388).

Experimental Diets

The basal diet was formulated to meet all nutritional requirements recommended by Aviagen, and the experimental diets were prepared by adding 15 and 30 mg/kg of curcumin as natural (CUR) or nanoparticles (Nano). The doses used were based on reports by Kazemizadeh et al., 2019. This resulted in 5 experimental diets, including control (unsupplemented curcumin), CUR15, CUR30, Nano15, and Nano30 (Table 1). The experimental diets were fed for 2 wk as an adaptation period (52–54 wk).

Table 1.

Composition of the basal diet.

Ingredient Amount (g/kg)
Corn 654.2
Soybean meal (42%) 65.0
Wheat bran 238.0
Corn oil 10.0
Di-calcium phosphate 13.0
Oyster 9.40
NaHCO3 1.00
NaCl 3.20
Mineral premix1 2.50
Vitamin premix2 2.50
DL-methionine 1.20
Nutrient composition
AME (MJ/kg) 11.30
CP (g/kg) 115.4
Methionine (g/kg) 3.10
Ca (g/kg) 7.30
Pavailable (g/kg) 3.40
Na (g/kg) 1.70
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1

The mineral supplement provided the following amounts per kilogram of feed: manganese (manganese oxide) 120 mg, iron (iron sulfate) 50 mg, copper (copper sulfate) 10 mg, iodine (potassium iodate) 2 mg, zinc (zinc oxide) 110 mg

2

The vitamin supplement provided the following amounts per kilogram of feed: vitamin A (vitamin A acetate) 12,000 IU, vitamin D3 3,500 IU, vitamin E (D-L-alpha-tocopherol acetate) 100 IU, riboflavin 12 mg, Niacin 50 mg, pantothenic acid 13 mg, pyridoxine (pyridoxine hydrochloride) 6 mg, folic acid 2 mg, cobalamin 0.03 mg, biotin 0.66 mg.

Preparation of Curcumin Nanoparticles

The preparation of niosomes containing curcumin involves the use of Tween 60, cholesterol, polyethylene glycol, and curcumin (Figure 1). The components were dissolved in chloroform at a temperature of 45°C on a rotary evaporator (Heidolf, Germany). A dry thin film is formed by evaporating the chloroform under vacuum conditions. This film is then hydrated with sterile water at 65°C for 1 h. The prepared nanoparticles are reduced in size by a sonicator probe (E-Chrome-Taiwan) for 30 min, based on the thin layer coating method (Haghiralsadat et al., 2017).

Figure 1.

Figure 1

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Niosomes nano-curcumin structure. Niosomes, an alternative to liposomes, are vesicles composed of non-ionic surfactants, which are biodegradable, relatively nontoxic, more stable, and inexpensive (Kazi et al., 2010).

Testosterone Measurement

The testosterone level was measured using the Monobind ELISA kit (Monobind Inc., Costa Mesa, CA) according to the manufacturer's instructions. In brief, the process involved adding 10 µL of serum or standard samples to the wells of a 96-well plate, followed by adding 50 µL of Testosterone Enzyme Regnet solution and shaking the plate (Monobind Inc.). Next, 50 µL of Testosterone Biotin Reagent solution was added, shaken, and incubated at room temperature for 60 min. Afterward, the plate contents were emptied by aspiration, and 350 µL of washing solution was added and then emptied thrice for washing. A working substrate solution of 100 µL was added to all wells, and the plate was allowed to incubate at room temperature for 15 minutes. Finally, 50 µL of Stop Solution was added to each well and gently mixed for 15 to 20 s to terminate the reaction. The optical absorbance of the samples was read at a wavelength of 450 nm via a plate reader, and the results were expressed in ng/mL units. The inter- and intra-assay coefficients of variation were 7.4% and 4.3%, respectively.

Antioxidant Enzymes and Measurement

To measure the activity of glutathione peroxidase (GPx) and superoxide dismutase enzymes (SOD) as the units per gram of hemoglobin, red blood cells were used after centrifugation of heparinized blood samples. For the measurement of SOD enzyme activity, Ransaud's kit was used according to the method of Williams et al. (1983). This method involves using xanthine and xanthine oxidase to produce superoxide radicals that react with 2-4-iodophenyl-3-4-nitrophenol-5-phenyl tetrazolium chloride (INT) and produce a red color in formazan, a color reaction indicative of the presence of superoxide dismutase. The activity of superoxide dismutase is measured by inhibition of this reaction, and a spectrophotometer was used to measure the activity at a wavelength of 505 nm at a temperature of 37°C. One SOD unit is the amount that inhibits INT regeneration by 50% under the test conditions. Ransel and Ransod kits (Randox. Laboratories, Crumlin, UK) were used to measure glutathione peroxidase activity based on the method of Paglia and Valentin (1967). The reaction involves the conversion of reduced glutathione to glutathione disulfide, using H2O2 as the substrate, and the produced H2O2 is converted to H2O by glutathione peroxidase. Using a spectrophotometer, the activity of glutathione peroxidase is measured by the consumption of NADPH at a wavelength of 340 nm.

Semen Collection

The roosters were habituated (for 2 wk) by abdominal massage for semen collection and were subjected to their experimental diets for 10 wk. Seminal characteristics were evaluated every 4 wk between 54 and 64 wk.

Sperm Volume

Semen volume was measured by collecting a sample from each rooster in graduated microtubes.

Sperm Count

Sperm cells were counted using a hemocytometer according to a standard method. Briefly, 10 µL of sperm suspension (1:200) was transferred to each of the counting chambers of the Neubauer hemocytometer and allowed to stand in a humid condition for 5 min to prevent drying. The cells were counted under a microscope at 400 x magnification, and the counted sperms were expressed as the number of sperm per milliliter (Partovi et al., 2020).

Sperm Motility

The process of evaluating sperm motility in roosters was performed using the method of Santiago-Moreno et al. (2009). The process involves diluting the semen of roosters 1:20 with a modified Beltsville diluent. After diluting the semen, sperm motility was evaluated subjectively using an optical microscope with 400 x magnification. Sperms with medium to fast progressive motility were considered as shunt sperms. The term “shunt” refers to the ability of sperm to swim forward and progress in a straight line.

Sperm Viability

A total of 20 µL of sperm suspension (at a dilution of 1:10) was mixed with an equal volume of 0.05% Eosin Y. Then, 20 µL Nigrosin was added to the slide to differentiate the dead and alive spermatozoa. The slide was examined using a light microscope with 400 x magnification after 2 min of incubation at room temperature. A sample of 200 spermatozoa was counted, and their viability percentage was recorded (Yarmohammadi Barbarestani et al., 2024). To evaluate the integrity of the sperm plasma membrane and sperm abnormalities in roosters by using the eosin-nigrosin staining method. The process involves diluting the semen of roosters 1:20 with 2.9% sodium citrate solution and mixing it with 4% eosin solution and 8% nigrosin solution. This mixture is called the eosin-nigrosin dye. To assess sperm plasma membrane integrity, 10 µL of eosin-nigrosin dye was added to a slide, followed by 10 µL of diluted semen (1:20), and the mixture was thoroughly pipetted for 30 seconds. After spreading the mixture onto another slide and incubating at 37°C, the dried slides were viewed under an oil lens at 1,000 x magnification. Dead sperm, identifiable by red staining due to membrane defects, contrasts with live sperm with intact membranes. Evaluation involves counting 200 sperm to determine the percentage of live and dead sperm. Additionally, abnormal sperm morphology, such as twisted or double tails and abnormal heads, can be identified by inspecting 200 sperm using the staining method (Silyukova et al., 2022).

Hypo-osmotic Swelling Test

This section outlines a method for assessing rooster sperm plasma membrane function using the hypo-osmotic swelling test (HOST) (Santiago-Moreno et al., 2009). The procedure involves combining 10 µL of semen with 500 µL of a 1:100 solution of sodium citrate and distilled water, followed by a 30-min incubation at 37°C. Subsequently, a drop of the sample was placed on a warm slide and spread with a stain solution containing eosin-nigrosin dye (Přinosilová et al., 2014). Following staining, the slide was viewed under a microscope equipped with an oil lens at 1,000 x magnification, with 200 sperm counted per slide. Sperm, with an optimal osmotic reaction range of 320 to 375 mmol, exhibit swift tail coiling in this environment, indicative of intact plasma membrane function. Conversely, sperm lacking this response are deemed non-viable, reflecting impaired plasma membrane integrity.

Fertility and Hatchability

Fertility and hatchability were evaluated using artificial insemination with the recommended technique (Akhlaghi et al., 2014b). A total of 75 broiler breeder hens (n = 15/group) were provided by Grant Parent Poultry Company, Mazandaran, Iran. Semen samples obtained from 4 roosters in each treatment were mixed and diluted with lake buffer, resulting in an inoculation dosage of 250 μL for each chicken. The inoculation was performed twice a wk at 4 pm. Artificial insemination (AI) was conducted during the 11th (first and second days) and 12th (first day) weeks of the experiment. Starting from 2 d after the first AI, eggs were collected daily for a period of 10 consecutive days. These eggs were then stored at a temperature of 13°C and a humidity level of 75% until they were ready for incubation. About 200 hatchable eggs (50 eggs per week), after disinfection with 15 mL of formalin and 0.6 g of permanganate, were placed in a fully automatic incubator (Victoria, Italy) at a temperature of 37.7°C. On the 10th day of incubation, the fertility rate of the eggs was evaluated with a candling device, and on the 21st day of hatching, the hatching rate of the treatments was calculated based on the number of fertilized eggs laid in the machine. For histopathological evaluation, both testes were excised and weighed that was fixed in neutral buffered formalin (10%), stored at -4°C, embedded in paraffin, sectioned (5–6 μm), and stained with hematoxylin-eosin. Morphological data were generated from 20 optical imagers randomly selected from 4 cross-sections of each testis using an optical microscope (Zeiss) equipped with an eyepiece camera (Labomed Inc., Los Angeles, CA) and analyzed using J Image software (National Institutes of Health, Bethesda, MD). The diameter and number of seminiferous tubules and thickness of the seminiferous epithelium were determined from 20 randomly selected seminiferous tubule segments (Islam et al., 2010).

Statistical Analysis

All data analyses, including one-way ANOVA, one-sample t test, repeated measurements, and slope ratio method were employed by SAS (2002). The NLIN procedure was used to estimate the relative bioavailability value (RBV) of curcumin nanoparticle to curcumin (Littell et al., 1997). Before estimating the RBVs, all data were tested for statistical and fundamental validity (Finney, 1978) The mean differences were analyzed by Tukey's multiple comparison tests at P < 0.05.

RESULTS

As shown in Table 2, the sperm motility, HOST, viability, sperm cell count, and sperm volume increased, either linearly or quadratically (P < 0.001), with increasing dietary curcumin regardless of the source. The effect of time was significant for all these traits except for sperm volume. The concentrations of testosterone (P < 0.001), GPx (P < 0.001), and SOD (P < 0.03) were linearly increased with increasing curcumin concentration (Table 3). As stated in Table 4, Table 5, DST, EpiTh, SPcount, fertility, hatchability, and RW-testis were linearly increased with increasing dietary curcumin levels (P < 0.001).

Table 2.

The effect of different concentrations of the natural (CUR) and nano-curcumin (Nano; 15 and 30 mg/kg) on sperm motility, hypo-osmotic swelling test (HOST), viability, sperm cell count, and sperm volume of rooster sperm.

Treatment Motility (%) HOST (%) Viability (%) Sperm cell count (sperm/mL) Volume (mL)
Control 68.38c 60.92e 67.38d 2.60d 0.26e
CUR15 72.58c 66.13d 71.46d 2.75d 0.30d
CUR30 80.83b 75.96b 81.96b 3.50b 0.38b
Nano15 78.13b 70.71c 77.42c 3.07c 0.33c
Nano30 87.08a 79.58a 86.71a 3.91a 0.42a
SEM 1.18 0.83 0.99 0.08 0.01
P-value
 Treat < 0.001 < 0.001 < 0.001 < 0.001 < 0.001
 Time < 0.001 0.003 < 0.001 < 0.001 0.408
 Treat × Time < 0.001 0.001 < 0.001 < 0.001 0.001
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Alphabetical superscripts in a row indicate a significant difference between the means (p <0.05 or p <0.01).

Table 3.

The effect of different concentrations of natural (CUR) and nano-curcumin (Nano; 15 and 30 mg/kg) on the concentration of testosterone, glutathione peroxidase (GPx), and superoxide dismutase (SOD).

Treatment Testosterone (ng/mL) GPx (U/mL) SOD (U/mL)
Control 3.52b 400c 43.3c
CUR15 3.68b 420c 46.0c
CUR30 4.75a 663ab 68.0a
Nano15 3.91b 600b 43.7c
Nano30 5.26a 821a 53.0b
SEM 0.14 45.2 2.32
Curcumin (mg/kg)
0.0 3.52c 400c 43.3b
15 3.80b 510b 44.8b
30 5.01a 742a 60.5a
SEM 0.09 29.6 1.29
Source
Natural 3.98b 494b 52.4a
Nano 4.23a 607a 46.6b
SEM 0.08 24.2 1.06
Probabilities
Curcumin < 0.001 < 0.001 < 0.001
Source 0.030 0.003 < 0.001
 Curcumin × Source 0.174 0.081 < 0.001
 Linear < 0.001 < 0.001 < 0.001
 Quadratic < 0.001 0.11 < 0.001
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Alphabetical superscripts in a row indicate a significant difference between the means (p <0.05 or p <0.01).

Table 4.

The effect of different concentrations of the natural (CUR) and nano-curcumin (Nano; 15 and 30 mg/kg) on the diameter of spermatogenic tubes (DST), and epithelium thickness (EpiTh), and spermatogonia count (SPcount).

Treatment DST (μM) EpiTh (μM) SPcount
Control 585e 136c 185c
CUR15 658d 177b 194c
CUR30 701b 214a 275b
Nano15 682c 148c 208c
Nano30 761a 217a 301a
SEM 3.61 3.00 4.70
Curcumin (mg/kg)
0.0 585c 136c 184c
15 670b 163b 201b
30 731a 216a 288a
SEM 2.53 1.98 3.20
Source
Natural 648b 176a 218b
Nano 676a 167b 231a
SEM 2.07 1.62 2.61
Probabilities
Curcumin < 0.001 < 0.001 < 0.001
Source < 0.001 < 0.001 < 0.001
 Curcumin × Source < 0.001 < 0.001 0.022
 Linear < 0.001 < 0.001 < 0.001
 Quadratic < 0.001 < 0.001 < 0.001
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Alphabetical superscripts in a row indicate a significant difference between the means (p <0.05 or p <0.01).

Table 5.

The effect of different concentrations of the natural (CUR) and nano-curcumin (Nano; 15 and 30 mg/kg) on the fertility, hatchability, and relative weight of testis (RW-testis).

Treatment Fertility (%) Hatchability (%) RW-testis (%)
Control 70.2c 71.0c 0.43e
CUR15 71.2b 71.0c 0.50d
CUR30 90.0a 88.3a 0.62b
Nano15 71.2b 72.0b 0.55c
Nano30 90.2a 89.0a 0.86a
SEM 1.28 1.34 0.03
P-value
 Model < 0.001 < 0.001 < 0.001
 Linear < 0.001 < 0.001 < 0.001
 Quadratic 0.73 0.95 0.03
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Alphabetical superscripts in a row indicate a significant difference between the means (p <0.05 or p <0.01).

Among the studied traits, 7 responses were statistically validated for slope-ratio analysis (Table 6). As depicted in Figure 2, the RBV for volume, viability, motility, HOST, RW-testis, GPx, and VLDL were estimated as 135 (CI: 115–156%), 143 (CI: 114–173%), 159 (CI: 122–196%), 132 (CI: 107–157%), 195 (CI: 126–264%), 176 (CI: 103–249%), and 178% (28–328%), respectively. Based on the estimated CI, the RBV of nano-curcumin for volume, viability, motility, HOST, RW-testis, and GPx was significantly higher than curcumin, however, the RBV of nano-curcumin for VLDL showed an increasing trend relative to curcumin.

Table 6.

Statistical validity of the bird responses for the relative bioavailability value (RBV) analysis.

Item Probability (α = 0.05)
Motility (%) HOST (%) Viability (%) Sperm cell count (sperm/mL) Volume (mL) Testosterone (ng/mL) GPx (U/mL) SOD (U/mL) DST (μM) EpiTh (μM) SPcount Fertility (%) Hatchability (%) RW-testis (%)
Average slope <.0001 <.0001 <.0001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001
Slope difference <0.001 0.003 0.001 0.001 <0.001 0.008 0.002 <0.001 <0.001 0.024 0.001 0.935 0.588 <0.001
Blank 0.496 0.424 0.240 0.012 0.091 0.001 0.163 0.001 0.002 <0.001 0.012 <0.001 <0.001 0.070
Intersection 0.232 0.159 0.080 0.458 0.218 0.918 0.172 0.111 0.257 <0.001 0.458 0.968 0.757 0.150
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HOST, hypo-osmotic swelling test; GPx, glutathione peroxidase; SOD, superoxide dismutase; DST, diameter of spermatogenic tubes; EpiTh, epithelium thickness; EpiTh, spermatogonia count; RW-testis, relative weight of testis.

The highlighted figures revealed that the model did not meet the statistical requirements for the validity of the RBV analysis.

Figure 2.

Figure 2

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Relative bioavailability of the nano-curcumin for volume, viability, motility, hypoosmotic swelling test, the relative weight of testis, and glutathione peroxidase (GPx) based on the slope ratio method.

DISCUSSION

In the present study, we investigated the effects of curcumin supplementation in different forms (natural and nanoparticles) on reproductive traits, antioxidant capacity, and nano-curcumin bioavailability in aged breeder roosters. The results showed that both sources of curcumin significantly improved most studied traits, including sperm characteristics, fertility, and hatchability, confirming the potential of curcumin as a beneficial supplement for improving the reproductive performance of aged roosters. Additionally, the bioavailability analysis revealed that nano-sized curcumin had higher bioavailability than its natural counterpart, showing its potential for improving the reproductive health of aging breeder roosters.

The higher estimated RBV of nano-curcumin for the studied variables in aged breeder roosters indicated that low concentrations of curcumin nanoparticles had a significant effect on the reproductive traits of the birds. In the poultry industry, male breeder birds are crucial because of their significant contribution to determining the genetics and production performance of a breeding stock. Therefore, it is essential to maintain their reproductive efficiency to meet the increasing demand for high-quality poultry products. However, with age, the reproductive efficiency of breeder roosters tends to decline, leading to reduced fertility, hatchability, and semen quality. The results of the present study indicate that curcumin supplementation can improve the reproductive performance of aged breeder roosters. The improved reproductive performance of the birds was attributed to the antioxidant effect of curcumin (Ak and Gulcin, 2008). Curcumin is a natural antioxidant that has been shown to have multiple benefits, including antioxidant and anti-inflammatory properties (Antony et al., 1999). It has been shown that dietary curcumin supplementation significantly decreased abnormal sperm rates in the semen, notably improved seminiferous tubules, increased testis scores, and serum testosterone levels, enhanced sperm count and motility, and reduced the abnormality percentage (Karimi et al., 2019). Curcumin supplementation could also improve the redox damage caused by H2O2 by enhancing the capacities of antioxidant enzymes (e.g., GPx and SOD), and dietary curcumin supplementation could relieve H2O2-induced oxidative damage, and reproduction decline through the Nrf2 signaling pathway and anti-apoptotic effects in roosters (Wu et al., 2023). In addition to the Nrf2 signaling pathway, which we have already discussed, curcumin has been shown to affect other signaling pathways that are crucial for sperm function and fertility. One such pathway is the Keap1-Nrf2 pathway, which is involved in the regulation of antioxidant responses. Curcumin has been shown to activate this pathway by inhibiting the Keap1 protein, leading to the stabilization and nuclear translocation of Nrf2. This activation of Nrf2 leads to the transcription of antioxidant genes, such as glutathione peroxidase 4 (GPX4), which is involved in the detoxification of reactive oxygen species (ROS) and the protection of sperm from oxidative damage. Another important signaling pathway affected by curcumin is the PI3K/Akt pathway, which is involved in the regulation of sperm motility and capacitation. Curcumin has been shown to activate this pathway by phosphorylating and activating the PI3K/Akt signaling cascade, leading to the enhancement of sperm motility and capacitation. Additionally, curcumin has been shown to affect the NOX5 gene expression, which is involved in the regulation of sperm reactive oxygen species (ROS) production. Curcumin has been shown to increase NOX5 gene expression, leading to the enhancement of sperm motility and progressive motility. Furthermore, curcumin has been shown to affect the NF-κB signaling pathway, which is involved in the regulation of inflammation and oxidative stress. Curcumin has been shown to inhibit the NF-κB signaling pathway, leading to the reduction of inflammation and oxidative stress in sperm (Aparnak and Saberivand, 2019).

There has been an increasing interest in nanotechnology, particularly in the areas of diagnosis, medicine, and nutrition. Specifically, the utilization of nanoparticles in poultry nutrition has drawn attention over the past few years. It has also been shown that nanoparticles can enhance bioavailability and reduce the effective dose needed (Nabi et al., 2020), which could have a remarkable effect on poultry industry, where efficient nutrient supplementation is essential for optimum production performance (Nabi et al., 2020). Nanoparticles present a high bioavailability, which allows for more efficient absorption and utilization of essential nutrients by the body, and also improvement of overall health and wellness. Additionally, the smaller size of nanoparticles allows them to reach areas of the body that larger particles cannot reach and it leads to better delivery and efficacy of important nutrients (Gonzales-Eguia et al., 2009; Nabi et al., 2020). Nanoparticles, like nano-curcumin, can easily pass through cell membranes in organisms and quickly interact with biological systems. Therefore, the use of nano-curcumin could be an effective strategy for improving the bioavailability of curcumin, leading to enhanced absorption (Reda et al., 2020).

More recently, Abdelnour et al. (2020) showed that curcumin nanoparticles had beneficial effects on sperm quality. They revealed that nano-curcumin at low concentrations had not only a positive effect on total antioxidant capacity (TAC), SOD, GPx, and sperm progressive motility, viability, and membrane integrity but also significantly reduced sperm abnormalities, apoptotic rate as compared with other treatments. In the present study, we showed that the bioavailability of nano-curcumin was higher in its natural form, which could be more effective in trace amounts on the reproduction traits of older adult breeder roosters than in the natural form. In fact, nanoparticles of curcumin may be effectively absorbed and used by the cells, even at low concentrations, indicating that nanobiotechnology provided an efficient tool for increasing the availability of curcumin. The exceedingly high RBV of nano-curcumin for GPx (176%) and RW-testis (195%) indicates its antioxidant attributes may have caused the positive effects of curcumin on the testis functionality, providing more evidence for the high potential of nanocurcumin in improving the reproduction of the aged roosters (Abdelnour et al., 2020).

Raoofi et al. (2021) reported that curcumin nanoparticles can improve the sperms and stereological variables such as round spermatid and Leydig cells by enhancing the level of serum testosterone. Ismail et al. (2020) also showed that the nanocurcumin supplement improved the progressive motility, vitality, TAC and plasma membrane integrity of sperm compared to the control.

The HOST is a commonly used test to assess sperm quality in various domestic animal species. Evaluating the plasma membrane integrity of spermatozoa is crucial in determining their fertilizing potential, with the HOST being a key indicator. This test has become a key parameter for evaluating semen owing to its strong correlation with semen evaluation parameters (Zubair et al., 2015). Since the cell membrane comprises phospholipids with a high susceptibility to oxidation, nanocurcumin could strengthen membrane integrity via its oxidant resistance (Zhou et al., 2020). Curcumin may help regulate Nrf2 levels in spermatozoa and enhance mitochondrial function (Zhou et al., 2020). In asthenozoospermia, curcumin caused a significant reduction in lipid peroxidation in spermatozoa. Curcumin can decouple the keap1-Nrf2 complex, leading to Nrf2 stabilization and transportation into cell nuclei. The event triggers the transcription of antioxidant genes like GPx and SOD, which play a role in the antioxidant response (Xie et al., 2018).

In summary, this study emphasizes the promising role of curcumin, specifically in its nanoparticle form, as a valuable supplement for enhancing the reproductive health of aged breeder roosters. Supplementation of curcumin improves sperm characteristics, semen quality, fertility, hatchability, and antioxidant status in aged breeder roosters. However, additional research is required to verify the impact of nano curcumin supplementation on various reproductive characteristics and determine the ideal levels and timing of supplementation for maximizing reproductive efficiency in aging breeder roosters.

DISCLOSURES

The authors declare no conflicts of interest.

ACKNOWLEDGMENTS

The authors gratefully thank Gorgan University of Agricultural Sciences and Natural Resources (Gorgan, Iran), Makian Darou Golestan (Gorgan, Iran), and MAMco broiler integration (Babol, Iran) for facility support.

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